Receptors of Cell Adhesion and Cellular Recognition

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BiOMEMBRANES A Multi-Volume Treatise Volumes • 1996 RECEPTORS OF CELL ADHESION AND CELLULAR RECOGNITION

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BIOMEMBRANES A Multi-Volume Treatise RECEPTORS OF CELL ADHESION AND CELLULAR RECOGNITION Editor. A.G.LEE Department of Biochemistry University of Southampton Southampton, England

VOLUME 3 • 1996

(jSItt) JAI PRESS INC. Greenwich, Connecticut

London, England

Copyright © 1996 byJAI PRESS INC. 55 Old Post Road No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London, England WC2E 7PB All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any way, or by any means, electronic, mechanical, photocopying, recording, filming or otherwise without prior permission in writing from the publisher. ISBN: 1-55938-660-6 Manufactured in the United States of America

CONTENTS

LIST OF CONTRIBUTORS

vii

PREFACE A.G. Lee

ix

MOLECULES OF CELL ADHESION AND RECOGNITION: AN OVERVIEW Randall Marsh and Robert Brackenbury

1

CELL RECOGNITION MOLECULES OF THE IMMUNOGLOBULIN SUPERFAMILY IN THE NERVOUS SYSTEM Georgi Gegelashvili and Elisabeth Bock

33

THE T CELL RECEPTOR Carol Morgan and John D. Fraser

77

THE MAJOR PRESENTATION AND HISTOCOMPATIBILITY COMPLEX J. Colombani CADHERINS: A REVIEW OF STRUCTURE AND FUNCTION Julie Wallis, Robert Moore, Paul Smith, and Frank S. Walsh THE INTEGRIN FAMILY Ron D. Bowditch and Randall J. Faull THE SELECTIN FAMILY Mark A. Jutila

105

127

159

183

vi THE CD44 FAMILY OF CELL ADHESION MOLECULES: FUNCTIONAL ASPECTS Charles B. Underbill MEMBRANE-ASSOCIATED MUCINS Hans L Vos, Jelle Wesseling, and John Milkens

CONTENTS

205

219

PLATELET MEMBRANE GLYCOPROTEINS Kenneth j. Clemetson

231

IMMUNOGLOBULIN Fc RECEPTORS: DIVERSITY, STRUCTURE, AND FUNCTION P. Mark Hogarth and Mark D. Hulett

269

INDEX

315


Robert Brackenbury

Department of Cell Biology, Neurobiology, and Anatomy University of Cincinnati Medical Center

Elisabeth Bock

Protein Laboratory University of Copenhagen

Ron D. Bowditch

The Scripps Research Institute Lajolla, California

Kenneth J. Clemetson

Theodor Kocher Institut Bern, Switzerland

J. Colombani

Laboratoire Universitaire d'Immunologie et d'Histocompatibilite Institut d'Hematologie Hopital Saint-Louis, Paris

Randall j. Faull

The Scripps Research Institute Lajolla, California

John D. Fraser

Department of Molecular Medicine University of Auckland School of Medicine Auckland, New Zealand

Ceorgi Gegelashvili

Protein Laboratory University of Copenhagen

John Milkens

Division of Tumor Biology Netherlands Cancer Institute

P. Mark Hogarth

Austin Research Institute Austin Hospital Victoria, Australia

VIII


Carol Morgan

Department of Health, Science, and Technology UNITEC Institute of Technology Auckland, New Zealand

Mark D. Hulett

Austin Research Institute Austin Hospital Victoria, Australia

Mark A. Jutila

Veterinary Molecular Biology Montana State University

Randall Marsh

Department of Cell Biology, Neurobiology, and Anatomy University of Cincinnati Medical Center

Robert Moore

Department of Experimental Pathology Guy's Hospital, London

Paul Smith


Charles B. Underhill

Department of Anatomy and Cell Biology Georgetown University Medical Center

Hans L. Vos


Frank S. Walsh


Julie Wallis


Jelle Wesseling


PREFACE

The quantity of information available about membrane proteins is now too large for any one person to be familiar with anything but a very small part of the primary literature. A series of volumes concentrating on molecular aspects of biological membranes therefore seems timely. The hope is that, when complete, these volumes will provide a convenient introduction to the study of a wide range of membrane functions. Volume 3 of Biomembranes covers receptors of cell adhesion and cellular recognition. Proteins in the plasma membrane of cells are heavily involved in processes of cell adhesion, but such proteins were not actually isolated and characterized until the mid-1970s. Since then, application of the methods of molecular biology has led to the recognition of four major classes of cell adhesion molecule (CAMs), the immunoglobulin superfamily, the cadherins, the integrins, and the selectins. A convenient system in which to study the importance of cell adhesion is in blood platelets where aggregation eventually leads to thrombus formation in a process involving a range of surface glycoproteins. Interaction with the extracellular matrix is exemplified by CD44, the receptor for hyaluronan, a complex carbohydrate that is a major component of the extracellular matrix surrounding migrating and proliferating cells. Membrane-associated mucins have a variety of effects on cell adhesion. The superfamily of immunoglobulin related proteins also include the T cell receptors and the major histocompatibility complex (MHC), which, together with the receptors for immunoglobulins (the Fc receptors),

Preface

x

are of fundamental importance in the processes of immunity. Volume 3 of Biomembranes explores the structures and functions of the molecules involved in these important functions of the cell. As editor, I wish to thank all the contributors for their efforts and the staff of JAI Press for their professionalism in seeing everything through to final publication. A.G. Lee Editor

MOLECULES OF CELL ADHESION AND RECOGNITION: AN OVERVIEW

Randall Marsh and Robert Brackenbury

I. Introduction II. Historical Background ofCell Adhesion and Recognition A. Differential Cell Adhesion as a Developmental Mechanism B. Discovery of Cell Junctional Complexes C. Cell-Cell Interactions in Immune Responses III. Isolation and Identification of Adhesion Molecules A. Functional Assays for Identification of Adhesion Molecules B. Immunologicalldentificationof Cell Surface Antigens C. Isolation and Analysis ofCellJunctional Complex Constituents D. Polymerase Chain Reaction-based Isolation of Related Molecules IV. Families of Adhesion Molecules A. Immunoglobulin Superfamily B. Cadherin Family C. Integrin Family D. Selectin Family V. Relationship Between Adhesion Molecules and Cell Junctions A. The Adherens Junction and Cadherins B. The Desmosome, Cadherins, and Cadherin-like Molecules C. The Hemidesmosome and Integrins

Biomembranes Volume 3, pages 1-32. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-660-6. 1

2 2 2 4 4 5 5 6 6 6 6 7 10 11 12 13 ; . . 15 18 20

I

RANDALL MARSH and ROBERT BRACKENBURY D. The Tight Junction E. The Gap Junction VI. Conclusion References

20 21 22 23

1. INTRODUCTION The aim of this overview is to outline the growth of knowledge about cell adhesion—^from early experiments implicating differential cell-cell adhesion as an important developmental mechanism, to the methods used to successfully isolate and characterize cell adhesion molecules (CAMs), to our current knowledge about the structure and function of CAMs. CAMs are cell surface proteins that mediate direct contact between the plasma membranes of adjacent cells. Binding of CAMs to one another promotes the formation of intercellular junctions that hold cells together. CAM interactions also serve to bring together a wealth of ligands and receptors on the plasma membranes of the apposed cells. During embryonic development, interactions between these ligands and receptors, including interactions between the CAMs themselves, generate signals that cause changes in gene expression, leading to differentiation and to the establishment of form. In mature tissues, interactions between the ligands and receptors are involved in maintaining structural integrity and normal physiological function. Because CAMs play an important role in promoting interactions between cells and in stabilizing tissue architecture, understanding the mechanisms of adhesion provides insight into how development occurs, how structures are maintained, and how alterations in normal processes lead to pathologies such as neoplasms.

IL HISTORICAL BACKGROUND OF CELL ADHESION AND RECOGNITION A. Differential Cell Adhesion as a Developmental Mechanism The importance of cell-cell associations in directing morphogenesis was first indicated by experiments in which tissues were dissociated and then allowed to reassemble into aggregates. H. V. Wilson (1907) observed that when sponge cells were dissociated by forcing them through silk mesh, they subsequently reaggregated into clusters that eventually formed into new, intact sponges. Reformation of new individuals depended upon movement of cells within the clusters so that their appropriate position within the mass was regained. Wilson's experiments thus revealed the adhesive quality of cells, as well as their ability to self-assemble into structures having a higher level of organization. Later experiments by Galtsoff (1925) showed that cells from different species of sponge form contacts with one

Cell Adhesion and Recognition: An Overview

3

another, but do not remain adherent. Instead, Galtsoff found that sponge cells preferentially aggregated with their own type in a mixture of cells from two or more species, showing that specific recognition and adhesion occurred at the level of the individual cell. Townes and Holtfreter (1955) provided the first evidence that cells from different germ layers of vertebrate embryos were able to specifically recognize one another and to sort-out. By using embryos from vertebrate species having cells of different sizes and colors, Townes and Holtfreter observed that cells within the aggregates sorted-out into defined regions in accord with their original position in the embryo —ectodermal cells on the outside of the aggregates, mesodermal cells in the middle, and endodermal cells on the inside. In addition, they found that the adhesive affinities of each cell population, as reflected by their position within the aggregates, depended upon the age of tissue used. This work demonstrated the ability of vertebrate cells to selectively recognize and adhere to one another, as well as to reconstitute original form, but did not address the underlying mechanisms responsible for these processes. Shortly after the work of Townes and Holtfreter, two general ideas regarding the mechanism behind cell-cell recognition and adhesion were proposed: Moscona's specific adhesiveness hypothesis (1962) and Steinberg's differential adhesiveness hypothesis (1963). Moscona proposed that cell-cell associations are mediated by specific adhesive interactions between a large number of different adhesion molecules that are selectively expressed on different cell types. He suggested that cells expressing the same adhesion molecules would recognize and adhere to one another, whereas cells expressing different molecules would not. In contrast, Steinberg suggested that adhesive specificity could be achieved with a small number of adhesive molecules. He proposed that the relative positions of different cell types in mixed aggregates was due to quantitative differences in adhesiveness, such that the cell type adhering the strongest would form the center of the aggregate, the cell type adhering the weakest would be forced to the exterior, and cell types with intermediate adhesiveness would lie in between. At successive stages in the development of the adhesion field, these two themes, the need for a large number of specific molecules versus the morphogenetic power of a few molecules, have recurred. Thus, Sperry's observations on the accurate reconnection of severed retinal ganglion cell connections to the optic tectum led him to propose the "chemo-affmity hypothesis," which posits the existence of a large number of "individual identification tags" for adhesive recognition (Sperry, 1963). Analysis of insect nervous system development revealed the existence of several such molecules, giving rise to the "labeled pathways hypothesis" (Goodman et al., 1984), virtually a modem restatement of the Moscona and Sperry views as applied to a particular developmental system. On the other hand, identification and characterization of a few widely distributed CAMs led Edelman to propose the "morpho-regulatory hypothesis," which is similar to the views of Steinberg in emphasizing the diversity of interactions that can be generated by the combinatorial

4

RANDALL MARSH and ROBERT BRACKENBURY

action of a few CAMs together with mechanisms for modulating their expression or function (Edelman, 1984). This hypothesis also points out that the role played by CAMs in directing morphogenesis makes them a prime target for evolutionary changes that lead to alterations in body form. In short, it now appears that different developmental situations dictate the participation of few or many CAMs together with varying levels of regulatory complexity, thus vindicating both points of view. B. Discovery of Cell Junctional Complexes

An additional mechanism that underlies the normal assembly of cells into complex tissues is the formation of specialized junctions between cells. With the advent of electron microscopy, it became possible to examine tissue and organ architecture at very high magnification. Such examination led to the realization that the membranes of neighboring cells were in contact with one another and with extracellular matrix through junctional complexes. These complexes were seen in similar histological sections of widely separated organisms, suggesting that they were a relatively universal feature among cells in contact with one another. Based on their structural appearance in the electron microscope, cell junctions were categorized into five types: the tight junction, the adherens junction, the desmosome, the hemidesmosome, and the gap junction. The identification of these junctions raised the important question of whether there existed a relationship between cell-cell adhesion and recognition and the formation of these specialized complexes. As discussed below, characterization of the components of these junctions has shown that CAMs are intimately connected with junction assembly and structure. C. Cell-Cell Interactions in Immune Responses

Intensive analysis of immune responses, driven by the significance for clinical care, has yielded much information regarding cell-cell interactions. In contrast to adhesion among cells of solid tissues, interactions involving leukocytes are transient. Two kinds of processes have been particularly well-studied. Lymphocyte "homing" refers to the recirculation of lymphocytes between blood vessels and peripheral lymphoid organs. This process involves specific interactions between lymphocytes and specialized endothelial cells located in the ileum (Peyer's patches) or in peripheral lymph nodes. Intense efforts also have focused on the interactions between effector cells and helper or suppressor cells that are crucial for regulating immune responses. The identification of molecules participating in these interactions has been greatly aided by the isolation and characterization of panels of monoclonal antibodies against cell surface antigens used in conjunction with a variety of precise assays for lymphocyte functions.


5

III. ISOLATION AND IDENTIFICATION OF ADHESION MOLECULES The above studies on recognition and adhesion of cells in vitro and on the nature of junctions between cells suggested how cells interacted and adhered to one another, but did not identify specific molecules that mediated intercellular adhesion. It was not until the mid 1970s that adhesion molecules were first isolated by means of functional assays, by characterization of cell surface antigens recognized by monoclonal antibodies, and by analysis of the constituents of cell-cell junctions. More recently, additional CAMs have been identified by taking advantage of the fact that related sequences can readily be amplified by the polymerase chain reaction, cloned, and analyzed. It is important to note that this latter method only identifies candidate CAMs that must then be characterized fiirther, in contrast to the identification of molecules via functional assays, which provides direct evidence for their role as CAMs. To be definitively classified as a CAM, each new molecule needs to fulfill several criteria: (a) the purified molecule should bind to the target cell; (b) expression of the molecule in nonadherent cells should confer adhesive ability; (c) the pattern of expression of the molecule should be consistent with its presumed adhesive role; (d) antibodies to the molecule should perturb development in vivo in a manner interpretable as resulting from disruption of adhesion; and (e) mutations that alter the function or expression of the molecule should produce direct effects on adhesion. A.

Functional Assays for Identification of Adhesion Molecules

Two types of functional assays have been used to identify molecules involved in adhesion: (a) direct binding assays and (b) indirect, immunological approaches. Direct binding assays test the ability of radiolabeled molecules to bind to cell surfaces, under the assumption that CAMs should interact with cell surface receptors. One of the first assays used, devised by Moscona and coworkers, was based on the ability of factors present in conditioned medium to promote cell aggregation when added to suspensions of single cells. This approach ultimately resulted in the identification of cognin (Krishna Rao and Hausman, 1993). In general, however, the direct-binding strategy has not proven very successful, evidently because many CAMs bind with low affinity. The majority of CAMs have instead been identified via indirect approaches, in which antibodies are used to perturb adhesion between cells. In these assays, cell-cell adhesion is measured by quantifying the ability of single-cell suspensions to collect into aggregates. These "aggregation assays" can be scored visually, by counting cells manually with a hemocytometer or, more conveniently, with an electronic particle counter. Other assays test the ability of single cells in suspension,

6


labeled by radioactivity or fluorescence, to bind to a monolayer of identical or distinct cells. B. Immunological Identification of Cell Surface Antigens

In the 1970s and 1980s, many new cell surface antigens were identified by shotgun screening of monoclonal antibodies. In some cases, investigators sought antibodies that recognized antigens with tissue-specific or other interesting distribution patterns; the neural cell adhesion molecule N-CAM was repeatedly identified by this strategy (Schachner et al., 1975; Jorgensen and Bock, 1974; Hirn et al., 1981; Lemmon et al., 1982). Such studies yielded substantial descriptive information, but major progress depended upon discovering an activity associated with the anonymous antigen or identifying it with a molecule isolated via a functional assay. Other monoclonal antibodies, however, were directly screened for their effects in functional assays. As an example, antibodies to the adhesion molecule uvomorulin (mouse E-cadherin) were assayed by their ability to prevent compaction of early mouse embryos (Hyafil et al., 1981). C. Isolation and Analysis of Cell Junctional Complex Constituents

In addition to the above methods used to isolate adhesion molecules, characterization of the molecular constituents of cell-cell junctions has also led to the discovery of several new adhesion proteins, described in Section V. Relationship Between Adhesion Molecules and Cell Junctions. D.

Polymerase Chain Reaction-based Isolation of Related Molecules

The advent of the polymerase chain reaction (PCR), an in vitro method of nucleic acid synthesis which allows specific segments of DNAto be amplified, has greatly accelerated the identification of new adhesion molecules. In this approach, oligonucleotide primers are synthesized that correspond to regions of high sequence homology among known adhesion molecules and used in PCR to selectively amplify mRNAs containing similar sequences that should encode additional, related adhesion molecules. One example of this approach is the identification of eight calcium-dependent adhesion molecules (cadherins) from nervous tissue using primers made from regions of high homology within the cytoplasmic domains of known cadherins (Suzuki et al., 1991).

IV. FAMILIES OF ADHESION MOLECULES This section introduces four basic families of CAMs, defined primarily by comparison of amino acid sequence data deduced from analysis of cDNA clones. The first hint, however, that such families might exist came from a series of cell binding studies carried out in the late 1970s by Takeichi and coworkers (Takeichi, 1977;


7

Urushihara et al., 1979). This work demonstrated that a variety of different cells express two kinds of cell-cell adhesion systems that can be distinguished by their requirement for divalent cations. Different methods of cell preparation preserved either "calcium-dependent" or "calcium-independent" adhesion systems (Urushihara et al., 1979). As described in the following section, later analysis revealed that molecules within each of these two functionally defined classes indeed are structurally related. A.

Immunogiobulin Superfamily

Molecules of the immunoglobulin superfamily are discussed in the chapter by Gegelashvili and Bock. A representative member of the immunoglobulin superfamily of CAMs is the neural cell adhesion molecule N-CAM, the first CAM to be identified and extensively characterized. N-CAM was identified via functional assays by Gerry Edelman's group at the Rockefeller University (Brackenbury et al, 1977; Thiery et al., 1977). N-CAM is highly antigenic and, consequently, was independently identified by several groups, including those of Schachner (Schachner et al., 1975), Goridis (Him et al, 1981), Jorgensen and Bock (1974), and Gottlieb (Lemmon et al., 1982), as an antigen enriched in the nervous system. N-CAM mediates calcium-independent adhesion (Brackenbury et al., 1981), employing both homophilic (N-CAM to N-CAM; Rutishauser et al., 1982) and heterophilic (N-CAM to heparan sulfate proteoglycans; Cole and Glaser, 1986; Cole and Akeson, 1989) modes of binding. As described below, many other CAMs that are calcium-independent have been found to be structurally related to N-CAM. Biochemical studies revealed that N-CAM consists of a series of related glycoproteins (Hoffman et al., 1982; Rougon et al., 1982) now known to be derived from alternatively spliced transcripts of a single gene (Murray et al., 1986a, b; Nguyan etal., 1986; Cunningham etal., 1987; Owens etal., 1987; Goridis and Wille, 1988). About 40% of the mass of N-CAM is carbohydrate, including a large amount of covalently associated sialic acid (Hoffman et al., 1982), present as long, unbranched chains (Finne et al., 1983). Biochemical analysis and characterization of cDNA clones has established that the three major polypeptide forms (180 kDa, 140 kDa, and 120 kDa) of N-CAM share a constant extracellular domain, but differ at their carboxyl-termini—the 180 kDa and 140 kDa forms span the plasma membrane and differ in the extent of their cytoplasmic domain (Murray et al., 1986a), whereas the 120 kDa form does not contain the membrane-spanning domain, but associates with the membrane via a phosphatidylinositol linkage (He et al., 1986; Hemperly et al., 1986a; Cunningham et al., 1987). In addition, analysis of one cDNA clone suggests the existence of secreted forms of N-CAM that are not membrane-associated (Gower et al., 1988). Besides these major structural variations, a variety of other alternative splicing events leads to small changes in the polypeptide structure of N-CAM (Dickson et al., 1987; Small et al, 1987; Santoni et al., 1989).

8


Homophilic binding mediated by N-CAM is weak relative to the cadherins, and some evidence suggests that N-CAM to N-CAM interactions may serve primarily signaling or regulatory roles rather than an adhesive function. For example, ligand binding to N-CAM triggers intracellular changes in second messenger systems (Schuch et al., 1989; Doherty et al., 1991; Doherty and Walsh, 1992). Because the major polypeptide forms of N-CAM differ primarily in the structure of the carboxyl-terminus, these variations in membrane attachment and cytoplasmic domains could mediate different effector functions stimulated by N-CAM-mediated cellcell adhesion. Additionally, variations in N-CAM sialic acid content appear to exert powerful regulatory influence over cell-cell interactions mediated by other CAMs (Rutishauser et al, 1988). Sequence analysis of N-CAM cDNA clones (Hemperly et al., 1986b; Barthels et al., 1987; Cunningham etal., 1987; Small etal., 1987) revealed that the extracellular domain, which contains the binding region, consists of five regions that show internal homology (see Figure 1). Comparison with other proteins indicated that these segments are significantly related to the immunoglobulin domain, a structural motif of approximately 100 amino acids folded into two P-pleated sheets connected by a disulfide linkage, that had been characterized as the basic structural and functional unit of immunoglobulins (Williams and Barclay, 1988). The discovery that N-CAM is a member of the immunoglobulin superfamily was followed by the recognition that a variety of neural cell surface molecules, including Thy-1 (Williams and Gagnon, 1982), PQ (Lemke and Axel, 1985), MAG (Arquint et al., 1987; Lai et al, 1987; Salzer et al., 1987), Ll/Ng-CAM (Moos et al, 1988; Burgoon et al., 1991), and contactin (Ranscht, 1988) all contain immunoglobulin domains. Many other immunoglobulin superfamily molecules, including I-CAM-1 and I-CAM-2, are expressed by immune cells (Springer, 1990) and the immunoglobulin superfamily member C-CAM is expressed by hepatocytes (Obrink, 1991). In most cases, the extent of homology among these CAMs is relatively low, but a few key residues are almost invariably conserved, and available evidence suggests that these residues are sufficient to preserve the characteristic three dimensional structure of the immunoglobulin domain (Williams and Barclay, 1988). These various CAMs contain differing numbers of Ig domains, from a single domain in PQ (Lemke and Axel, 1985) and Thy-1 (Williams and Gagnon, 1982) to six domains in LI (Moos et al., 1988; Burgoon et al., 1991). Like N-CAM, several other Ig superfamily CAMs also contain variable numbers of fibronectin type III repeats. Most of the Ig superfamily molecules are integral membrane proteins, but some, such as MAG and Thy-1, also include forms that are linked to the membrane via phosphatidylinositol linkages. The cytoplasmic domains of various Ig superfamily molecules vary significantly in size and do not generally show any sequence similarity. Some Ig superfamily molecules, such as the PDGF receptor, c-kit, and the CSF-1 receptor, contain tyrosine kinase domains within their cytoplasmic segments (Yarden et al., 1986). Other Ig superfamily molecules contain tyrosine phosphatases within their cyto-

Cell Adhesion and Recognition: An

Overview

IMMUNOGLOBULIN SUPERFAMILY - N-CAM

OOOOOo^ CADHERINS-

l-£

E-CADHERIN

D-C

>-C

>-C

DH

^sssa-

INTEGRINS

a

SELECTINS liJBiiiiyiyuiMiyiiUi^k^^

P-SELECTIN r~nm

n n m m m m n m

extracellular domain

cytoplasmic domain

Figure 1, Representatives of the four major classes of cell adhesion molecules. All of these molecules are transmembrane glycoproteins, shown with the extracellular domain to the left, a transmembrane segment (the plasma membrane is indicated by two vertical lines), and cytoplasmic domains. The top portion of the figure shows one of the forms of N-CAM, which consists of five immunoglobulin domains (omega figures) and two fibronectin type III repeats (open boxes). Cadherins consist of several homologous domains (open boxes), a region close to the interior of the membrane (grey box) whose function has not been well-defined, and a - 7 0 amino acid segment (stippled box) that mediates binding to a-, p-, and y-catenins. The integrins consist of a and p polypeptides that combine In various combinations. The amino-terminal portion of both chains (grey boxes) combine to form the ligand binding site. Some a-chains are cleaved, but held together by disulfide bonds. The P-chain contains several Ca^-'-bind ing regions (light grey boxes). The selectins contain a lectin domain at the amino terminus (large grey box), an EGF-domain, and varying numbers of domains that share homology to complement regulatory proteins (small grey boxes).

10


plasmic domains (Brady-Kalnay and Tonks, 1994). In general, many Ig superfamily molecules appear to mediate transmembrane signaling events (Williams and Barclay, 1988), and the variability in cytoplasmic domain structure may mirror the variety of effector responses. Thus, many "adhesion molecules" may primarily serve signaling functions (Thiery and Boyer, 1992; Bosenberg and Massague, 1993). B. Cadherin Family

A second major family of adhesion molecules is the calcium-dependent CAMs, or "cadherins" (Takeichi, 1988), discussed in the chaper by Wallis et al. The existence of this class of molecules was first suggested by Takeichi's experiments demonstrating calcium-dependent and calcium-independent adhesion mechanisms on several types of cells (Takeichi, 1977; Urushihara et al., 1979). Specific calciumdependent CAMs derived from epithelia were subsequently identified by Edelman and Cunningham (Gallin et al., 1983), Takeichi's group (Yoshida and Takeichi, 1982), Jacob's group (Hyafil et al., 1981), and Damsky and Buck (Damsky et al., 1983). Similarities in biochemical properties and tissue distribution suggested a close similarity among these molecules isolated from diverse species, and subsequent cloning studies have revealed that these molecules are indeed homologues of the molecule now generally referred to as E-cadherin. All cadherins are glycoproteins that include a large extracellular domain consisting of several repeated domains that contain calcium-binding motifs (Ringwald et al., 1987; see Figure 1). Cadherins mediate calcium-dependent cell-cell adhesion by a homophilic mechanism (Nagafuchi et al., 1987) and the binding region is centered around the peptide HAV, located in the first of the cadherin domains (Blaschuk et al, 1990; Nose et al., 1990). Like the Ig superfamily molecules, the cadherins are encoded by single genes. There is no evidence for alternative splicing leading to variations in cadherin polypeptide structure. With one exception, classical cadherins contain a highly conserved cytoplasmic domain. Deletions within the cytoplasmic domain abolish the ability of the cadherin to mediate adhesion and simultaneously abolish the binding of cytoplasmic cadherin-associated proteins termed "catenins" (Nagafuchi and Takeichi, 1988,1989; Ozawa et al, 1989,1990). Several catenins have been identified and characterized and there appears to be diversity in the catenins expressed by different cell types (Hirano et al., 1992). The a-catenins contain regions with significant sequence similarity to vinculins (Herrenknecht et al, 1991; Nagafuchi et al., 1991), suggesting that the catenins may provide the known link between cadherins and the cytoskeleton (Hirano et al, 1987; Nelson et al., 1990). The use of PCR-based strategies and pan-cadherin antibodies have led to the identification of almost 30 cadherins. N-cadherin is concentrated in nervous tissue, but also found in cardiac muscle and lens (Hatta and Takeichi, 1986). B-cadherin (Napolitano et al., 1991), R-cadherin (Inuzuka et al, 1991), and T-cadherin


11

(Ranscht and Dours-Zimmerman, 1991) are also found in neural tissue. Interestingly, T-cadherin lacks a cytoplasmic domain, and thus does not associate with catenins, yet mediates adhesion as effectively as other cadherins (Vestal and Ranscht, 1992). P-cadherin is enriched in the extraembryonic membranes of developing embryos, but is also found in epidermis, retinal pigmented epithelium, and mesothelium (Nose and Takeichi, 1986). Other cadherins have been detected, but not well characterized (Suzuki et al., 1991). Thus, while there are broad differences in the distribution of cadherins, some tissues and even individual cells may express several cadherins. The functions of multiply expressed cadherins and the ways in which their activities are coordinated are not yet known. Cadherins play important roles during normal development and in pathological conditions (see Takeichi, 1991). E-cadherin-mediated adhesion appears to be the primary mechanism for initial adhesion of epithelial cells and is a necessary prerequisite for the formation of gap junctions and desmosomes (Kanno et al., 1984; Meyer et al., 1992). A series of recent studies suggests that cadherins may function as "invasion suppressors" inasmuch as loss of cadherins in tumor cells leads to increased invasiveness that can be reversed by restoration of cadherin expression (Behrens et al, 1989; Frixen et al, 1991; Vleminckx et al., 1991). One report (Chen and Obrink, 1991) indicates that E-cadherin-mediated contact inhibits membrane activity and cell movement. N-cadherin promotes neurite extension and fasciculation (Bixby and Zhang, 1990; Doherty and Walsh, 1992). PCR methods were also used to identify a series of molecules related in structure to cadherins, but which differ substantially in their cytoplasmic domains (Sano et al, 1993). These components, termed "protocadherins," contain extracellular domains composed of variable numbers of repeated cadherin homology domains, but have cytoplasmic domains that are not represented in any other known molecules. These molecules are, in some cases, known to mediate adhesion in in vitro assays, but their in vivo functions are unknown. C. Integrin Family

The family of receptors termed integrins, originally identified as cell surface receptors for extracellular matrix components, has more recently been found to be involved in cell-cell interactions as well (see chapter by Bowditch and Faull). The first integrin to be characterized was identified as the antigen recognized by a monoclonal antibody that blocked attachment of myoblasts to fibronectin-coated surfaces (Neff et al., 1982). Cloning of this molecule (Tamkun et al., 1986) and comparison of the deduced amino acid sequence with other molecules revealed the existence of a family of related molecules (Hynes, 1987). Other early members of this family included molecules first identified as a fibronectin receptor (Pytela et al., 1985), the platelet aggregation molecule gpIIb/IIIa, and several lymphoctye molecules identified as "lymphocyte function antigens" or LFAs (Springer et al., 1987) or VLAs, for "very late antigens" (Hemler et al., 1983).

12


Integrins are heterodimers consisting of an a-chain and a P-chain, which can combine in various combinations (see Figure 1). At present, 12a subunits and 7(3 subunits have been described. Initially, it appeared that each a subunit could combine only with a single p subunit; together with some similarities in distribution, this pattern of association led Hynes (1987) to classify the integrins into subfamilies, based upon a common p subunit. More recently, however, it has been found that some a subunits can combine with more than one P subunit. The extracellular domains of the a- and p-chains, which are both transmembrane glycoproteins, associate to form the ligand binding region. All a subunits contain several sites that bind Ca^"^, which is required for integrin—ligand interactions. While some heterodimers are relatively specific for a single ligand (such as a^Pj, which recognizes fibronectin), other combinations (such as a3pj) recognize several ligands. Many of the ligands that bind to integrins, including fibronectin, vitronectin, laminin, and type I collagen, contain a tripeptide, arg-gly-asp (RGD), that is essential for binding to integrins (Ruoslahti, 1988). Other ligands, however, do not contain the RGD sequence and bind via other motifs. Most of the ligands recognized by integrins are extracellular matrix components, but the a^^^ integrin binds either I-CAM-1 or I-C AM-2, which are cell surface molecules of lymphocytes, and the a4P ^ integrin binds V-CAM-1 onendothelialcells(see Springer, 1990). I-CAM-1, I-C AM-2 and V-C AM-1 are all immunoglobulin superfamily members. The cytoplasmic domains of both integrin subunits are required for association of integrins with the cytoskeleton. This linkage occurs via interactions with talin, vinculin, and a-actinin, which localizes integrins to focal adhesions, points of close contact between cultured cells and substratum. Integrins thus provide a structural link between the extracellular matrix and the interior of the cell. In addition, binding of ligands to integrin receptors triggers changes in gene expression and differentiation (Werb et al., 1989). The name "integrins" emphasizes the key role played by these molecules in mediating cellular responses to extracellular cues. D. Selectin Family

The second maj or role played by cell-cell interactions in immune function relates to the recirculation or "homing" of lymphocytes. Go wans and Knight (1964) showed that lymphocytes taken from a localized site and reintroduced into the circulation would preferentially repopulate their site of origin, suggesting that the lymphocytes recognized and were directed by specific regions of endothelium. Development of in vitro assays that mimicked preferential lymphocyte binding (Stamper and Woodruff, 1976) provided a system for analyzing the molecular basis of homing. This system revealed that binding was calcium-dependent and could be blocked by addition of specific sugars (see Yednock and Rosen, 1989). Eventually, complex carbohydrates containing sialic acid were shown to block binding efficiently, and monoclonal antibodies were raised that blocked this binding.


13

One such monoclonal antibody, MEL-14, blocked binding of lymphocytes to specialized endothelium of peripheral lymph nodes without affecting binding to endothelium of Peyer's patches (Gallatin et al, 1983). The antigen recognized by MEL-14 was identified by cDNA cloning as a novel adhesion molecule consisting of an amino-terminal lectin-like domain, followed by an epidermal growth factor motif and several domains related in structure to regions of complement-regulating proteins (Bowen et al., 1989; Lasky et al., 1989; Siegelman et al., 1989; Tedder et al, 1989; see Figure 1). Two additional lectin-like molecules involved in trafficking of leukocytes were also identified. One, initially termed ELAM for endothelial leukocyte adhesion molecule, was expressed by endothelial cells within hours of activation. This molecule promoted the binding of monocytes and neutrophils. The other molecule, termed GMP-140 (granule membrane protQin, 140 kDa) or PADGEM (platelet activation-rfependent granule external membrane protein), is localized within vesicles of platelets and endothelial cells, but is released to the cell surface within minutes of activation. This molecule also mediates binding to neutrophils and monocytes. Cloning of ELAM (Bevilacqua et al., 1989) and GMP-140/PADGEM (Johnston et al, 1989) revealed that both contained aminoterminal lectin domains followed by an EGF domain and several complement-associated domains. Just as the presence of the lectin domain suggests, recent work supports the idea that these molecules bind carbohydrate moieties (see Rosen, 1993). These three molecules are now referred to as "selectins"—the MEL-14 antigen is L-selectin, ELAM is E-selectin, and GMP-140/PADGEM is P-selectin (Bevilacqua et al., 1991). The selectins are discussed in the chapter by Jutila. The selectins play key roles in the initial phase of leukocyte attachment to endothelial cells. During this phase, the leukocytes remain attached, but roll along the endothelial surface. After activation, firm attachments are mediated by the interaction between leukocyte integrins and I-CAMs or V-C AM on the endothelial cell (see Lasky, 1992). Both phases are essential for subsequent extravasation (passage of the leukocyte through the endothelial wall). The physiological significance of these events is underscored by the effects of the inherited disease leukocyte adhesion deficiency, in which congenital absence of [31 integrins abolishes the ability of neutrophils to extravasate, resulting in repeated bacterial infections (Anderson and Springer, 1987).

V. RELATIONSHIP BETWEEN ADHESION MOLECULES AND CELL JUNCTIONS Cell junctions are specialized membrane domains that provide direct mechanical intercellular coupling and that regulate a variety of cellular processes including locomotion, proliferation, and differentiation (Edelman and Crossin, 1991; Geiger, 1993; Hedrick et al, 1993). Originally identified and defined by electron microscopy (Farquhar and Palade, 1963; Stevenson and Paul, 1989), their molecular organization and function have attracted increasing interest among investigators

14


Tight Junction

Actin Filaments

Adherens Junction

Actin Filaments

Desmosome

intermediate Filaments

Hemidesmosome

Intermediate Filaments

W//MW////////////^^^^^^ TSl Figure 2. Association of CAMs and cytoskeletal elements with epithelial junctions. The figure shows two adjacent epithelial cells, with the microvillar brush border at the top (apical region) and basement membrane at the bottom (basal region). Although the drawing is not to scale, the junctions are depicted in their correct sequence and general location. Thus, tight junctions, adherens junctions and belt desmosomes form so-called "junctional complexes'' near the apical portion of the cells and hemidesmosomes anchor the cells to the basement membrane. Gap junctions and spot desmosomes may be found in many regions of the plasma membrane.


15

studying cell-cell interaction and communication. In mammalian cells, intercellular junctions are categorized into five major types: the adherens junction, the desmosome, the hemidesmosome, the tight junction, and the gap junction. Figure 2 is a schematic illustration of the location and structure of these different junctions as they are organized on a typical epithelial cell. The following summarizes our current knowledge about the composition of these junctions, particularly as it relates to the role of CAMs in these structures. The molecular constituents of intercellular junctions have been characterized primarily by subcellular fractionation and analysis of semipurified preparations, and by the generation of cDNA and immunological probes to junctional components. As predicted from their diverse functions, cell junctions have been found to be heterogeneous in structure, involving a variety of transmembrane molecules, cytoskeletal networks, and anchoring proteins that link the two (Geiger et al., 1992; Legan et al, 1992; Birchmeier, 1993; Citi, 1993; Kaiser et al., 1993). Over the last few years, it has become evident that many of the major glycoproteins present in intercellular junctions are members of two families of adhesion molecules: the cadherins and the integrins. This realization implies that adhesion molecules may play direct roles in the formation, structure, and function of cell-cell junctions. A. The Adherens Junction and Cadherins Adherens junctions are sites of firm adhesion between cells where actin microfilaments are anchored to the plasma membrane, most typically as a subapical girdle (zonula adherens) in polar epithelial cells, but also as localized streak-like junctions (fasciae adherens) or small discs (puncta adherentia) in other cell types (Staehelin, 1974; Geiger et al., 1985; Geiger and Ginsberg, 1991; Schmelz and Franke, 1993). Adherens junctions physically appear as a dense plaque on the cytoplasmic faces of apposed plasma membranes, with an intercellular gap of 15—20 nm filled with fibrillar protein (Fawcett, 1986; see Figure 1). The cytoplasmic plaque serves as the attachment site for actin filaments that lie beneath each plasma membrane, and which form a continuous band around each cell. These actin filaments may be involved in the folding of epithelial sheets during morphogenesis, and in the transmission of forces and morphogenetic signals between the cytoskeletons of adjacent cells (Cunningham and Edelman, 1990). Characterization of the composition of adherens junctions has revealed that the major extracellular and transmembrane components are cadherins, and that the cytoplasmic plaque is composed of proteins involved in connecting the cadherins to the actin cytoskeleton (Boiler et al., 1985; Geiger et al., 1985, 1990; Gumbiner and Simons, 1986; Tsukita et al, 1990; Geiger and Ginsberg, 1991; Takeichi, 1991; Ozawa and Kemler, 1992; see Table 1 for summary). E-cadherin (uvomorulin, L-C AM) and N-cadherin (A-C AM) have been definitively localized to the junction (Boiler et al., 1985; Volk and Geiger, 1986), but it is likely that other members of the cadherin family (Geiger and Ayalon, 1992) are also present (Liaw et al., 1990;


16 Table 1. Cell Junction

A d h e s i v e C o m p o n e n t s of C e l l - C e l l Junctions References

. Adherens^ A. Integral Membrane Components Volk & Geiger (1986). J. Cell Biol. 103, 1441-1450^. N-Cadherin (130 kDa)^ E-Cadherin (125 kDa) Boiler et al. (1985). J. Cell Biol. 100, 327-332. B. Peripheral Components HIrokawa et al. (1982). J. Cell Biol. 94, 425-443. Actin (42 kDa) Drenckhahn & Dermietzel (1988). J. Cell Biol. 107, Myosin (476 kDa) 1037-1048. Tropomyosin (70 kDa) Geiger et al. (1981). J. Cell Biol. 9 1 , 614-628. a-Actinin(IIOkDa) Lazarides and Burridge (1975). Cell 6, 289-298. Vinculin(130kDa) Geiger (1979). Cell 18, 193-205. Ankyrin-like Protein (220 kDa) I t o h e t a l . (1991). j . Cell Biol. 115, 1449-1462. Zyxin (82 kDa) Crawford & Beckerle (1991). j . Biol. Chem. 266, 5847-5853. Radixin (82 kDa) Tsukita et al. (1989). J. Cell Biol. 108, 2369-2382. Ezrin (85 kDa) Sato et al. (1992). J. Cell Sci. 103, 131-143. Moesin (75 kDa) Sato e t a l . (1992). J. Cell Sci. 103, 131-143. a-Catenin(102kDa) Nagafuchi et al. (1991). Cell 65, 849-857. p-Catenin (93 kDa) McCrea et al. (1991). Science 254, 1 3 5 9 - 1 3 6 1 . y-Catenin/Plakoglobin (83 kDa) Cowin e t a l . (1986). Cell 46, 1063-1073. ZO-1 (220 kDa) Itoh et al. (1993). J. Cell Biol. 121, 491-502. APC (300 kDa) Hulsken et al. (1994). J. Cell Biol. 127, 2061-2069. EGF-Receptor(180kDa) Hoschuetzky et al. (1994). J. Cell Biol. 127, 1 3 7 5 1380. II. Desmosome'^ A. Integral Membrane Components Desmoglein Dsg 1 (150-165 kDa) Giudice et al. (1984). J. Cell Biochem. 26, 35-45. Desmoglein Dsg 2 (120 kDa) Koch et al. (1991a). Eur. J. Cell Biol. 55, 200-208. Desmoglein Dsg 3 (130 kDa) Amagai e t a l . (1991). Cell 67, 869-877. Desmocollin Dsc1a(130 kDa) Koch et al. (1992). Proc. Natl. Acad. Sci. USA 89, 3 5 3 357. Desmocollin Dsc 1b(115 kDa) Giudice et al. (1984). j . Cell Biochem. 26, 35-45. Desmocollin Dsc 2a (107 kDa) Koch et al. (1992). Proc. Natl. Acad. Sci. USA 89, 3 5 3 357. Desmocollin Dsc 2b (101 kDa) Koch et al. (1992). Proc. Natl. Acad. Sci. USA 89, 3 5 3 357. Desmocollin Dsc 3a (22kDa) Garrod (1993). Curr. Opin. Cell Biol. 5, 30-40. Desmocollin Dsc 3b (16 kDa) Garrod (1993). Curr. Opin. Cell Biol. 5, 30-40. E-Cadherin (125 kDa) Parker et al. (1991). J. Biol. Chem. 266, 10438-10445. Polypeptide of 22 kDa Cowbin & Garrod (1983). Nature 302, 148-150. Polypeptide of 140 kDa Jones et al. (1986). Proc. Natl. Acad. Sci. 83, 7 2 8 2 7286. B. Peripheral Components Desmoplakin I (240 kDa) Mueller & Franke (1983). J. M o l . Biol. 163, 6 4 7 - 6 7 1 . Desmoplakin II (210 kDa) Mueller & Franke (1983). J. M o l . Biol. 163, 6 4 7 - 6 7 1 . Plakoglobin (83 kDa) Cowbin & Garrod (1983). Nature 302, 148-150. Band 6 Protein (75 kDa) Cowbin & Garrod (1983). Nature 3 0 2 , 1 4 8 - 1 5 0 . Desmocalmin (240 kDa) Tsukita & Tsukita (1985). J. Cell Biol. 101, 2070-2080. Keratocalmin (250 kDa) Fairley et al. (1991). J. Clin. Invest. 88, 315-322. (continued)

Cell Adhesion

and Recognition:

An

Overview

Table 1. Cell Junction

17

(Continued) References

Nuclear Lamin B-IIke Protein Cartaudetal. (1990). J. Cell Biol. I l l , 581-588. (140kDa) Skalli et al. (1994). J. Cell Biol. 125, 159-170. IFAP300(300kDa) III. Hemidesmosome A. Integral Membrane Components Stepp et al. (1990). Proc. Natl. Acad. Sci. USA 87, a6P4 integrin (350 kDa) 8970-8974. BPAG2(180kDa) Diaz et al. (1990). J. Clin. Invest. 86, 1088-1094. Polypeptide of 125 kDa^ Klatte et al. (1989). J.Cell Biol. 109, 3377-3390. B. Peripheral Components BPAC1 (230 kDa) O'Keefe et al. (1989). J. Biol. Chem. 264, 8310-8318. Polypeptide of 200 kDa Cartaud et al. (1990). J. Cell Biol. I l l , 581-588. HD1 (500 kDa) Hieda et al. (1992). J. Cell Biol. 116, 1497-1506. Skalli et al. (1994). J. Cell Biol. 125, 159-170. IFAP 300 (300 kDa) IV. Tight Junction A. Integral Membrane Components Furuse et al. (1993). J. Cell Biol. 123, 1777-1788. Occludin (65 kDa) B. Peripheral Components ZO-1 (220 kDa) Stevenson et al. (1986). J. Cell Biol. 103, 755-766. ZO-2(160kDa) Gumbiner et al. (1991). Proc. Natl. Acad. Sci. USA 88, 3460-3464. Cingulln(140kDa) CIti et al. (1988). Nature 333, 272-275. 7H6 Antigen (155 kDa) Zhong et al. (1993). J. Cell Biol. 120, 477-483. p i 30 (130 kDa) Balda et al. (1993). J. Cell Biol. 123, 293-302. Spectrin (a-240 kDa; P-220 kDa) I t o h e t a l . (1991). J.Cell Biol. 115, 1449-1462. Actin (42 kDa) Madara (1987). Am. J. Physiol. 253, c171-c175. /. Gap Junction'^ A. Connexins Rat Connexin 26 (26 kDa) Nicolson & Zhang (1988). Modern Cell Biol., Vol 7, 207-210. Rat Connexin 32 (32 kDa) Paul (1986). J. Cell Biol. 103, 123-134. Rat Connexin 43 (43 kDa) Beyer e t a l . (1987). J. Cell Biol. 105, 2621-2629. Xenopus Connexon 38 (38 kDa) Eblharaetal. (1989). Science 243, 1194-1195. Xenopus 30 kDa Protein (30 kDa) Gimllch et al. (1988). J. Cell Biol. 107, 1065-1074. Sheep Lens MP70 (70 kDa) Kistler et al. (1988). Nature 3 3 1 , 721-722. Notes: ^Components listed are representative of zonula adherens. Îtalicized proteins are known to function as adhesion molecules. '^References are intended to be representative rather than comprehensive, and should be used as sources for further information. Listed components represent molecules definitively localized to a given junction. Molecular weights presented are generalized figures. •^Desmosomal components have various names. Nomenclature presented is from Buxton et al. (1 ^93), J. Cell Biol. 121, 481-483. For clarification of earlier names see Legan et ai. (1992), Bioessays 14,

385-393. ^May represent 125 kDa chain of ae integrin subunit. ^Not all known connexins are listed. For a table of connexins expressed in mammalian cells see Dermietzel and Spray (1993), TINS 16, 186-192 and Dermietzel et al. (1990), Anat. Embryol. 182, 517-528.

18


Lampugnani et al, 1992; Ayalon et al, 1994). It is well documented that catenins bind directly to the cytoplasmic domain of these cadherins and play a role in coupling this domain to actin filaments (Ozawa et al, 1990; McCrea et al., 1991; Nagaflichi et al., 1991; Kintner, 1992; Hirano et al., 1992; Hinck et al., 1994a, 1994b). In addition to the catenins, a number of other actin-binding proteins (see Table 1) are also present in the plaque. It is becoming clear that all of these molecules form part of a multiprotein complex that links the cadherins to actin. The detailed structure of this complex, in terms of exactly what molecules bind to what other molecules, remains to be worked out. Interestingly, the most recently identified constituents of the adherens junction have turned out to be the EGF receptor and the APC tumor suppressor gene product (Hiilsken et al., 1994; Hoschuetzky et al., 1994). The presence of these molecules in the adherens junction, along with the known fact that P-catenin shares significant homology with armadillo, a, protein that is part of a cell signaling pathway in Drosophila (McCrea et al., 1991; Peifer et al., 1992), strongly suggests a potential role for the adherens junction not only in cell adhesion but also in signal transduction and in tumor suppression. B. The Desmosome, Cadherins, and Cadherin-like Molecules

Like the adherens junction, the desmosome also mediates firm adhesion between cells, and represents a site where a cytoskeletal network is connected to the cell surface. The desmosome consists of a dense plaque on the cytoplasmic face of each apposed plasma membrane, and an intercellular gap of 15—20 nmfilledwith fibrillar material (Fawcett, 1986; see Figure 1). Unlike the adherens junction plaque, however, the plaque of the desmosome serves as an attachment site for intermediate filaments of mostly the cytokeratin-type, although attachment of desmin and vimentin have also been reported (Kartenbeck et al., 1984; Green and Jones, 1990; Schwarz et al, 1990). Desmosomes therefore serve to link the intermediate filament networks of adjacent cells. In so doing, desmosomes may confer structural continuity and tensile strength to epithelia, as well as transmit signals between the cytoskeletons of adjacent cells (Cunningham and Edelman, 1990). The results of recent cDNA cloning and sequencing experiments have established that the major glycoproteins of the desmosome, called desmocollins and desmogleins, are members of the cadherin family (Koch et al., 1990,1991a, b; Collins et al., 1991; Mechanic et al., 1991; Legan et al, 1992; see Table 1). Originally, the term "desmoglein" was used to refer to all desmosomal glycoproteins (Gorbsky and Steinberg, 1981), but the term "desmocollin" was introduced (Cowin et al, 1984) to distinguish between two types of desmosomal glycoproteins with different properties. Of the two, the desmocollins have a higher degree of homology with the "classical cadherins" described above, but both show homology in their extracellular domain to the classical cadherins, and both are specialized for calcium-


19

dependent adhesion (Steinberg et al., 1987; Holton et al., 1990; Koch and Franke, 1994). The extracellular domains of desmocollins are the same size as those of cadherins and have the same calcium binding sites, conserved sites for N-linked glycosylation, and conserved cysteine residues (Collins et al, 1991; Legan et al, 1992). There are three desmocoUin genes (designated " 1 , " "2," and "3"), each encoding two products ("a" and "b") differing by about 6 kDa due to alternative splicing that leads to an in-frame stop codon in the transcript of the smaller "b" form (Collins et al., 1991; Parker et al., 1991). For the desmogleins (see Table 1), the first four extracellular domains have the same structure as those of the desmocollins and classical cadherins, but all desmogleins are distinguished in having an extra carboxyl-terminal domain containing a number of repeats of a -29 residue sequence not present in other cadherins nor in any other known proteins (Schafer et al., 1994). There are three desmoglein genes, each with a single gene product (Amemann et al., 1991, 1992a, b). At present, desmocollins and desmogleins appear confined to the desmosome type of junction (Karpati et al., 1992). It is thought that the extracellular domains of these molecules interact on adjacent cells, but it is not clear whether binding is homophilic or heterophilic. It is known, however, that the homophilic binding site used by classical cadherins is not conserved in the desmosomal proteins (Collins et al., 1991; Koch et al., 1991b; Mechanic et al., 1991). The desmosomal plaque is composed of the cytoplasmic domains of the desmocollins and desmogleins, as well as a number of other proteins involved in linking these cadherins to intermediate filaments (see Table 1). The cytoplasmic domains of the desmocollins and desmogleins have only limited homology with cadherins, are much larger, and have many unique features (Troyanovsky et al., 1993; Koch and Franke, 1994). It is likely that these differences reflect adaptations for binding to intermediate filaments rather than to actin. The organization of the plaque proteins and the nature of the linkage to intermediate filaments is not well understood. Plakoglobin is thought to associate with the desmocollins and desmogleins through short cadherin-like regions in their cytoplasmic domains (Korman et al., 1989), and thus may serve as the first link of a multiprotein complex. The desmoplakins are located between the plaque and the attached intermediate filaments, presumably forming the middle of the complex, but do not bind directly to the filaments, suggesting additional proteins are involved (Garrod, 1993). These proteins are likely represented by desmocalmin, band 6 protein, keratocalmin, nuclear lamin-B like protein, and/or IFAP 300, all of which are known to bind intermediate filaments (Tsukita and Tsukita, 1985; Kapprell et al., 1988; Cartaud etal, 1990; Koch and Franke, 1994; Skallietal, 1994). Desmoyokin is not believed to play a role in this linkage complex, but rather to function in stabilizing the periphery of the desmosomal plaque (Heida et al., 1989).

20

RANDALL MARSH and ROBERT BRACKENBURY C. The Hemidesmosome and Integrins

The hemidesmosome represents a site of cell-substratum adhesion located on the basal surfaces of epithelial cells where the cells are exposed to underlying basement membrane (Fawcett, 1986). Morphologically, the hemidesmosome resembles half of a desmosome in that it is composed of a cytoplasmic plaque linked to intermediate filaments (Kelly, 1966; see Figure 1). The molecular composition of these two junctions is quite different, however, suggesting that their structural similarity is fortuitous (Legan et al., 1992). The difference in composition is mirrored by a difference in function of the two junctions; whereas the desmosome links intermediate filament networks between cells, the hemidesmosome links the filaments to extracellular matrix of basement membrane. This linkage presumably rivets cells to the membrane, serving to hold each cell down and to distribute tensile forces through the entire epithelium and its underlying connective tissue. The principle adhesive protein in the hemidesmosome is not a cadherin, but rather an a^^^ integrin complex that binds to a component in the basement membrane, most likely kalinin (Stepp et al., 1990; Jones et al, 1991; Rouselle et al., 1991; Sonnenberg et al, 1991). The a^^^ complex is unique in that its |34 subunit has a cytoplasmic tail that is over 1000 amino acids long and that is largely unrelated to other known proteins (Hogervorst et al., 1990; Tamura et al., 1990). This extremely long tail is thought to contribute to the plaque of the hemidesmosome, and likely represents a specialization for binding to intermediate filaments and for assembly of the plaque (Sonnenberg et al., 1991). It is not clear if the tail of the P4 subunit binds directly to the intermediate filaments, or if additional proteins are involved (see Table 1). There is evidence that BPAGl (bullous pemphigoid antigen) has homology to desmoplakins, and that it occupies a similar position and may have a similar intermediate filament-linking role in the hemidesmosome as desmoplakins do in the desmosome (Stanley et al., 1988; Sonnenberg et al., 1991). The function of BPAG2, the 200 kDa polypeptide, IFAP 300, and HDl in the hemidesmosome is not clear, but the last three may couple intermediate filaments to the P4 subunit (Guidice et al., 1991). Because members of the integrin family participate in transmembrane signaling (Du et al., 1991), it is also possible that these proteins form part of a signaling pathway between epithelial cells and the extracellular matrix of the basement membrane. D. The Tight Junction

The tight junction is the apical-most juncfion of epithelial cells (see Figure 1), representing a site where the membranes of adjacent cells appear to touch each other at intervals and even to fuse (Farquhar and Palade, 1963). The tight junction forms a continuous permeability barrier between adj acent cells, and this barrier selectively regulates the flow of molecules through the paracellular space (Madara, 1988; Cereijido et al., 1988). In addition, the tight junction serves to maintain the polarity


21

of the epithelial cell, namely the biochemically and functionally distinct apical and basolateral domains of the plasma membrane, by preventing lateral diffusion of transmembrane proteins from one domain to the other (Gumbiner, 1987; Rodriguez-Boulan and Nelson, 1989; Rabito, 1992). Despite significant progress on the identification and characterization of peripheral membrane proteins associated with the tight junction (Citi, 1993; Anderson et al., 1993), it was only recently that a transmembrane protein, named occludin, was isolated (Furuse et al, 1993). Given the nature of the tight junction, such a protein would be expected to function simultaneously as a homophilic adhesion molecule and as a selective pore-forming protein. Interestingly, however, occludin does not show any significant sequence homology to any known CAM nor, in fact, to any other protein (Furuse et al., 1993). Analysis of its primary structure reveals that it contains four putative membrane-spanning segments in its amino-terminal half, a pattern very similar to that of the connexins of the gap junction, proteolipoprotein of myelin, and synaptophysin of synaptic vesicles (Furuse et al., 1993). This general structure may be functionally important for proteins that form adhesions between membrane surfaces and that locally restrict permeability. Relatively little is known about how occludin interacts with the other known proteins of the tight junction, all of which are peripheral proteins present on the cytoplasmic face (see Table 1). It is known that both ZO-1 and ZO-2 are located just beneath the plasma membrane and are thus closest to the cytoplasmic domain of occludin (Gumbiner et al., 1991; Jesaitis and Goodenough, 1994). In contrast, cingulin and 7H6 antigen are located farther inside the cell (Stevenson et al., 1989; Citi, 1993; Zhong et al., 1993). It has recently been shown that the cytoplasmic domain of occludin interacts directly with ZO-1, and that ZO-2 likely associates with occludin by binding to ZO-1 (Furuse et al., 1993). Spectrin tetramers are associated with ZO-1 (Itoh et al., 1991), suggesting a molecular linkage between occludin and the actin-based cytoskeleton known to underlie the junction (Madara, 1987). Cingulin may also mediate interactions with the actin network because it is structurally similar to the rod portion of myosins, a family of actin binding proteins (Citi et al., 1989). More specific information on the manner in which the tight junction proteins are assembled is not yet available. As with the adherens junction, the desmosome, and the hemidesmosome, it seems likely that the tight junction also has a role in signal transduction. While no specific function has been determined for any of the tight junction proteins, there is homology between the amino-terminal domain of ZO-1 and the discs-large (dig) tumor suppressor, a member of a family of proteins implicated in signal transduction at specialized cell-cell contacts in Drosophila (Willott et al., 1993). E. The Gap Junction The gap junction is perhaps the most widespread of the cell-cell junctions, being found in large numbers in most animal tissues and in practically all animal species.

22


It appears as a patch where the membranes of two adjacent cells are separated by a uniform narrow gap of about 2-4 nm (Fawcett, 1986). Within this patch is a dense aggregation of up to several hundred channels, each individual channel called a connexon. Each connexon is a hexagonal oligomer of six related or identical proteins in one membrane that is joined head-to-head with a similar complex in the membrane of the adjacent cell, forming a pore that allows inorganic ions and other small water-soluble molecules (< 1-2,000 daltons) to diffuse between the coupled cytoplasms (Loewenstein, 1979; see Figure 1). The proteins that comprise each connexon are members of a family of related gene products called connexins, which are classified according to their respective calculated molecular mass (Revel, 1988; Dermietzel et al., 1990; see Table 1 for examples). About a dozen connexins have been identified to date, each showing a distinct, but often overlapping, tissue distribution (Bennet et al., 1991; Haefliger et al., 1992). While connexins have long been thought to carry out the adhesive function of the channels, they do not show any homology to other families of adhesion molecules. Interestingly, however, their hydropathy plot is similar to that of occludin, the transmembrane protein of tight junctions thought to mediate adhesion between apposed membrane surfaces (Furuseetal, 1993).

VI. CONCLUSION As described in the chapters of this volume, knowledge about CAMs has exploded over the last 15 years. More than 50 CAMs have been identified and structural characterization has allowed these molecules to be classified into families. The initial definition of CAMs was operational, based on their ability to mediate cell-cell adhesion in assays in vitro. As expected, many of the CAMs, notably the desmogleins and desmocoUins of desmosomes and the cadherins found within adherens junctions, do appear to function as adhesive molecules in vivo. Recent studies suggest, however, that CAMs do not serve simply as intercellular glue. For example, contacts involving N-cadherin, Ll/Ng-CAM, or N-CAM all trigger changes in intracellular second messenger systems that affect neurite extension (Doherty and Walsh, 1992) and contact mediated by E-cadherin affects cell motility (Chen and Obrink, 1991). The structure of the membrane-associated protein tyrosine phosphatase fi, which includes an immunoglobulin domain that mediates cell-cell adhesion and a cadherin-related segment in its cytoplasmic region is strongly suggestive of a role in cell-contact regulation of cellular differentiation (Brady-Kalnay and Tonks, 1994). In the case of some integrin receptors, ligand binding is known to affect gene expression (Damsky et al, 1983). These observations suggest that the field of cell adhesion is moving beyond identifying and structurally characterizing CAMs and into the more exciting realm of precisely defining their roles in cellular physiology and differentiation.


23

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CELL RECOGNITION MOLECULES OF THE IMMUNOGLOBULIN SUPERFAMILY IN THE NERVOUS SYSTEM

Georgi Gegelashvili and Elisabeth Bock

I. Introduction 34 A. Cell-Cell Recognition 34 B. Families of Cell Recognition Molecules 35 II. The Immunoglobulin (Ig) Gene Superfamily: General Design 35 A. Ig-homology Domain 35 B. Fibronectin (Fn) Type Ill-like Domains 37 III. Classification of Neural Recognition Molecules of the Ig Superfamily . . . . .37 A. Ig-Like Glycoproteins Without Fnlll Domains or Any Other Structural Motifs 37 B. Ig/FNIII Glycoproteins 40 C. Other Ig-like Proteins Expressed in the Vertebrate Nervous System . . . . 44 IV. The Neural Cell Adhesion Molecule, NCAM 45 A. Diversity of NCAM 45 B. Localization and Organization of the NCAM Gene , . . 46


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GEORGI GEGELASHVILI and ELISABETH BOCK C. Regulation of NCAM Gene Expression D. The Functions of NCAM E. Structural Determinants of NCAM Function Acknowledgments References

48 52 54 60 61

I. INTRODUCTION A. Cell-Cell Recognition One of the fundamental features of a living cell is a prompt and adequate reaction to various environmental cues as illustrated by the complex cellular behavior during tissue formation, regeneration, wound healing, and so forth. Thus, during development of the nervous system, cells seem to respond to an immense number of physico-chemical signals in various combinations, and this "tuning" generates the brain with its characteristic symmetry, polarity, and periodicity (Edelman and Crossin, 1991). Little is known about the nature of these factors, although the extracellular matrix and the surface of interacting cells are recognized as essential components of the tuning machinery (Linnemann and Bock, 1989; Crossin et al., 1990; Bixby and Harris, 1991; Hynes and Lander, 1992). Acquisition of complex and variable information derived from these sources requires the presence of specific primary sensors. Indeed, all cells so far tested appear to express membraneassociated glycoproteins characterized by affinities for constituents of the extracellular matrix and cell-surface molecules expressed by other cells. These glycoproteins were originally characterized by their capacity for creating adhesion between cells or between cells and the extracellular matrix (Thiery et al, 1977; Hoffinan and Edelman, 1983; Sadoul et al., 1983). Hence, the adhesive potential of these molecules determined their general designations—substrate adhesion molecules (SAMs) and cell adhesion molecules (CAMs). However, these operational designations do not always accurately reflect the biological functions of these molecules. Thus, an increasing body of evidence suggests that the morpho-regulatory activities of several CAMs may be accounted for by a direct activation of second messenger systems (for review, see Schachner 1992; Walsh and Doherty, 1992) probably leading to gene regulation. Other interactions between cell adhesion molecules primarily affect intracellular cytoskeletal structures and, thus, cytoskeleton-dependent local transient processes (e.g., locomotion; Atashi et al., 1992; Gumbiner, 1993; Maxfield, 1993). Certain CAMs exhibit capacity for ATP hydrolysis (Aurivillius et al., 1990; Dzhandzhugazyan and Bock, 1993), or influence the extracellular ionic milieu via the regulation of transporter ATPases (e.g., adhesion molecule on glia, AMOG; Gloor et al., 1990). The initiation and proper performance of adhesion, signal transduction, and other functions require a precise, reciprocal recognition by apposed "sticky" molecules. Hence, the term "recognition mole-

Ig-CAMs in the Nervous System

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cule" seems to be a more appropriate general designation for these glycoproteins, although their conventional names often incorporate the orthodox abbreviation "CAM" and this term will also be used in this review. B. Families of Cell Recognition Molecules

Initially, cell recognition molecules were divided into calcium dependent and calcium independent CAMs (Edelman, 1987). However, this classification was only justified in the case of homophilic binding (i.e., CAM on one cell surface binds to an identical CAM on the surface of an opposed cell). However, certain adhesion molecules bind to different partners (heterophilic binding) and may fall into either group, depending on the nature of the ligand. For example, binding of I-CAM to rhinovirus is Ca^^-independent, whereas I-CAM-LFA-1 interaction requires Ca^"^ (Staunton et al, 1990). In the last decade, a large number of cell surface molecules implicated in recognition events have been classified more accurately, based on structural similarities and related functions. Currently, the CAMs so far discovered are grouped into the following families: the immunoglobulin (Ig) superfamily (Edelman, 1987; Bock, 1991; Yoshihara et al., 1991; Brummendorf and Rathjen, 1993), the cadherin family (Takeichi, 1991), the integrin superfamily (Reichardt and Tomaselli, 1991; Hynes, 1992), the selectin (LEC-CAM) family, and the H-CAM family (Obrink, 1991). These families are continuously growing and new families seem to be emerging. AMOG may thus be considered as a founding member of a distinct group of cell recognition molecules. In this article, we will focus on the members of the Ig superfamily of cell recognition molecules which are predominantly or exclusively expressed in the vertebrate nervous system, with special emphasis on the neural cell adhesion molecule, NCAM.

II. THE IMMUNOGLOBULIN (Ig) GENE SUPERFAMILY: GENERAL DESIGN A.

Ig-homology Domain

The inclusion of a protein into a gene superfamily is generally based on similarities in overall gene design, amino acid sequence, tertiary structure, and frequently, but not necessarily, function. With regard to the immunoglobulin superfamily, the structural features of the founding members, the immunoglobulins (Hill, 1966; Edelman, 1970; Edmundson et al, 1975; Hunkapiller and Hood, 1986), served as paradigm for the selection of subsequent members. The hallmark of the immunoglobulin gene superfamily is a structural element designated the immunoglobulin (Ig) homology unit or domain (Hunkapiller and Hood, 1986). The number of these characteristic domains in an individual molecule may vary from one to several and they are typically arranged in series in the extracellular portion of a molecule.

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GEORGI GEGELASHVILI and ELISABETH BOCK

Figure /. Arrangement of exons in the NCAM gene. Variably expressed exons are drawn as inserts between exons 7 and 8, 12 and 13, and 17 and 18. The bifurcation of the linear diagram following exon 14 symbolizes incompatibility of exon 15 and exons 16-19 within a single mRNA (for more details, see text).

The Ig domain is a globular structure composed of approximately 70-110 amino acids. Although the members of the superfamily display considerable diversity in amino acid composition of Ig-domains, these structural blocks contain well-conserved amino acid clusters which seem to ensure a homologous three dimensional organization of the unit (Hill et al, 1966; Edelman, 1970; Hunkapiller and Hood, 1989). Each domain consists of two P-sheets that are connected by disulfide bonds formed between highly conserved cysteine residues believed to stabilize the overall domain structure (Edmundson et al., 1975; Amzel and Poljak, 1979). In immunoglobulins, the so-called variable (V) domains contain 65-75 amino acids between the bond-forming cysteines. In constant (C) domains, the distance between the conserved cysteines is shorter, approximately 55-65 amino acids. In V domains, each P-sheet is composed of 4 antiparallel P-strands complemented by a short connecting p-strand stretch, while p-sheets in C domains contain 3 and 4 strands (Figure 1). The interior of the globular domain is mainly formed by hydrophobic amino acid residues, whereas hydrophilic residues are oriented outwardly. Based on the predicted mode of folding and statistical analysis of conserved amino acids, a modified classification for Ig-like domains was introduced by Williams (1987). The sequences fitting the general design of the Ig domain were categorized into 3 sets: V, CI, and C2. V-set sequences are supposed to be folded in a manner analogous to V domains of immunoglobulins. The predicted three dimensional structures of both CI and C2 sequences are presumably very similar. However, at least two putative


37

P-strands of a C2 domain display a higher resemblance to V sequences than to CI sequences (Williams, 1987; Williams and Barclay, 1988). CI domains are found in immunoglobulins (Williams and Barclay, 1988). C2 sequences are typical for cell adhesion molecules of the immunoglobulin superfamily, although V-type domains also are found in several recognition molecules. Furthermore, a structural shift from C-type towards V-type folding may occur, due to alternative splicing (see NCAM, below). Determination of tertiary structure by X-ray crystallography or NMR has not been performed for most of the recognition molecules of the Ig-superfamily except in a few instances (Becker and Reeke, 1985; Bjorkman et al., 1987; Davis et al., 1993; Brady etal., 1993). B. Fibronectin (Fn) Type Ill-like Domains

Cell recognition molecules of the Ig-superfamily frequently express another structural feature, repetitive elements homologous to the fibronectin type III (Fnlll) domain. These repeats are generally expressed in the extracellular portion of the molecule, between the Ig-like domains and the plasma membrane (Cunningham et al., 1987; Moos et al., 1988; Brtimmendorf and Rathjen, 1993). The prototype of this domain was found to be repeated up to 16 times in the extracellular matrix protein, fibronectin (Kornblihtt et al, 1985; for review, see Engel, 1991). Recent structural data suggest that the Fnlll domain and related structural units might be composed of two (J-sheets with three and four antiparallel P-strands, thus exhibiting a remarkable resemblance to the C-type structure (Baron et al., 1992; Leahy et al, 1992). An evolutionary and functional relationship between Ig and Fnlll-like domains has not been established.

III. CLASSIFICATION OF NEURAL RECOGNITION MOLECULES OF THE Ig SUPERFAMILY The Ig superfamily recognition molecules so far characterized in the nervous system may be grouped into three subfamilies, based on their structural features (Burgoon et al., 1991; Grumet et al., 1991; Bnimmendorf and Rathjen, 1993): molecules containing Ig-like domain(s), but lacking Fnlll-like repeats, may constitute one group, and molecules combining Ig- and Fnlll-type units fall into a second category (Ig/FnIII) (Figure 2). Athird group is composed of molecules that combine Ig-domains with structural units other than Fnlll-like domains. A.

Ig-Like Glycoproteins Without Fnlll Domains or Any Other Structural Motifs

The following glycoproteins may be allocated to this first group: MRC OX-2 (Clark et al., 1985), OBCAM (Schofield et al., 1989), PVR (Mendelhson et al..


38

"¥ N-CAM

TAG-1

F3/F11

Po

Thy-1

MRC OX-2

MAG

Figure 2. General design of cell recognition molecules of the Ig superfamily expressed In the vertebrate nervous system. Typical members containing (A) both Ig- and Fnlll-like domains (Ig/FnIII molecules) and (B) Ig-like domains only. Loops labeled "C2" and ' V represent distinct types of Ig-like domains, and boxes represent Fnlll-like domains. Among the possible splice variants, the Isoforms with the largest number of Fnlll-IIke domains are drawn. The putative sites of N-llnked glycosylation are marked by symbol (T). Asterisks indicate locations of the Arg-Gly-Asp sequences. Arrows symbolize attachment to the plasma membrane via a GPI anchor. The existence of the transmembrane version of F11 (designated contactin, see Ranscht, 1988) Is currently debatable (Wolff et al., 1989; Zisch et al., 1992; modified from Yoshihara et al., 1991; reprinted with permission of Elsevier Scientific Publishers).

1989), HT7 (Seulberger et al, 1990), myelin/oligodendrocyte glycoprotein (Gardiner et al, 1992), Thy-1 (Seki et al, 1985), P^ (Lemke and Axel, 1985; Filbin and Tennekoon, 1992), DM-GRASP, also known as BEN, JC7, or SCI (Pourquie et al., 1990; Burns et al, 1991; Tanaka et al., 1991; El-Deeb et al., 1992), myelin-associated glycoprotein, MAG, and the related (approximately 40% identity in amino acid sequences) Schwann cell myelin protein, SMP (Arquint et al., 1987; Salzer et al., 1987; Fujita et al., 1989; Dulac et al., 1992). The latter two glycoproteins exhibit a specific structural feature—â disulfide bridge linking the first and second immunoglobulin-like domains (Pedraza et al, 1990; Dulac et al, 1992). Thus, MAG and SMP seem to belong to a special subgroup. It should be emphasized that no adhesive or morphoregulatory functions have yet been attributed to OX-2, OBCAM, PVR,


39

HT7, or myelin/oligodendrocyte glycoprotein. It is noteworthy that a small domain in the multifunctional amyloid precursor protein, APP, exhibits sequence homology to the first Ig-like domain of OX-2 (Golde et al., 1990). Thy-1 The Thy-1 cell surface antigen was initially identified in thymus. Subsequently, Thy-1 was found to be ubiquitously expressed in the nervous system, particularly in all neurons after cessation of axonal growth (Morris, 1985). The Thy-1 gene was mapped to the q23 band of human chromosome 11, in the vicinity of other immunoglobulin gene superfamily members: NCAM and CDS (see review by Williams and Barclay, 1988). Thy-1 is a relatively small protein (17.5-18.7 kDa) with a single Ig-like domain of V type. It is linked to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor (Williams and Gagnon, 1982). The expression of Thy-1 in rat brain increases dramatically during the first three postnatal weeks (Morris, 1985). In cell cultures, the expression of Thy-1 can be modified by cAMP (Theveniau et al., 1992). The functions of Thy-1 are largely unknown, although recently, Thy-1-mediated adhesion of lymphoid cells to stromal cells has been demonstrated (Irlin and Peled, 1992). In the nervous system, Thy-1 forms complexes (via a homophilic mechanism), which inhibit neurite sprouting (Mahanthappa and Patterson, 1992b). In contrast, antibody-induced clustering of Thy-1 promotes neurite outgrowth (Lipton et al., 1992; Mahanthappa and Patterson, 1992a) by activating calcium influx into neurons via N- and L-type calcium channels (Doherty et al., 1993). Perturbations of Thy-1 expression are suspected to affect the growth of abnormal neurites in Alzheimer's disease (Leifer and Kowall, 1992). Po

More than 50% of the total protein content of peripheral myelin consists of P^ (Ishaque et al., 1980). P^ (29 kDa) contains a single V-type Ig domain, and unlike Thy-1, possesses transmembrane and cytoplasmic domains (Lemke and Axel, 1985). There is only one site for N-linked glycosylation and the attached carbohydrate moiety carries several epitopes: HNK-1, L3, L4, and Turp-27 (Filbin and Tennekoon, 1991; Krog and Bock, 1992). P^ seems to be involved in both heterophilic and homophilic binding. In the latter case, glycosylation of the two P^ molecules constituting the homophilic pair is essential (Filbin and Tennekoon, 1993). The polypeptide backbone and the HNK-1 epitope, but not the L3 epitope, appear to play pivotal roles in homophilic recognition and binding (Griffith et al, 1992). Heterophilic partners for P^ have not been identified yet. Homophilic PQ-PQ bonds are established between apposed loops of myelin sheath, whereas heterophilic P^-mediated recognition occurs between axons and Schwann cells. The latter interaction is believed to be involved in neurite outgrowth (Schneider-Schaulies et al, 1990). In vivo, P^ has been shown to be essential for the normal spiraling and

40


compaction of the myelin sheath and for the maintenance of axonal integrity (Giese etal, 1992). MAG

The myelin-associated glycoprotein, MAG, is a relatively minor component of the myelin sheath proteins (Quarles et al., 1973). Two distinct mRNAs, generated by differential splicing of a single primary transcript, are translated into two isoforms designated L-MAG (72 kDa) and S-MAG (67 kDa) (Arquint et al., 1987). The overall amount as well as the ratio of MAG isoforms are developmentally regulated (Frail and Braun, 1984). The principal structural difference between these two isoforms has been mapped to the cytoplasmic part which appeared to be 45 amino acid residues shorter in S-MAG than in L-MAG (Arquint et al., 1987). Only the latter isoform seems to be phosphorylated in vivo, but the functional consequences of this modification are so far unknown. Eight potential glycosylation sites are identified in MAG and several carbohydrate epitopes (HNK-1, L3, L4, and Turp-27) have been detected (Krog and Bock, 1992). The extracellular portion of MAG contains five Ig-like domains (C2-type) and is believed to be involved in heterophilic binding. The tripeptide Arg-Gly-Asp is found between the first and second Ig-like domains, and integrins, which frequently recognize this sequence in other proteins, are suspected to be MAG receptors (Salzer et al., 1987; Ruslahti and Pierschbacher, 1987). In addition, heparin and different coUagens may also interact with MAG (Fahrig et al, 1987). MAG is predominantly expressed at the axonmyelin interface and seems to maintain an apposition of the myelin sheath to the axon (Attia et al., 1989). B. Ig/FNIII Glycoproteins

The second category (Ig/FnIII) of neural cell recognition molecules includes NCAM, mouse LI (Moos et al., 1988) and its rat homologue, nerve growth factor-inducible large external glycoprotein, NILE (Bock et al., 1985; Prince et al., 1991), the closely related chicken NgCAM (Moos et al., 1988; Burgoon et al., 1991), also designated as G4 (Rathjen et al., 1987) or 8D9 (Lemmon and McLoon, 1986), chicken NrCAM /Bravo (de la Rosa et al., 1990; Grumet et al., 1991; Kayyem et al., 1992), chicken neurofascin (Volkmer et al., 1992), chicken Fll (Wolff et al., 1989; Zisch et al., 1992) and its mouse homologue F3 (Gennarini et al., 1989), chicken axonin-1 and its rat homologue TAG-1 (Furley et al, 1990; Zuellig et al., 1992). NCAM contains five immunoglobulin-like domains of C2type, whereas the rest contain six C2-type Ig-like domains. F3/F11 and axonin1/TAG-1 are very similar to each other with regard to amino acid sequence (approximately 50% identity), mode of attachment to the membrane (exclusively via a glycosylphosphatidylinositol (GPI) anchor), type of binding (preferentially heterophilic), and, thus, may fall into a separate subgroup, designated the Fl 1-subgroup. The transmembrane glycoproteins LI/NILE, NgCAM, NrCAM/Bravo, and


41

neurofascin constitute another subgroup, designated the LI-subgroup, which display both homophilic and heterophilic binding (Brummendorf and Rathjen, 1993). NCAM consists of a group of closely related isoforms characterized by five Ig-like domains, different modes of attachment to cell membrane, and other structural peculiarities (e.g., polysialylation). The NCAM isoforms may be regarded as a distinct subgroup of the Ig/FnIII glycoproteins, and will be described separately in the following section. The L1 Subgroup These molecules exhibit apparent similarity in their overall domain organization, distribution, and biological functions. Particularly, all members of the LI subgroup are transmembrane glycoproteins with 6 Ig-like domains and 3-5 Fnlll-like repeats in the extracellular portion (Burgoon et al, 1991; Grumet et al, 1991; Volkmer et al, 1992). In the central nervous system, these molecules are predominantly expressed in postmitotic neurons, being located at the sites of neuron-neuron contacts, on growth cones, and on developing axons (Rathjen and Schachner, 1984; Shiga et al, 1993). In addition, LI is expressed by Schwann cells in the peripheral nervous system (Martini and Schachner, 1986) and is induced by NGF in PC 12 cells (McGuire et al., 1978) and cultured immature astrocytes (Saad et al., 1991). With regard to amino acid sequences, the overall resemblance between the subgroup members is not striking: there is only 40% identity between NgCAM and LI, and 36% identity between NgCAM and the NgCAM-related cell adhesion molecule, NrCAM (Burgoon et al., 1991; Grumet et al., 1991). However, the cytoplasmic portions of LI, NgCAM, NrCAM, and neurofascin exhibit a remarkable homology in amino acid sequences. Particularly, two highly conserved stretches of unknown function are located within this region (Grumet et al., 1991; Volkmer et al., 1992). Another site of similarity between LI, NrCAM, and neurofascin is a short sequence, Arg-SerLeu-Glu, variably expressed in the middle between the above-mentioned conserved stretches. This small insert is a translation product of a differentially spliced exon and constitutes the only site of variation in the two existing isoforms of LI (Miura et al., 1991; Reid and Hemperly, 1992). In contrast, several isoforms of NrCAM and neurofascin have been identified and this additional diversity is due to splicing events altering the extracellular portions of these two molecules. The optional segments are located between the second and third Ig-like domains, and between the sixth Ig-like domain and the battery of Fnlll-like domains. It is noteworthy that only the first, second, and fourth Fnlll-like domains are present in all isoforms of NrCAM and neurofascin, whereas the third and fifth Fnlll-like domains are encoded by alternatively spliced exons (Grumet et al., 1991; Kayyem et al., 1992; Volkmer et al., 1992). Only neurofascin (but not LI, NgCAM, or NrCAM) expresses a unique sequence enriched in proline, alanine, and threonine. This segment, localized in close proximity to the ^ran^-membrane portion of the molecule contains sites for 0-linked glycosylation (Volkmer et al., 1992). Analogous

42


structures, found in other glycoproteins including NCAM, are proposed to regulate the orientation of the molecule relative to the plasma membrane (Jentoft, 1990). N-linked carbohydrates are present in all members of the LI subgroup. However, glycosylation of the individual members seems to be heterogeneous. For example, only 35% of total LI contains the HNK-1 epitope (Faissner, 1987; Kucherer et al., 1987). Additional modifications of the molecules by phosphorylation and sulfation may also occur (Linnemann et al., 1988). Thus, the individual members of the LI subgroup exhibit further diversity due to alternative splicing and post-translational modifications, although the functional significance of these modulations is unclear. The structural variants may presumably regulate the efficacy of recognition and binding, as well as the choice of binding mode and partner. The molecules in the LI subgroup are engaged both in homophilic and heterophilic binding. Furthermore, LI c/^-interacts with NCAM within the plane of membrane. This kind of heterophilic interaction appears to enhance the efficacy of Ll-Ll binding between apposed membranes. Chicken NgCAM seems to be a binding partner for several recognition molecules, including axonin-1 (Kuhn et al., 1991), Fll (Brtimmendorf et al., 1993), neurocan and 3F8 proteoglycan (Grumet et al., 1993). The related molecule, NrCAM, employs homophilic binding in neuron-neuron interaction, but also binds to an unidentified partner expressed on non-neuronal cells. Interestingly, the latter mechanism utilizes divalent cations— calcium or magnesium (Mauro et al, 1992). Recently, a heterophilic interaction between NrCAM and F11 has been demonstrated (Morales et al, 1993). Sequence data provide some hints concerning possible interaction between LI, NgCAM, and neurofascin and recognition molecules belonging to other gene superfamilies. Particularly, each of these molecules contains the Arg-Gly-Asp sequence which is known to bind to various integrins (Ruslahti and Pierschbacher, 1987). This tripeptide is duplicated in the sixth Ig-like domain of LI, whereas in NgCAM and neurofascin, a single Arg-Gly-Asp sequence is located within the third Fnlll-like domain (note, that in neurofascin, this domain is expressed optionally, see above). However, it is not known whether these molecules actually interact with integrins. So far, functional domains responsible for different modes of recognition and binding are poorly elucidated. In addition to adhesive activity, LI seems to be capable of signal transduction. In neural cells, triggering of LI with specific antibodies elevates the intracellular calcium concentration and, depending on the cell type, decreases or increases intracellular pH and the level of inositol phosphates (Schuch et al., 1989; von Bohlen und Halbach, 1992). Certain carbohydrate moieties, distinct from the HNK-1 epitope, seem to be involved in LI-dependent opening of calcium channels (Itoh et al, 1992). The calcium influx into neurons, rather than adhesion per se, presumably accounts for the LI-stimulated neurite outgrowth in vitro (Williams et al., 1992). Regulation of neurite outgrowth, axonal elongation, fasciculation, and guidance are considered to be functions of LI and related molecules. LI and NgCAM

Ig'CAMs in the Nervous System

43

participate in both neuron-neuron and neuron-glia interactions (Grumet and Edelman, 1988; Grumet et al, 1984), but neurite outgrowth on astrocytes seems to be independent of LI (Ard et al, 1991). When employed as a substrate, LI was found to enhance migration of cerebellar neurons (Asou et al.,1992; Miura et al., 1992). Participation of NrCAM in induction of axonal growth has also recently been demonstrated (Morales et al., 1993). The Fl 7 Subgroup The molecules comprising this subgroup exhibit an overall amino acid similarity of approximately 50%. The species homologues (axonin-1 versus TAG-1, and Fl 1 versus F3) display a higher degree of resemblance than the related molecules within the same evolutionary group. Thus, human and rat TAG-1 are 91% identical, whereas human TAG-1 and chicken axonin-1 exhibit 75% similarity, and the homology between rat TAG-1 and chicken axonin-1 constitutes 75 %. The sequence similarities determine the shared structural design of the subgroup members: typically, they contain six Ig-like domains followed by four Fnlll-like repeats and attach to the plasma membrane via a GPI-anchor. Unlike other Ig/FnIII glycoproteins, no alternative splicing, hence no multiplicity of isoforms is found within the Fll-subgroup. The observed diversity of the corresponding mRNA species is presumed to be due to differential polyadenylation (Gennarini et al, 1989; Furley et al, 1990; ZueUig et al, 1992; Hasler et al, 1993). Variations in electrophoretic mobilities between the species homologues may be attributed to differences in glycosylation (Wolff et al, 1987; Hasler et al, 1993). However, the HNK-1 carbohydrate epitope is expressed by all members of the group (Krog and Bock, 1992). TAG-1 contains the Arg-Gly-Asp sequence, whereas its species homologue, axonin-1, does not (Zuellig et al, 1992). The Fll-subgroup members are predominantly expressed on membranes in developing nerve fiber tracts (Stoeckli et al, 1991). Up-regulation of F3 seems to be a hallmark of differentiation of neurons in culture (Theveniau et al, 1992). However, in brain, only a subset of neurons displays F3 immunoreactivity. Purkinje and stellate neurons in the cerebellum appear to be devoid of F3, whereas other cerebellar neurons, Golgi and granule cells, express this recognition molecule. On F3-positive cells, the molecule was found either to be restricted to axonal membranes (granule neurons), or to be uniformly distributed all over the cell surface (Golgi neurons). Immunostaining was observed on pre- or postsynaptic membranes, but never on both (Faivre-Sarrailh et al, 1992). The terminal parts of axons release axonin-1 which undergoes post-translational processing into soluble form in the intracellular compartments (Stoeckli et al, 1991). Soluble forms may also arise from the cleavage of GPI anchors of Fll-related molecules expressed on the cell surface. Phospholipases exhibiting GPI-degrading activity have recently been demonstrated in brain (Fouchier et al, 1990; Hoener et al, 1990). It is notewothy that the major part of axonin-1/TAG-1 exists in soluble form.

44


whereas only a minor fraction of F11/F3 is secreted (Stoeckli et al, 1989; Moss and White, 1992). Soluble forms interfere with cell-cell bonds established by the membrane-bound forms. For example, soluble axonin-1 exhibits anti-adhesive properties and seems to disturb fasciculation of axons (Stoeckli et al, 1991). On the other hand, secreted F11 -related glycoproteins are capable of mimicking other morphoregulatory activities exhibited by the GPI-associated forms in vitro. For example, in neuronal cultures, soluble F3 enhances neurite outgrowth in a dose-dependent manner, and thus mimics the action of membrane-linked F3 (Durbec et al., 1992). In vivo, F11/F3 and axonin- l/TAG-1 are believed to play central roles in neurite outgrowth, elongation, fasciculation, pathway selection, and possibly, in target recognition (Sonderegger and Rathjen, 1992; Brummendorf and Rathjen, 1993; Briimmendorf et al, 1993). However, the precise mechanisms whereby F11 -related glycoproteins function in vivo are still unclear. Nevertheless, there is no doubt that specific recognition and binding precede the chain of morphoregulatory events elicited by these molecules. The members of the Fll subgroup are engaged both in heterophilic and homophilic binding. However, the latter mechanism has only been demonstrated for axonin-1 and TAG-1 (Rathjen and Jessel, 1991; Rader et al., 1993). So far, only a single heterophilic binding partner (NgC AM) has been revealed for axonin-1 (Kuhn etal., 1991), whereas Fll additionally interacts withNrCAM (Morales etal., 1993) and several constituents of the extracellular matrix: restrictin (Brummendorf et al., 1993), tenascin (Zisch et al., 1992), and the oligodendrocyte-derived ECM protein Jl-160/180 (Pesheva et al., 1993). It is noteworthy that Jl-160/180 causes a dramatic repression of cell-substrate adhesion and inhibition of neurite outgrowth (Pesheva et al., 1993). Functional sites responsible for the binding of Fll to NgCAM, NrCAM, restrictin, and tenascin have been mapped to Ig-like domains Mil (Zisch et al, 1992; Briimmendorf et al., 1993; Morales et al., 1993). However, it is still unclear whether these sites are overlapping, or whether simultaneous binding of several ligands to Fll may occur. C. Other Ig-like Proteins Expressed in the Vertebrate Nervous System

The major representatives of this third group are the neurotrophin receptors related to the ^rA:proto-oncogene product. These receptor tyrosine kinases combine two extracellular C2-type Ig-domains with a characteristic leucine-rich repeat flanked by cysteine clusters (for review see Chao, 1992). The FGF tyrosine kinase receptors that express 1-3 Ig-like domains may also fall into this category (Johnson and Williams, 1993). Recently, a brain-specific chondroitin sulfate proteoglycan neurocan has been cloned and characterized. It consists of a single V-type Ig-domain followed by several structural motifs such as hyaluronic acid binding regions, EGF-like repeats, lectin-like domains, and complement regulatory-like domains (Ranch etal., 1992)


45

IV. THE NEURAL CELL ADHESION MOLECULE, NCAM NCAM is the most extensively studied cell-cell recognition molecule. NCAM was initially described as a synaptic membrane protein termed the D2 antigen (Jorgensen and Bock, 1974) and later shown to mediate cell-cell adhesion (Rutishauser et al., 1976; Thiery et al., 1977). cDNA and genomic clones have provided detailed information on the molecular structure of NCAM (Murray et al, 1984; Goridis et al, 1985; Barbas et al., 1986; Cunningham et al., 1987; Owens et al, 1987; Small et al., 1988; Thompson et al, 1989; Barton et al, 1990; Hirsch et al, 1990, 1991; Chen et al., 1990) and enabled transfection experiments elucidating the functions of the molecule (Edelman et al., 1987; Doherty et al, 1989,1990,1992a,b; Walsh and Doherty 1992; Edvardsen et al., 1993). The exon-intron organization of the NCAM gene supports the hypothesis that the entire Ig superfamily emerged from an early NCAM-like gene (Edelman, 1987; Williams and Barclay, 1988; Edelman and Crossin, 1991). Thus, it is obviously justified to describe NCAM in detail as a suitable prototype of the immunoglobulin superfamily of cell recognition molecules. A.

Diversity of NCAM

NCAM is the common designation of a group of closely related glycoproteins. In the aduh brain, it appears as three distinct classes of Mj. 190 kDa, 135 kDa, and 115 kDa. The corresponding molecular weights of the polypeptide backbones deduced from mouse cDNA sequence data are 117 kDa, 91 kDa, and 79 kDa. In the following, these major NCAM classes will be referred to as NCAM-A, NCAMB, and NCAM-C, respectively. The isoforms mainly differ in the mode of attachment to the plasma membrane and the length of the cytoplasmatic domains. Thus, NCAM-C is linked to the plasma membrane via a GPI anchor, whereas NCAM-B and NCAM-A are integral transmembrane proteins. The largest isoform, NCAM-A, contains 267 more amino acid residues than NCAM-B in the cytoplasmic tail (Santoni et al., 1987). In addition to the membrane-bound isoforms, a small fraction of soluble NCAM has also been identified and characterized (Krog et al, 1992; Olsen et al., 1993). Soluble NCAM consists partially of the lytic fragments of membrane-bound NCAM (He et al., 1987; Nybroe et al., 1989a), and partially of full-length forms with preserved transmembrane and cytoplasmic domains (Olsen et al., 1993). A distinct truncated isoform may be a third source of soluble NCAM (Goweretal, 1988). There are several potential sites for N- and 0-linked carbohydrate attachment, although the actual sites of glycosylation are not yet defined. Some N-linked carbohydrate moieties carry sulfate residues and linear chains of a, 2-8-N-acetylneuraminic acid homopolymers (polysialic acid, PSA, see Finne et al., 1983). Another type of post-translational modification—^phosphorylation—âlso contributes to the number of diverse variants of NCAM.

46


The NCAM polypeptides are translated from five classes of mRNA of 7.4-, 6.7-, 5.2-, 4.3-, and 2.9-kb, respectively (Goridis and Wille, 1988; Walsh and Dickson, 1989). B. Localization and Organization of the NCAM Gene The NCAM gene is a relatively large and complex transcriptional unit which spans more than 85 kilobases (Owens et al., 1987; Walsh, 1988, Colwell et al., 1992). The gene is mapped to the ncam locus on chromosome 9 in the mouse (D'Eustachio et al., 1985), chromosome 8 in the rat (Kobayashi et al., 1992), and to band q23 in the distal portion of the long arm of chromosome 11 in man (Nguyen et al., 1986; Walsh et al., 1986). Interestingly, the same region comprises the genes for two other immunoglobulin superfamily members, Thy-1 and the T cell receptor a chain (D'Eustachio et al., 1985). Further studies on genetic mapping have revealed a linkage between NCAM and the esterase-6 and apolipoprotein C-III genes on rat chromosome 8 (Kobayashi et al., 1992). In addition to the NCAM gene, band q23 on the human chromosome 11 contains a cluster of genes encoding apolipoproteins CIII, Al, and AIV (Mietus-Snyder et al., 1990). Recently, the NCAM gene was shown to be situated 150 kilobases downstream of another occupant of the llq23 band, the dopamine D2 receptor gene, which probably is associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism (Eubanks et al, 1992). However, no neurological disease has so far been shown to be directly linked to the NCAM gene. It is assumed that the entire NCAM family is exclusively encoded by a single gene in the eukaryotic genome and that all distinct forms so far identified arise from post-transcriptional and/or post-translational modifications (for reviews, see Edelman and Crossin, 1991; Walsh and Doherty, 1991b; Goridis and Brunet, 1992). However, it has recently been established that the genome of Xenopus laevis contains two functional copies of the NCAM gene, and the amino acid sequences of their translation products seem to diverge slightly (approximately 8%). Both NCAM genes are expressed at similar levels throughout embryonic development and in all adult tissues examined (Tonissen and Krieg, 1993). Thus, the functional significance of this finding, as well as the occurrence of the NCAM gene duplication in other species, have yet to be investigated. At least 25 exons have been so far recognized in the NCAM gene (Figure 3). The number, order, size, and junctional sites of the NCAM exons appear to be highly conserved among the species studied, whereas intron sizes are more variable (Walsh and Doherty, 1991). The NCAM gene is believed to be transcribed into a pre-mRNA and subsequent removal of introns and optionally used exons, combined with differential polyadenylation, generates a variety of the mature mRNA species (Walsh, 1988; Goridis and Wille, 1988; Walsh and Dickson, 1989).


0-6

47

I 7 I 8 I 9 I 10 I 11 I 12 I 13 I 14 CTIAAGI

\ . |

16

I 17

I 19

I

18 Figure 3. A putative folding pattern of the C2-type Ig domain. The segments ((3strands) constituting two opposing layers (p-sheets) are marked in alphabetical order: A-G, from N- to C-terminal (the directions are indicated by arrowheads). The four-chain p-sheet is composed of A, B, D, and E antiparallel P-strands. The opposing three-chain layer consists of C, F, and G p-strands (striated). A disulfide bond (thick black line) is formed between the B and F strands (modified from Edmundson et al., 1975; reprinted, with permission of American Chemical Society).

Exon 0, coding the initiation methionine and most of the signal peptide, and exons 1 to 14, are expressed constitutively, regardless of the site(s) of splicing. Exons 1 to 10 encode 5 Ig-like C2-type domains in the N-terminal part of NCAM. The pattern—^two exons encoding each Ig-like domain—îs typical for most Ig CAMs. Optional insertion of a 30 bp exon (designated VASE, variable domain alternatively spliced ^xon) between exons 7 and 8 may take place, and this addition probably transforms the fourth Ig-like domain from a C2- into a V-type (Small et al., 1988; Small and Akeson, 1990). Another splice site is located between exons 12 and 13. Five exons may be inserted into this region: exons a (15 bp), b (48 bp), c (42 bp), SEC (239 bp), and an AAG triplet (Dickson et al., 1987; Gower et al., 1988; Thompson et al., 1989). It is an open question whether all possible combinations of the exons a, b, c, and AAG are expressed in all tissues and species, although most variants are present in skeletal muscle and brain (Barthels et al., 1992). The expression of exon a introduces a set of proline residues which possibly changes the local configuration into a hinge-like structure (Santoni et al., 1989). Exons b and c encode amino acid stretches enriched in threonine and serine residues constituting potential sites for 0-linked glycosylation (Walsh et al., 1989). Insertion of the AAG triplet changes an arginine to glutamine-glycine, but the significance of this substitution has not yet been defined. Insertion of AAG between exons 13 and 14 has also been detected, although the particular mechanism of such an edition is unclear (Barthels et al., 1992). Exon SEC, which so far only has been identified in a human NCAM cDNA clone, contains a stop codon, and may thus result in the synthesis of a truncated, soluble NCAM form (Gower et al., 1988). The exons downstream to exon 14 determine the mode of attachment of NCAM to the plasma membrane and specify the major size classes of NCAM mRNA and polypeptides routinely observed by Northern- and immuno-blotting (Owens et al.,

48

GEORGl GEGELASHVILI and ELISABETH BOCK

1987; Walsh and Dickson, 1989). The choice of exon 15 introduces a stop codon followed by two distinct polyadenylation sites and this determines the generation of the 5.2 kb and 2.9 kb mRNA species which are translated into the 120 kDa NCAM-C isoform. Exon 15 encodes a C-terminal sequence which contains information for association with glycosylphosphatidylinositol (GPI) situated in the plasma membrane (Walsh and Doherty, 1991a, 1991b). Skipping of exon 15 is a permissive event for expression of exons 16-19. Exon 16 encodes a transmembrane sequence and the following exons contain information about the cytoplasmic part of the molecule. The reading frame can only be closed by exon 19 and a single polyadenylation site is also mapped to this exon. The size of mRNAs expressing all three terminal exons (17, 18, and 19) is 7.4 kb and these are translated into proteins of 180 kDa (NCAM-A). Omission of exon 18 results in expression of the 6.7 kb mRNA species and translation into polypeptides of 140 kDa (NCAM-B). Thus, if all possible combinations of splicing events take place, a striking number of mRNAs and corresponding polypeptides might be generated. However, the existence of relatively few transcripts and polypeptide versions has so far been reported (Dickson et al., 1987; Gower et al., 1988; Small et al., 1988; Santoni et al., 1989; Thompson et al., 1989; Andersson et al., 1990; Barthels et al., 1992). C. Regulation of NCAM Gene Expression The developmental, regional, tissue, and cell type-specific patterns of NCAM seem to be precisely regulated, although little is known about the nature of primary environmental cues and consequent events responsible for thefinetuning of NCAM expression (for reviews, see Linnemann and Bock, 1989; Edelman and Crossin, 1991; Walsh and Doherty, 1991b). However, transcription and splicing are probably independent targets for these regulatory signals. Transcriptional regulation is responsible for quantitative changes in NCAM expression, whereas alternative splicing contributes to qualitative variations (the pattern of NCAM isoforms). Promoter Region of NCAM Gene The efficacy of the NCAM gene transcription is controlled by a single 5' promoter region of the gene, which has recently been isolated and characterized (Barton et al., 1990; Chen et al., 1990; Hirsch et al., 1990, 1991; Colwell et al., 1992). Apart from the major transcription initiation site, some other minor sites are suspected to function. However, no TATA- or CAAT-like elements have been found in any species analyzed, and this peculiarity assigns NCAM to the so-called "housekeeping" genes. Another typical feature of the promoter region is a remarkable enrichment in GC residues. Several DNA segments are capable of interaction with nuclear proteins and may thus be responsible for regulation of transcriptional activity. A comparison of the chicken NCAM promoter region with the analogous regions of the mouse, rat, and human genes has revealed different degrees of conservation of


49

the regulatory elements, including two putative homeodomain binding sites. The most preserved segments appeared to correspond to one of the homeodomain binding sequences, an octomer element, and to SP-1 and AP-1 elements (Colwell etal, 1992). Homeotic Genes and Regulation of NCAM

Transcription

The molecular nature of the proteins that bind to specific sequences within the promoter region of NCAM gene has yet to be clarified. However, some of them are predicted to be products of the so-called homeotic genes which are believed to play crucial roles in the establishment of body design (Kessel and Gruss, 1990; Hirsch et al., 1991; Hunt and Krumlauf, 1991). Recent studies have provided convincing proofs that homeotic gene products from Xenopus are able to control transcription of the NCAM gene, independently from other genes. Particularly, the product of the transfected homeotic HOX2.5 gene appeared to induce NCAM transcription in NIH 3T3 fibroblasts, and this process was found to be mediated via the homeodomain binding site II (HBS-II) located in the promoter region of the NCAM gene. This activity could be blocked by another related homeoprotein—^HOX2.4 (Jones et al, 1992). The promoter region is also a target for the H0XC6 (HOX3.3) homeoprotein which seems to control transcriptional activity via binding to a conserved sequence CCTAATTATTAA (homeodomain binding site-I, HBS-I), although an adjacent potential homeodomain binding site (HBS-II) may also play a modulatory role (Jones et al, 1993). However, little is known about the involvement of homeotic genes in NCAM expression, although it has been shown that deletion within the Pax homeoprotein correlated with an altered expression of NCAM and malformation of the neural tube in mutant mouse embryos (Epstein et al., 1991; Moase and Trasler, 1991). Regulation of Alternative

Splicing

So far, little is known about the general molecular mechanisms underlying alternative splicing. With regard to NCAM, the requirements for the skipping of exon 18 have been investigated in a single study utiUzing N2a cells, which expressed an artificial minigene modified by systematic deletions and replacements. The central part of exon 18 appeared to lack specific elements essential for the rejection of this exon, whereas the 5' flank of the exon seemed to be crucial for the differentiation state-dependent choice of this splice site (Tacke and Goridis, 1991). However, putative trans-diCtmg factors that may operate at this site and undergo quantitative and/or qualitative changes during development have not been identified. Only a few substances, NGF, cAMP, and osteogenic protein-I, which exhibit pronounced morphoregulatory activities, have been shown to affect the ratio of different splice forms (Prentice et al., 1987; Perides et al., 1992; Gegelashvili et al., 1993a, 1993b). Thus, treatment (1-4 days) of NG108-15 cells with osteogenic

50


protein-I results in an induction of NC AM-C which is undetectable in control cells, a 29-fold up-regulation of NCAM-A, and sixfold increase of NCAM-B, the isoform with the highest constitutive expression in control cells (Perides et al, 1992). In PC-12 cells, NGF up-regulates NCAM-B and induces expression of the NCAM-A isoform which is not produced in untreated cells (Prentice et al, 1987). Astrocytes subjected to a long-term treatment with cAMP develop a mature phenotype and selectively increase the expression of 6.7 kb mRNA class (encoding NCAM-B), primarily due to an up-regulation (approximately sevenfold) of exon VASE-containing transcripts (Gegelashvili et al., 1993a). Regulation of NCAM Expression by Biological and Pharmacological Agents

The molecular events which link environmental signals with the expression and/or activation of putative nuclear factors that, in turn, regulate the NCAM gene promoter activity, are poorly understood. Most of the exogenous substances so far shown to modulate NCAM synthesis, act at the starting sites of the signaling pathways, although the outcome (up- or down-regulation of NCAM) seems to depend on cell type and differentiation state. For example, in myotube cultures, the relative NCAM levels were demonstrated to be specifically increased by NGF (Lyles et al., 1993). Similarly, NGF appeared to increase the expression of the 140 kDa NCAM isoform in PC-12 cells within 24 hours (Prentice et al., 1987). In contrast, another research group did not find any. effect of NGF on NCAM expression in the same cell line (Friedlander et al., 1986). In a neuronal cell line, ND26, as well as in primary cultures of astrocytes, the synthesis of NCAM was also NGF-insensitive (Theveniau et al., 1992; Saad et al., 1991). TGF-|3 decreased the expression of NCAM in immature, but not in mature astrocytes (Saad et al., 1991), whereas the same growth factor up-regulated NCAM in embryonic cells and the 3T3 fibroblast cell line (Roubin et al., 1990). As mentioned above, another member of the TGF-P superfamily, osteogenic protein-I (OP-I), was also found to increase NCAM synthesis in a neuroblastoma-glioma hybrid cell line, NG 108-15 (Perides et al, 1992). bFGF induces NCAM expression in PC-12 cells (Mann et al., 1989), but not in astrocytes (Saad et al., 1991) or myotube cultures (Lyles et al., 1993). The stimulatory effects of both NGF and bFGF on NCAM synthesis in PC-12 cells are enhanced by cholera toxin and forskolin—âgents that increase intracellular cAMP concentrations (Mann et al., 1989). Up-regulation of NCAM mRNA and polypeptides has been observed in astrocytes (Gegelashvili et al, 1993a) and myotube cultures (Lyles et al., 1993) after treatment with a dibutyryl derivative of cAMP. Interestingly, in the latter study, forskolin had no effect on the levels of NCAM. An opposite situation was demonstrated in Schwann cell precursors, which responded to forskolin, cholera toxin, and cAMP analogues by acquisition of myelin-producing phenotype characterized by down-regulation of several marker proteins, including NCAM (Morgan et al, 1991). No effect of dibutyrl cAMP was detected on the level of NCAM expression in the neuronal cell line


51

ND26 (Theveniau et al, 1992). In contrast, differentiation of the human neuroblastoma cells, SK-N-BE, toward a neuronal phenotype by another morphogen, retinoic acid, resulted in a marked induction of NCAM (Melino et al, 1991). Similar effects were obtained with an anti-neoplastic agent, neocarzinostatin, when applied to murine neuroblastoma cells (Lowengrub and Schor, 1993). Elevated intracellular Ca^"^ did not change the quantity of NCAM in PC-12 cells (Doherty et al., 1988), whereas the calcium channel agonist, nisoldipine, increased the relative levels of NCAM in serum-free myotube cultures (Lyles et al, 1993). Removal of NCAM-C in astrocytoma C6 cells by means of phosphatidylinositol-phospholipase C (PIPL-C) provoked down-regulation of another isoform, 140 kDa NCAM-B, and of the corresponding 6.7 kb transcripts as well. In contrast, steady-state levels of the 2.9 kb NCAM mRNAclass remained unaffected, whereas the rate of translation into NCAM-C was dramatically increased (Theveniau et al., 1991). However, the mechanism of this remarkable regulation of NCAM expression remains unclear. One explanation is that PIPL-C may generate diacylglycerol (DAG). However, the possibility of DAG translocation to the internal side of the lipid bilayer, where it acts as a second messenger, has not yet been demonstrated. Moreover, DAG, as well as other activators of protein kinase C, phorbol esters, appeared unable to induce NCAM in PC-12 cells (Doherty et al., 1988). On the other hand, induction of c-fos after treatment of cells with exogenous PIPL-C has recently been demonstrated (Rahman et al., 1992). Fos is a constituent of the AP-1 transcription factors and the binding site for this complex is conserved in the NCAM gene promoter region (Colwell et al., 1992). Some of the above-mentioned growth factors and activators of second messenger pathways are known to induce immediate early genes (fosjun, myc, etc., families) that may regulate the NCAM gene transcription (Bartel et al., 1989; Sheng and Greenberg, 1990; Vaccarino et al., 1993). However, dramatic changes in NCAM expression (down-regulation) following activation of an early response gene (N-myc) have only been demonstrated in a single study (Akeson and Bernards, 1990). A list of substances shown to affect relative or total amount of NCAM may be supplemented by GABA (Meier and Jorgensen, 1986), laminin (Pollerberg et al, 1985), thyroxine (Thompson et al., 1987; Levi et al., 1990), absorbed horse serum, veratridine, insulin-like growth factor II, fetuin, calcitonin gene-related peptide (Lyles et al., 1993), undefined autocrine factors derived from N2a neuroblastoma and 3T3 fibroblast cell lines (Roubin et al., 1990), and the anti-neoplastic agent, suramine (Fantini et al., 1990). It is noteworthy that most of the above-mentioned data were obtained in vitro and the only agent ever shown to regulate NCAM expression in vivo is thyroxine (Levi et al., 1990). Further studies are needed to determine whether some of the many endogenous substances supposed to act as developmental cues actually modulate NCAM expression in vivo.

52


D. The Functions of NCAM Binding Properties of NCAM Promotion of cell-cell aggregation was initially considered to be the sole function of NCAM. NCAM-mediated cell-to-cell binding was thought to be executed via a calcium-independent homophilic mechanism and this view was supported by a series of in vitro binding assays utilizing solubilized NCAM, or NCAM presented on a solid phase or on liposomes (Rutishauser et al.,-1982; Hoffman and Edelman, 1983; Sadoul et al, 1983; Moran and Bock, 1988; Hall et al., 1990). This concept was confirmed in studies utilizing NCAM-negative L-cells and subclones transfected with NCAM cDNAs (Edelman et al., 1987). In addition, the ability of NCAM to interact with heparin/heparan sulfate (Cole and Glaser, 1986; Cole et al., 1986; Nybroe et al., 1989; Reyes et al, 1990), collagens (Probstmeier et al, 1989,1992), LI (Kadmon et al., 1990 a,b; Simon et al., 1991), and chondroitin sulfate proteoglycans, 3F8 and neurocan (Grumet et al., 1993), have been demonstrated. The discovery of heparin-binding properties of NCAM has raised doubts about the homophilic nature of NCAM-mediated adhesion. Thus, deletions in a putative heparin-binding site, as well as application of soluble peptides corresponding to this region, abolished NCAM binding to heparin and, concomitantly, reduced cell-cell adhesion (Cole and Akeson, 1989; Reyes et al., 1990). Moreover, in contrast to other studies (see above), Kadmon et al. (1990a) demonstrated that heparin is essential for the aggregation of NCAM-coated latex beads. These data suggest two possible explanations of heparin's role in binding events: (1) heparin may induce conformational change(s) in NCAM and, thus activate, unmask, or even create a distinct site responsible for homophilic binding; and (2) the heparin-binding site, upon occupation by ligand, may become competent for /ra«5-association with an apposing NCAM molecule. However, no experimental data supporting or rejecting either of these two hypotheses are yet available. On the other hand, c/s'-interaction of NCAM with LI significantly enhances ("assists") homophilic binding of LI on apposed cells or beads (Kadmon et al., 1990a, 1990b). Furthermore, data hinting at the probability of NC AM—APP interaction have recently been presented (Schubert et al., 1993). In vitro interactions of NCAM with heparan sulfates, different types of collagens, and with chondroitin sulfate proteoglycans have raised some yet unanswered questions concerning NCAM's ability for mediating cell-to-extracellular matrix binding in vivo and the physiological relevance of such interactions. NCAM-dependent recognition events seem to be modulated by changes in the overall cell surface density of NCAM (Hoffman and Edelman, 1983), redistribution of different isoforms on the cell surface (e.g., targeting to specialized areas, see PoUerberg et al, 1987; Persohn et al., 1989; Powell et al, 1991), and by post-translational modifications, such as glycosylation, and possibly, phosphorylation (Rutishauser etal., 1988;Kunemundetal., 1988; Acheson etal., 1991). However, both


53

homophilic and heterophilic NC AM-mediated binding events are characterized by low affinities (Moran and Bock, 1988; Probstmeier et al., 1989, 1992; Nybroe et al., 1989) and these data indicate that adhesionp^r ê may not be the most important biological function of NCAM. Signal Transducing Properties of NCAM and Regulation of Neurite Growth An increasing body of evidence suggests that many CAMs, including NCAM, are able to act as signal transducing receptors. In a pioneering study, Schuch et al. (1989) showed that addition of anti-NCAM antibodies, presumed to mimic natural NCAM ligand(s), can influence second messenger systems in PC-12 cells. Particularly, an enhanced calcium influx, reduced levels of D-myo-inositol-l,4-biphosphate and D-myo-1,4,5-triphosphate, and decreased intracellular pH were detected upon triggering of NCAM with antibodies (Schuh et al, 1989). A similar approach revealed decreased K^ currents in gHal precursor cells (Sonthheimer et al., 1991). In both cases, involvement of pertussis toxin-sensitive G proteins were suggested. However, in a subsequent study, it appeared that the intracellular effects elicited by addition of antibodies and/or purified NCAM depended on cell type and, in some cases, on the nature of the substrate. The complexity of the picture was increased by the failure of cultured cerebellar neurons to respond to anti-NCAM antibodies, whereas the same cells were effectively triggered by purified NCAM (Von Bohlen und Halbach et al., 1992). In these studies, no morphological changes were detected in response to antibodies or solubilized NCAM, although the changes in signaling systems were pronounced. However, in a model system utilizing monolayers of parental or NCAM-transfected 3T3 cells as substrate for neuronal cells, a crucial role of NCAM in neurite outgrowth has clearly been demonstrated. The morphoregulatory activity of NCAM could be abolished by blockers of L- and N-type calcium channels, pertussis toxin, and by a protein kinase inhibitor of broad specificity—^K-252a (Doherty et al., 1991). Conversely, opening of voltage-gated calcium channels by potassium depolarization or by a specific agonist was sufficient to promote the differentiation of PC-12 cells cultured on a monolayer of NCAM-deficient 3T3 fibroblasts and, thus to mimic the morphoregulatory effects of NCAM (Saffel et al, 1992). Therefore, an increase in intracellular Ca^"^ concentration may be considered a pivotal step in NC AM-dependent neurite outgrowth in this model system. Rearrangement of cytoskeletal elements, including microtubules, is supposed to take place in growing processes (Mitchison and Kirschner, 1988). Hence, the NCAM-sensitive decrease in tyrosine phosphorylation of tubulin, detected in isolated nerve growth cone membranes, may be of physiological significance (Atashi et al, 1992). Other In Vitro Effects of NCAM NC AM-dependent recognition and adhesion seem to be responsible for up-regulation of choline acetyltransferase in sympathetic neurons (Rutishauser et al., 1988),

54


and for inhibition of growth of fibroblast cells in vitro (Aoki et al., 1991). The expression of transmembrane NCAM-B, but not of GPI-anchored NCAM-C, has been shown to down-regulate secretion of matrix metalloproteinases (MMPs) in glioma cell lines (Edvardsen et al, 1993). Both the antiproliferative activity and regulation of MMP secretion may be of functional importance in embryonal development as well as in tumor growth and invasion. Recently, ecto-(Ca^^-Mg^'*')-ATPase activity tightly associated with solubilized NCAM has been demonstrated (Dzhandzhugazyan and Bock, 1993). A similar activity has been proposed to be an intrinsic property of another member of the immunoglobulin superfamily—Cell-CAM (Lin and Guidotti, 1989; Aurivillius et al., 1990). However, no experimental data are yet available explaining the role of ecto-ATPase activity in CAM-triggered processes. In Vivo Roles of NCAM

A bulk of data elucidating in vivo functions of NCAM have been obtained by direct injection of antibodies into living animals. This approach has revealed the involvement of NCAM in outgrowth, bundling, branching, and guidance of axons during development and regeneration (Silver and Rutishauser, 1984; Thanos et al., 1984; Tosney et al., 1986; Rieger et al., 1988; Landmesser et al., 1990), innervation of tectum (Fraser et al, 1988) and of muscle (Landmesser et al., 1988), and migration of neural crest cells (Bronner-Fraser et al., 1992). On the other hand, an altered expression of NCAM was found to be without effect on the formation of the neural tube inXenopus laevis embryo (Kintner, 1988). Recently, a homozygous mouse unable to express NCAM-A has been created and the consequences of such genetic deletion were analyzed. The deficiency led to severe malformations in the olfactory bulb, presumably due to defects in cell migration. Other regions (retina, cerebellum, hippocampus) were also affected, however, to a lesser extent (Tomasiewicz et al., 1993). NCAM-A is concentrated at post-synaptic sites (Persohn et al., 1989), and a role for NCAM in memory consolidation (via stabilization of newly established synapses) has been suggested (Scholey et al, 1993). Indeed, a complete inactivation of the NCAM gene in transgenic mice led to deficits in spatial learning and explorational behavior, whereas motor activity appeared to be unaffected (Cremer et al, 1994). E. Structural Determinants of NCAM Function

Recent advances in molecular and cell biological techniques have boosted an extensive scrutiny of the structural units essential for the execution of NCAM function(s). We here briefly review the present status of the problem, and attempt to draw a functional map of NCAM based on the modular arrangement of the molecule.


55

The First Ig-like Domain

The functions of this structural unit have recently been elucidated utilizing the corresponding recombinant protein synthesized in a bacterial expression system (Frei et al., 1992). When used as a substratum, the first Ig-like domain appeared able to promote adhesion of neuronal cell bodies, modify cellular migration pattern from cerebellar microexplants, and to increase the spreading of cell bodies. By addition of the recombinant domain to intact cells, it was shown that regulation of intracellular Ca^^, pH, and inositol phosphates turnover was another pivotal function of this domain. Also, the first Ig-like domain appeared to be the only NCAM fragment capable of decreasing neurite fasciculation, but no significant role in neurite outgrowth has been assigned to this domain (Frei et al., 1992). The Second Ig-like Domain

The functional relevance of this domain is primarily determined by two neighboring stretches (amino acids 133-137 and 142-146) containing clusters of basic amino acids—SL motif typical of heparin-binding sites (Cardin and Weintraub, 1989). Deletions within this sequence abolish the binding of heparin and heparan sulfates, and significantly reduce the adhesive potential of NCAM (Cole and Akeson, 1989; Reyes et al, 1990). On the other hand, occupation of this site by heparin results in inhibition of NCAM-collagen interactions (Probmeister et al., 1989, 1992). This hints at a possible location of the collagen-binding sequence(s) within the second Ig-like domain. When used as a substratum, a recombinant protein corresponding to the second Ig domain was shown to promote adhesion of neuronal cell bodies (however, less pronounced than in the case of the first Ig domain). Addition of the second Ig-like domain to intact neurons had a marked effect on intracellular pH values and Ca^"^ concentrations, but in contrast to the first Ig-like domain, not on the metabolism of inositol phosphates (Frei et al., 1992). The Third Ig-like Domain

The data on the roles of the third Ig-like domain are rather controversial. Studies employing blocking antibodies indicate that this domain is involved in the homophilic binding mechanism (Frelinger and Rutishauser, 1986; Cunningham et al., 1987). Recently, a stretch often amino acid residues (KYSFNYDGSE) located in the third Ig-like domain of chicken NCAM has been proven essential for homophilic binding. Site-directed mutagenesis employed to substitute individual amino acid residues within this sequence revealed the importance of electrostatic and hydrophobic interactions, as well as of the predicted p-strand configuration, in homophilic NCAM binding (Rao et al, 1992,1993). However, when a recombinant protein corresponding to the third Ig-like domain was used as a substratum, it was found to be unable to promote attachment of neurons (Frei et al, 1992). The striking discrepancy between the studies may primarily be due to lack of glycosylation in

56


recombinant protein fragments. Indeed, the third Ig-like domain harbors a putative site for N-Hnked glycosylation, which may carry the HNK-1 epitope expressed on several CAMS, including NCAM (KruseetaL, 1984). The HNK-1 epitope has been presumed to participate in cell adhesion (Keilhauer et al, 1985; Ktinemund et al., 1988; Cole and Schachner, 1987). The Fourth Ig-like Domain

A remarkable feature of the fourth Ig-like domain is a developmentally regulated insertion of a stretch often amino acids encoded by exon VASE (Small et al, 1988; Small and Akeson, 1990). The postulated consequence of this inclusion is a structural shift in the Ig-like domain from a C2- to a V-type configuration. Since the product of exon VASE is located in close proximity to a putative glycosylation site (another candidate for the HNK-1 epitope), changes in the orientation of carbohydrate chains are also possible (Walsh and Doherty, 1991b). However, the role of the insert may not merely be limited to the above-mentioned structural changes. The highly conserved and unique product of exon VASE is externally oriented and, may therefore, be a potential candidate for specific regulatory interaction(s) with yet unknown counter-receptor(s). Involvement of this insert in the regulation of cell-cell binding has been surmized (Santoni et al., 1989). An interesting effect of the VASE product is a dramatic down-regulation of the neurite growth-promoting activity of NCAM (Doherty et al., 1992b). The axonal elongation is impaired when VASE-containing NCAM is expressed on either the growth substratum or the growing axon (Liu et al., 1993). The molecular mechanism by which the product of the VASE exon diminishes neurite outgrowth is so far unclear. One possibility is that binding of a ligand to the fourth Ig-like domain is responsible for the increased calcium influx which is essential for neurite outgrowth (Doherty et al., 1991), and this process may therefore be inhibited by the modification of the domain by the product of exon VASE. However, no detectable influence on calcium homeostasis in neurons was observed after addition of a recombinant fourth Ig-like domain (without VASE) and the only considerable morphoregulatory activity of this fragment was promotion of neurite fasciculation (Frei et al., 1992). Recently, a sequence with a certain resemblance to a carbohydrate recognition motif of lectins, including plant lectins, was found to contribute to the neurite growth-regulating potential of the fourth Ig-like domain. This distinct subdomain apparently binds to oligomannosidic carbohydrates expressed by other neural recognition molecules, including LI. This cz^-association between NCAM and LI seems to modulate the morphoregulatory properties of both partners (Horstkorte et al., 1993). The Fifth Ig-like Domain and Polysialic Acid Chains

Three potential sites for N-linked glycosylation are the hallmarks of this domain (Santoni et al., 1988). Each of these is competent to carry a,2-8-linked linear


57

N-acetylneuraminic acid homopolymers, designated polysialic acid, PSA (Finne et al., 1983; Regan, 1991;Rougon, 1993). This type of post-translational modification is very unusual: Besides NC AM, the sodium channel a-subunit is the only molecule shown to be polysialylated in mammals (Troy, 1990; Zuber et al., 1992). Removal of PSA has two functional consequences: it increases in the efficacy of the homophilic binding (Hoffman and Edelman, 1983; Rutishauser et al, 1985; Moran and Bock, 1988; Acheson et al., 1991) and it dramatically down-regulates the neurite outgrowth-promoting activity of NCAM (Doherty et al., 1990). Hydrated PSA chains occupy a substantial volume and this is presumed to restrict the interaction of other recognition molecules located on apposed membranes (Rutishauser et al., 1988). Indeed, the removal of PS A has been shown to decrease the distance between two interacting cells (Yang et al., 1992). The morphoregulatory potential of polysialic acid may also be executed via a recently discovered signaling pathway: PSA appeared to mediate specific binding and internalization of a peptide corresponding to the Antennapedia homeoprotein, which induces neuronal differentiation in culture (Joliot et al., 1991). Since the homologous homeoprotein (HOX2.4) is capable of regulating the promoter activity of the NCAM gene, this pathway may operate as a feedback mechanism for NCAM expression. Obviously, PSA is a key modulator of NCAM function and, not surprisingly, its level is under a fine control throughout development, especially during the processes of neuronal plasticity (Nybroe et al., 1988; Doyle and Regan, 1993). In contrast, the polypeptide backbone of Ig-like domain V, produced in a bacterial expression system (i.e., without addition of carbohydrate moieties), was practically incapable to display any functional activity (Frei et al., 1992). Immunogenic Subregion Located Between Ig Domain V and the First Fnlll Domain

A highly hydrophilic segment (mainly encoded by exon 11), located between the fifth Ig loop and the first Fn Ill-like domain of NCAM, harbors epitope(s) recognized by a large group of monoclonal antibodies: Leu 19, UJ 13 A, Eric 1, Lu 243, Lu 246, and 5.1 HI 1 (Patel et al, 1989). An attempt to elucidate the functional significance of this domain has recently been made utilizing a new monoclonal antibody HR 1. This antibody recognizes a sequence of 15 amino acids located just C-terminal to the fifth Ig-like domain. In chicken retinal organ culture, application of the antibody resulted in a severe disturbance of axonal fasciculation and path-finding (Pollerberg and Beck-Sickinger, 1993). Fnlll-like

Domains

The designation of these structural units is probably only justified for chicken and rodent NCAM, whereas the corresponding region in human NCAM displays

58


a very remote resemblance with fibronectin type III repeats (Walsh and Doherty, 1992). The combined two Fnlll domains produced in a bacterial expression system exhibited an ability (comparable to that of intact brain NCAM) for induction of neurite outgrowth, spreading of neuronal cell bodies, and to a lesser extent, adhesion to cell bodies (Frei et al., 1992). However, none of the second messenger systems tested (Ca^"^, inositol phosphates, cAMP) were affected and thus, the mechanism of morphoregulatory activity of the Fnlll-like domains remained unexplained. Moreover, a recent study utilizing cells transfected with NCAM constructs of different lengths and compositions, showed that the Ig-like domains were required for intercellular adhesion, whereas the Fnlll-like repeats were dispensable (Zhou et al, 1993). The translation products of four optionally expressed exons (a, b, c, and the AAG triplet) inserted between the two Fn Ill-like domains may contribute to significant changes in NCAM orientation relative to the membrane. Apart from a hinge-like flexible structure encoded by exon a (discussed below), the translation products of exons b and c harbor potential sites for O-linked glycosylation. These carbohydrate chains are presumed to act as spacers that increase the distance between the membrane and the functional sites of NCAM (Jentoft, 1990; Walsh and Doherty, 1991b). So far, O-linked glycosylation has been demonstrated in muscle cells, but not in cultured neurons (Lyles et al, 1984;. Nybroe et al., 1988) or brain (Walsh et al., 1989), although the b and c exons certainly are expressed in neural tissue (Barthelsetal., 1992). Hinge Structure

Electron microscopy utilizing rotary shadowing has visualized NCAM as a bent structure containing a flexible hinge-like segment (Hall and Rutishauser, 1987; Becker et al., 1989). The sequence corresponding to the hinge region is situated between the Ig-like domain IV and the site of attachment to membrane, but the precise location of this structure as well as its function is still controversial. Within this part of NCAM, two areas may participate in the formation of a hinge (Hall and Rutishauser, 1987; Becker et al., 1989): a region of general protease sensitivity located N-terminal to the Ig-like domain V (Cunningham et al., 1987), and a string of prolines encoded by exon a, located between the two Fn Ill-like domains (Thompson et al., 1989). So far, the functional significance of this hinge-like structure is unclear. Attachment to the Plasma Membrane

As discussed above, two modes of attachment to the plasma membrane (anchorage via GPI versus transmembrane insertion) result from alternative splicing of NCAM pre-mRNA: expression of exon 15, or a skipping of this exon with concomitant expression of exons 16-19.


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Exon 16 encodes a putative membrane-spanning sequence enriched with hydrophobic residues, which possibly possesses an a-helical conformation—â typical feature of transmembrane domains. Exon 15 encodes a sequence which is quite conserved between various species (68% similarity between human, mouse, and chicken) and which determines a covalent linkage to GPI (Walsh and Doherty, 1991a). The GPI anchor serves as a sorting signal for trafficking and sequestration of NCAM-C to certain areas in polarized cells (Powell et al, 1991). GPI-linked NCAM does not obviously differ from the transmembrane forms with regard to efficacy of homophilic binding, but may provide a distinct mode of signal transduction. Although lacking direct access to the cytoplasmic compartment, GPI-linked proteins can be complexed (via unknown adapter molecules) with signal transducing molecules such as heterotrimeric G proteins, and serine- or tyrosine-kinases (Stefanova et al, 1991; Sargiacomo et al., 1993). In some cells, the release of GPI-anchored polypeptides by means of PIPL-C triggers an unidentified signaling pathway leading to an early expression ofc-fos (Rahman et al., 1992). However, it is not established whether these features of GPI-linked proteins are applicable to NCAM-C. Naturally occuring enzymatic cleavage of the GPI anchor with subsequent release of NCAM-C may trigger an array of regulatory events such as: 1. Production of diacylglycerol and consequent activation of protein kinase C (for discussion, see Sontheimer et al., 1991); 2. Control of membrane-associated NCAM amount (including the activation of transcriptional and translational mechanisms, Theveniau et al., 1991); 3. Remote control of recognition events by binding of released NCAM-C to membrane-associated complementary receptors (NCAM, LI, heparan sulfates, etc.); 4. Creation of a substrate path for migrating cells by immobilization of soluble NCAM-C to the extracellular matrix, an so on. Another suggested feature of GPI-linked NCAM-C is an increased lateral mobility within the plane of membrane, but the functional consequences arising from this putative property have not been experimentally tested. In general, NCAM-C seems to constitute a dynamic and readily adjustable pool. Cytoplasmic Part of NCAM The role of the cytoplasmic "tail" of NCAM is unclear. The intracellular part of NC AM-A is encoded by exons 17,18, and 19, whereas the NCAM-B isoform lacks the product of the optionally expressed exon 18. The insertion of 267 amino acids encoded by this exon has at least three major biochemical consequences: 1. Introduction of additional phosphorylation sites (Sorkin et al., 1984; Linnemannetal., 1985);

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2. Introduction of sequences responsible for association with the cytoskeletal actin-binding protein spectrin (Pollerberg et al., 1987); and 3. Introduction of a specific target site for the calcium-dependent protease calpain (Covault et al, 1991). At the functional level, NCAM-A apparently differs from NC AM-B with regard to a neurite outgrowth-promoting capacity: When expressed on a monolayer of 3T3 cells serving as a substrate for developing neurons, NCAM-A, unlike NCAM-B, appeared to act as a very poor activator of neurite elongation (Doherty et al., 1992a). This may be due to a limited lateral mobility of NCAM-A, provided by the association with cytoskeletal elements. Anchoring to the cytoskeleton has been suggested to stabilize NCAM-A-mediated cell-cell contacts, such as synapses (Persohn et al., 1989). Conversely, Ca^'^-dependent proteolytic cleavage within the NCAM-A-specific domain has been predicted to release the molecule from the restrictive control performed by cytoskeletal elements. Since the synaptic compartment is enriched with calpain, calcium channels, and presumably, with NCAM-A, this mechanism may play a role in synaptic plasticity (Sheppard et al. 1991). However, a recent study does not support the idea that NCAM-A plays an exclusive role at stable cell-cell contact sites: in neuroblastoma cells, these zones were found to be equally enriched both with NCAM-A and NCAM-B (Woo et al., 1993). Phosphorylation of NCAM is known to be developmentally regulated (Gegelashvili et al., 1993a; Linnemann et al., 1985), although the significance of this process remains unclear. The incorporation of phosphate into the NCAM-Aspecific domain, by analogy with another CAM, P-cadherin (Matsuyoshi et al., 1992), may regulate the association with cytoskeletal elements (or other intracellular components). Another possible role of phosphorylation is modulation of the signal-transducing abilities of NCAM. However, these hypotheses are so far not supported by experimental data. The creation of deletion mutants lacking phosphorylation sites, or the "knocking out" of appropriate protein kinase(s) may elucidate the role of NCAM phosphorylation. In summary, the available data suggest that the different parts of the molecule apparently play important roles in the performance of specific activities in various in vitro systems. However, thefiiU-rangebiological responses may be dependent on a concerted action of all NCAM domains and isoforms composing a functional entity. Eukaryotic cellular systems utilizing deletion mutations in the NCAM gene and the corresponding transgenic animals will shed more light on the NCAM structure/function relationships.

ACKNOWLEDGMENTS This work was supported by grants from the Novo Nordisk Foundation, the Danish Biotechnology Program (1991-1995), the DANVIS program, the Carl and Ellen Hertz


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Foundation, and the Carlsberg Foundation. The authors are grateful to Mrs. Tea Gobronidze and Mrs. Heile Hehn for secretarial assistance.

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THE T CELL RECEPTOR

Carol Morgan and John D. Fraser

I. Introduction 78 11. Structure of the T cell Receptor 79 A. The a/p T cell Antigen Receptor 80 B. The CD3 Complex and i; Dimer 81 C. The Y/6 T cell Receptor 82 IL Specificity for Antigen and MHC 82 A. Diversity oftheTcR Allows Specificity for Antigen 82 B. Antigen Presentation by MHC 83 C. Recognition of Peptide:MHC by TcR . 85 D. TcR:MHC Interactions 87 E. CD4 and CD8 are Involved in T cell Recognition 87 IV. Structural Basis ofT cell Recognition 88 A. TcR Recognition of Superantigens Differs from Recognition of Peptide Antigens 88 B. TcellRecognitionMay Involve Dimerization of the MHCiTcR Complex .89 V. Cellular Signaling through the TcR 90 VL The Role ofthe TcR in T cell Development 93 VII. Conclusion 95 Acknowledgments 95 References 96


78

CAROL MORGAN and JOHN D. FRASER

I. INTRODUCTION The T cell receptor (TcR) is probably one of the most well studied and most complex of all cell surface receptors. This multicomponent receptor consists of ligand binding structures which have extraordinary amino acid diversity, as well as invariant chains which are linked to cellular protein tyrosine kinases for signal transduction. In the immune system, the TcR is responsible for the recognition of foreign antigens as "non-self and this ultimately leads to the destruction of cells infected with foreign microorganisms. In T cell responses, protein antigens are proteolytically processed within antigen presenting cells (APC) in order to be able to bind to molecules of the major histocompatibility complex (MHC). The resulting peptide:MHC combinations are then displayed on the surface of the APC. For example, when a cell is infected with a virus, the majority of peptides bound to that cell's MHC molecules will be of viral origin, so the immune system very quickly will recognize that infected cell as "foreign," and it will be destined for destruction. The ability of the T cells to recognize peptides in association with particular MHC molecules is called MHC restriction. The peptideiMHC complex is recognized by the TcR in much the same way an antibody recognizes its antigen. The TcR consists of six interactive transmembrane chains associated with another surface molecule, one of the two "coreceptors," CD4 or CDS. The function of CD4 or CDS molecules is to bind to conserved regions of MHC and facilitate the TcR:peptide:MHC interaction. T cells expressing CD4 recognize exogenous peptides bound to class II MHC and are usually of the T helper phenotype. CDS T cells recognize endogenous peptides in context with class I MHC, and are most often cytotoxic. Thus, the process of antigen recognition by T cells actually involves eight separate polypeptide chains on the T cell. The six chains of the TcR can be functionally divided into two groups; the antigen specific a/p heterodimer, and the signal transducing proteins, y, 8, £, (^, and r\. The a/p heterodimer is an immunoglobulin-like molecule, responsible for antigen plus MHC recognition. After triggering of the T cell through the a/p heterodimer and other costimulatory molecules, signal transduction occurs through the CD3 chains of the TcR. These transmembrane proteins all contain sequence motifs for tyrosine or serine phosphorylation and have been shown to associate with the intracellular protein tyrosine kinases, p56'^^ p59^", ZAP-70, and the oncogene product. She. In responding to foreign antigens, T cell activation through the TcR causes cell proliferation and differentiation, and secretion of cytokines which are involved in the regulation of other immune cells, such as B lymphoc5ês. In addition to its role in immune surveillance, the TcR plays a major role in T cell development. Immature T cells with self-reactive TcRs are selectively deleted in the thymus. Cells bearing functional TcRs with lower affinity for self MHC are positively selected to mature and seed the peripheral lymphoid organs. While the functions of the TcR have been studied for decades, the structure of the TcR is still an emerging field. Despite a lack of physical data on the TcR and its

The T Cell Receptor

79

interaction with peptide:MHC, structural models have been proposed for the TcR. Although no crystallographic data are yet available for the TcR, its sequence similarity to immunoglobulins allows the use of molecular modeling to predict the three dimensional structure of the TcR. Biochemical characterization of the TcR components and of partial TcR complexes provides evidence for association between the a/p heterodimer and the CD3 chains. This chapter summarizes the recent advances in our understanding of the structure and function of the T cell receptor. We look forward to further elucidation of the TcR structure to provide answers to many questions concerning this complex receptor.

II. STRUCTURE OF THE T CELL RECEPTOR The T cell receptor has been studied intensely since the early 1980s (van Agthoven etal., 1981;Borstetal., 1982,1983a, 1983b; Meueretal., 1983). The cDNA of the a- and p-chains of the T cell antigen receptor were cloned in 1984, and the deduced amino acid sequences of the a- and p-chains described (Chien et al., 1984; Hannum et al., 1984; Hedrick et al., 1984; Patten et al., 1984; Saito et al., 1984b; Yanagi et

CDS

Figure 1. Individual chains of the T cell receptor. This schematic diagram of the multimeric T cell receptor complex shows the immunoglobulin-like features of the Individual chains.


80

Table 7. Components of the Human T cell Receptor Chain

MW(kDa)'

a

36-46 (32)

P

40-44 (34)

CD3Y

25-28(16)

CD36

20(16)

CD3s

20 (20)

C

16(16)

n

22 (22)

Y

45-60 (29)

5

40-60(31)

Glycosylation

Function

Complex, N- Antigen, MHC recognition 1 inked High mannose, Antigen, MHC recognition complex, N1 inked Signal N-l inked transduction Signal N-linked transduction Signal None transduction Signal None transduction Signal None transduction N-linked Antigen recognition N-linked Antigen recognition

Phosphorylation PTK associated No

No

No

No

Yes, serine

No

Yes, serine

No

Yes, serine Yes, tyrosine

p59^y", p56'^^ ZAP-70 p59^y", ZAP-70

Yes, tyrosine

No

No

No

No

No

Note: ^Values in parentiieses indicate MW of polypeptide backbone.

al, 1984; Loh et al., 1987). In the last decade, our understanding of this complex of molecules has grown enormously, and several excellent reviews have been published (AlHson and Lanier, 1987; Clevers et al, 1988; Jorgensen et al, 1992b). The TcR and its accessory molecules belong to the immunoglobulin gene superfamily. These proteins are characterized by globular, disulfide-linked extracellular domains with conserved (Ig-like) folds of P sheets (Novotny et al., 1986). The multichain TcR is composed of an a/p heterodimer, associated on the cell surface with s, 8, and Y transmembrane proteins, called CD3, and a C^/r\ heterodimer or a ^2 homodimer (see Figure 1 and Table 1). A. The a/p T cell Antigen Receptor The a/p components of the TcR are unique for each T cell clone, whereas the CD3 subunits are identical in all T cells. The highly polymorphic a/p chains are responsible for antigen and MHC recognition. The 36-46 kDa acidic a chain is disulfide-linked to the more basic 40-44 kDa P chain. Both have immunoglobulin (Ig)-like primary amino acid structures, with variable (V) and constant (C) regions (Hannum et al., 1984; Hedrick et al., 1984; Saito et al., 1984a; Yanagi et al, 1984). The V regions are 102-119 amino acids (aa) in length and are homologous to Ig V regions (Patten et al., 1984). As for antibodies, the diversity in TcR is limited to distinct hypervariable loop regions. These regions form the contact points for

The T Cell Receptor

81

interaction with antigen and MHC and are termed complementarity determining regions 1,2, and 3 (CDRl, CDR2, CDR3). TcR Vp regions have additional hypervariable regions (Patten et al, 1984; Novotny et al., 1986), one of which, HV4 (Chothia et al., 1988; Claverie et al., 1989; lores et al., 1990), will be discussed later. The constant regions are 138-179 aa in length and contain four functional regions. There is an Ig-like extracellular domain which is disulfide-bonded. An additional cysteine just outside the transmembrane region forms a disulfide bond between the a- and p-chains. The 20-residue transmembrane region is distinctly hydrophobic with a conserved lysine near the center of the region. This positive charge may be important for interaction with the CD3 chains, all of which have negatively charged residues in their transmembrane regions. The fourth C region consists of a very short (4-12 aa) cytoplasmic tail. B. The CDS Complex and C, Dinner The CD3 chains are homologous to each other both in sequence and structure but are antigenically distinct (van Agthoven et al., 1981; Borst et al., 1983a, 1983b, 1984). The y- and 5-chains are glycoproteins, whereas the s chain is not glycosylated (see Table 1). These chains have much longer cytoplasmic regions than the a/p chains, and contain sequence motifs characteristic of regions that are responsible for signal transduction. The CD3 8 cytoplasmic region is different from its CD3 Y and 8 counterparts; the cytoplasmic portions of the latter two chains are 63% homologous. The CD3 probably exists as two heterodimers, 6/E andy/s (Blumberg et al., 1990), and is associated with a (^2 or (^/r| dimer. The C, chain is 16 kDa, nonglycoslyated, and has no sequence homology to the other subunits of the TcR. The major part of this protein is intracellular with only a short (6-9 aa) extracellular region. In the majority of TcR, the C, chain exists as a 32 kDa homodimer, disulfide-linked in the extracellular region. About 5—20% ofC^ chains associate with a 22 kDa r| chain to form a 38 kDa disulfide-linked heterodimer. Efficient expression of the TcR on the cell surface requires all of the components; however, the individual chains can interact in the endoplasmic reticulum or Golgi apparatus without requiring the presence of all the subunits. Partially assembled complexes have been identified (Alarcon et al., 1987a; Berkhout et al., 1988) and examination of these indicates that CD3 chains can pair in the absence of the aand P-chains. Similarly, the ^ chain can dimerize when transfected in fibroblasts in the absence of all other chains (Berkhout et al, 1988), and the a- and P-chains can pair without CD3 (Lippincott-Schwartz et al., 1988). Partial detergent solubilization of TcR reveals complexes of ySs, ye, 5s, and aP5 (Bonafacino et al., 1988). Also, in a- or P-deficient cell lines, the CD3 chains were found to be complexed with either P- or a-chains, respectively, in the absence of the other component (Alarcon et al, 1987a). From the pairing of the chains observed in the partial complexes, the following assumptions can be made about the association of the subunits in the intact TcR. The p chain probably interacts directly with the y chain

82


of CD3. Similarly, the a chain most likely interacts with CD38. CD38 interacts with both the y- and 8-chains, and C, associates with the a/p dimer. The TcR components are assembled in the endoplasmic reticulum (ER). Although there have been many studies on the details of the assembly process, the exact order of assembly is not known. Within the ER, the newly synthesized a-, P-, and CD3-chains are transiently associated with a 26 kDa nonglycosylated protein called CD3co, or T cell receptor associated protein (TRAP; Alarcon et al, 1987a; Pettey et al., 1987; Bonafacino et al., 1988). This chain binds noncovalently to the nascent subunits within the ER, but is not present in the fully assembled complex. It may act as a chaperone to facilitate the proper association between the subunits. The C^ chain dimerizes rapidly after synthesis. Association of the C^ chain with the a/p:CD3 complex is required for efficient surface expression of the oligomeric complex (McCleod et al, 1986). In the absence of any one of the subunits, the partial complex can assemble in the ER, but will not be expressed on the surface, and is either degraded or disassembled in the ER or lysosomes (Weiss and Stobo, 1984; McCleod et al., 1986; Alarcon et al., 1987a). C. The y/6 T ceil Receptor

About 1-10% of peripheral T cells, as well as dendritic cells, possess a TcR in which the CD3 chains are associated with the alternative antigen receptor chains, Y and 6. Like the a/p TcR, the y/5 TcR is comprised of V- and C-immunoglobulinlike regions (Chien et al., 1984; Saito et al., 1984b). Unlike a/p TcR, the y/5 TcR is expressed in several forms. The Cy2 gene encodes a 55 kDa glycoprotein which has a 29 kDa protein backbone (Littman et al., 1987). This C region lacks the cysteine which normally forms the disulfide bond between the two chains of the heterodimer. In contrast, the Cyl gene encodes a 35 kDa glycoprotein which does form disulfide bonds with C8. In addition, some clones express functional TcR y/y homodimers (Alarcon et al., 1987b; Borst et al, 1987; Moingeon et al., 1987). The 5 chain is a 45 kDa glycoprotein (Chien et al., 1987; Pardoll et al., 1987). Unlike a/p TcRs which recognize peptideiMHC, y/8 TcRs recognize antigen, but do not restrict for MHC (O'Brien, 1989; Holoshitz et al., 1989), and the true ligand for y/8 TcR has yet to be established unequivocally. Both a/p T cells and y/8 T cells have similar effector fiinctions, that is, cytolysis and cytokine production. The physiologic role of the y/8 TcR is very much a field of active investigation (Raulet, 1989; Born et al, 1990; Haas et al., 1990; Itohara et al., 1993).

n. SPECIFICITY FOR ANTIGEN AND MHC A.

Diversity of the TcR Allows Specificity for Antigen

Antigen recognition and MHC restriction by cell surface TcR is achieved through the a/p (or y/8) heterodimer (Dembic et al., 1986b). An extraordinary diversity of

The T Cell Receptor

83

antigen recognition is permitted by the rearrangement of different coding segments in the genome (variable (V), diversity (D), and joining (J) genes). The genomic arrangement of TcR genes is like that of immunoglobulins, with hundreds of interchangeable V-, D-, and J-genes (Toyonaga et al., 1985; Yoshikai et al., 1985; Davis and Bjorkman, 1988; Wilson et al., 1988). Both the sequence and genetic organization of these genes bear considerable homology to immunoglobulin genes. The human p chain locus contains about 50 V-, 2 D-, and 12 J-genes. There are 25 V^ and up to 100 J^^ genes. The y/8 genomic region contains 10 Vg, 2 Dg_, and 2 Jg-segments, 7 V^-, and 2 J^-segments. There are 2 Cp genes, 1 C^^, 1 Cg-, and 4 Cy-genes, of which at least one is nonfunctional (Cy3). Figure 2 shows the genomic organization of the TcR genes of mice. The human TcR locus is similarly organized. Like Ig heavy-chain genes, functional TcR P genes are made up from the genomic splicing of one each of V^-, Do-, and Jo-gene segments, and additional N-regional diversity is created at the junctions. Similarly, the TcR a chains consist of rearranged V^^- and J^^-genes. The rearrangements are facilitated by an as yet unidentified VDJ recombinase common to both Ig gene and TcR gene rearrangement. Two recombinase-associated genes, RAG-1 and RAG-2, are known to be required for this process (Schatz and Baltimore, 1988; Schatz et al., 1989; Oettinger et al., 1990). Genetic knockout experiments in mice show that in both RAG-1 deficient and RAG-2 deficient mice there is no rearrangement of TcR or Ig genes, and that these mice have no mature T or B cells (Mombaerts et al, 1992; Shinkai et al, 1992). The enzymes involved in the genetic rearrangement and the specific mechanism of recombination are not yet known. The use of a particular Va or Vp gene segment does not correlate with phenotype (CD4 or CD8), function, specificity, or MHC restriction (Goverman et al, 1985; Hedrick et al., 1985; Garman et al., 1986). TcRs with the same Vp genes can be either MHC class I or class II restricted. Both the a- and p-chains of the TcR are required for MHC specificity, but individual a- or P-chains may bias the MHC specificity (Blackman et al., 1988). Additionally, there are no convincing data that particular V genes correlate with antigen specificity (Goverman et al., 1985; Rupp etal., 1985; Fink etal., 1986). The germline potential for creating a diverse repertoire of y/6 TcRs is considerably lower than that of a/p TcRs. Furthermore, while all of the Vy genes are rearranged in immature thymocytes, mature y/8 T cells express only a limited number of Vy genes, notably Vyl. Thus, the diversity in y/5 TcRs is generated mainly by the junctional regions (PardoU et al., 1987). B. Antigen Presentation by MHC The a- and p-chains of the TcR recognize foreign peptide antigen bound to the major histocompatibility complex (MHC) on the surface of an APC. Given the random generation of peptides by the APC and the role of the MHC in shaping the T cell repertoire, it is not surprising that the MHC features structurally in TcR

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recognition. The MHC class I and class II molecules have similar heterodimeric structures. The extracellular region consists of two a helices arranged over a platform of p sheet (Bjorkman, 1987; Brown et al., 1993). Processed peptides fit into a groove between the a helices, and come into contact with residues on the helices as well as with residues on the floor of the platform. A fiill description of the MHC structure and function will be presented in a separate chapter of this volume. Analysis of the peptides bound by class I and class II MHC molecules and their abilities to stimulate T cells has led to our current understanding of the interactions between TcR, antigen, and MHC (Rothbard and Gefter, 1991; Jorgensen et al., 1992b). The optimum peptide length for MHC class I is 8-11 aa. Class II MHC can bind peptides up to 30 aa in length because the ends of the peptide groove are open and the peptide can extend beyond each end of the groove (Brown et al., 1993). The list of peptides with known MHC and/or TcR contacts is continually being expanded and from the available data, much information can be gathered about TcR:peptide:MHC interactions. By comparing the amino acid sequences of peptides which bind to known MHC molecules, it can be demonstrated that in most cases, only two or three residues on the peptide actually contact the MHC. There is evidence that peptide residues not directly involved in MHC binding may maintain a degree of flexibility, allowing different conformational states of the peptide within the MHC and allowing interaction of peptide residues with the TcR (Quo et al., 1992; Silver et al, 1992). C. Recognition of PeptideiMHC by TcR

An MHC molecule with a peptide in its groove presents a flat surface which the TcR can recognize and bind (Figure 3). Evidence shows that the TcR interacts with only a few residues of the peptide (Schwartz, 1985; Guillet et al., 1986; Lorenz et al., 1989) and the interaction is sensitive to the spatial configuration of the critical residues (reviewed in Jorgensen et al., 1992b). Thus, different MHC molecules could present the same peptide in different configurations resulting in differential TcR recognition. Perhaps when the TcR interacts with the peptide it supports a particular peptide conformation and this contributes to the stability of the TcRiMHC interaction. A number of studies have implicated the CDR3 regions of the TcR a- and P-chains as contact points for the peptide. The CDR3 region is comprised of the V-D and D-J junctions of the rearranged V-, D-, and J-genes and as such, these regions exhibit greater diversity than the CDRl and CDR2 regions which consist of genomic V region sequences. Also, the TcR genes, unlike immunoglobulin genes, do not undergo somatic hypermutation, so the diversity of the CDRs-1 and -2 is limited to what is encoded in the genome. It seems likely that the CDRs-1 and -2 serve to interact with the polymorphic a helices of the MHC, and the much more variable CDR3 provides the antigen specificity. Analysis of the CDR3 regions in

86


Figure 3. Model for TcR:peptide:MHC interaction. The figure shows a side-view model of the TcR:peptide:MHC interaction. The V domains of immunoglobulin FAb were used to model the V domains of a TcR (top of figure). The carbon backbone of VL and VH is shown for TcR-a and -p, respectively. The CDR1-, CDR2-, and CDR3regions and the p chain HV4 loop are labeled 1-4, respectively, and are shown with Van der Waals densities to distinguish them from the remainder of the molecule. For the MHC molecule (bottom of figure), the carbon backbone of the a helical domains of HLA-A2 was used to model the class II structure and is shown with a circle indicating the position of a hypothetical peptide extending perpendicular to the plane of the paper. The surface of the TcR-V region combining site is complementary to that of the MHC:peptlde complex, with CDR1 and CDR2 domains fitting over the MHC a helices. The CDR3s from each TcR-V domain are aligned over the peptide.

The T Cell Receptor

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TcRs induced in the immune response to several antigens indicates these regions to be of major importance for antigenic reactivity (Engel and Hedrick, 1988; Danska et al., 1990; Lai et al., 1990; Wither et al., 1991). A strategy to examine specific TcR:peptide interactions utilized substituted peptides to elicit T cell responses in TcR a- or P-transgenic mice restricted by the mouse MHC class II, I-E^. Examination of the sequences of the TcRs showed compensatory changes in the CDR3 regions to account for the substitutions in the immunogenic peptide (Jorgensen et al, 1992a). In this way, the binding specificity between the TcR and moth cytochrome C peptide (MCC 88-103) was clearly shown to involve contact between aa 99 (position 12 in the peptide) and residues in the a chain CDR3 and between residue 102 (position 15 in the peptide) and the P chain CDR3. D. TcR:MHC Interactions

MHC restriction of T cell recognition implies that in addition to specificity for peptide, the TcR also has specificity for the MHC molecule. There is some debate as to whether or not the TcR physically contacts the MHC molecule during TcR activation (Chain, 1987), but many studies indicate a distinct interaction between the TcR and MHC molecules. Mutations made in either class I or class II molecules at sites predicted by three dimensional models to point out towards the TcR resulted in disruption of T cell recognition (Ajitkumar et al, 1988; Peccoud et al, 1990). Mutations in the TcR molecule provide evidence that the amino-terminus of the TcR a chain interacts with MHC class II (Hong et al., 1992). From the data at hand, a structural model of the TcR:peptide:MHC complex can be devised. The model uses a TCR structure based on the crystal structure of immunoglobulins (Davis and Bjorkman, 1988), with the MHC class II structure (Brown et al., 1993). When the above mentioned peptide:TcR interactions and the proposed sites of MHC:TcR interaction are taken together, the TcR model can be positioned on top of the peptide:MHC such that the TcR a- and P-chain CDRl and CDR2 loops come into contact with the MHC a helices. This leaves the centrally located CDR3 loops in a position to contact the peptide in the center of the MHC groove (Figure 3). E. CD4 and CD8 are Involved in T cell Recognition

A complicating feature of how T cells see antigen involves the T-cell "coreceptors", CD4 and CD8. These membrane glycoproteins influence the TcR:MHC interaction by acting as a receptor on the T cell for conserved regions of the MHC molecules on the antigen presenting cell (Doyle and Strominger, 1987; Travers, 1990). T cells expressing CD4 are mainly of the T helper phenotype and interact with MHC class II antigen presenting cells. CD8 cells are mainly cytotoxic T lymphocytes and interact with class I MHC. CD4 and CD8 are 55Kd and 32Kd, respectively, and have sequence homology to the immunoglobulin and TcR V regions (Littman et al., 1985; Maddon et al, 1985; Sukhatme et al., 1985). While these molecules may not be absolutely necessary for T cell function (Marrack et

88


al., 1983), gene transfer studies showed that T cell stimulation is markedly increased in the presence of these components (Dembic et al., 1986a; Gabert, 1987; Sleckman, 1987). It has been postulated that the binding of CD4 and CD8 to MHC on the APC enhances the "avidity" of T celliAPC interaction (Dialynas et al, 1983; Marrack et al., 1983; Shaw et al, 1985), possibly by stabilizing the TcR:MHC complex. However, recent studies support a different role for CD4; to facilitate the phosphorylation of CD3 by the tyrosine kinases of the signaling pathways in T cell activation (Ravichandran et al., 1993).

IV. STRUCTURAL BASIS OF T CELL RECOGNITION A. TcR Recognition of Superantigens Differs from Recognition of Peptide Antigens Superantigen is a term used to describe a growing family of potent immunostimulatory molecules consisting of bacterial toxins, viral antigens, and other molecules which can stimulate up to 20% of T cells in peripheral blood. The molecules include the staphylococcal entertoxins (SEA, SEB, SEC, SED, SEE), toxic shock syndrome toxin (TSST), antigens of the mouse mammary tumor virus (MMTV), and other endogenous retroviral antigens in the Mis loci. In most cases, these proteins bind directly to class II MHC without processing by an APC, and stimulate characteristic subsets of T cells, depending on the particular TcR Vp which is expressed (Table 2). Unlike recognition of peptide antigen, recognition of most superantigens has been shown to be independent of the TcR P chain CDR3 sequence and the entire TcR a chain. Mutational analysis of all of the components involved, that is, MHC, TcR, and the superantigens (SEA, SEB, TSST) has revealed much information on the sites of interaction between the molecules. The binding of the toxins to MHC occupies sites away from the peptide groove, which are specific for the toxin (Dellabona et al., 1990). For example, while SEA can compete for binding against both SEB and TSST, the binding of SEB and TSST is noncompetitive (Eraser, 1989; Scholl et al., 1989; Pontzer et al, 1991; Purdie et al., 1991). Binding of SEA, but not SEB, to MHC requires Zx^^ (Eraser et al., 1992). The SEAiMHC interaction involves a Zn^"*" bridge between H187, H225, and D227 on the SEA, and H81 on the MHC class II p helix (Herman et al., 1991; Eraser et al., 1992; Irwin et al., 1992; Hudson et al, 1993). TSST binding to MHC utilizes residues M36 and K39 on the class II a chain, as well as residues in the p chain (Karp et al., 1990; Braunstein et al., 1992; Panina-Bordignon et al, 1992). Amodel can be proposed such that SEA, based on the known interaction sites, can be positioned at the end of the MHC molecule across the end of the groove. This is supported by evidence that binding of the toxin to MHC does not interfere with peptides binding in the groove (Karp et al, 1990). Despite differences in MHC binding positions, it appears that all the superantigens bind TcR at a common site on the p chain. A number of studies have

The T Cell Receptor

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Table 2. Characteristics of Superantigens and Conventional Antigens Characteristic Size Presentation by MHC Recognition by TcR Processing required % peripheral T cells stimulated Source

Superantigen

Peptide antigen

25-30 kDa Class II Vp HV4 No Up to 20%

-0.3 kDa Class II or class 1 Vp CDR3 and Va CDR3 Yes

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50% y/8 T cells, we found that low doses of lipopolysaccharide (suboptimal for neutrophil accumulation) increased the accumulation of y/5 T cells in the skin by 10-fold within 4-6 h. In association with the influx of y/8 T cells, there was a dramatic increase in the percentage of venules that express E-selectin. Thus, E-selectin expression in vivo correlates with extravasation of all the maj or leukocyte types that bind this molecule in in vitro assays.

The Selectin Family

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Regulation ofE-selectin Expression Optimal E-selectin expression on the surface of endothelial cells requires 2-4 h following cytokine stimulation. Expression requires transcriptional control of E-selectin gene expression and de novo mRNA generation (Bevilacqua et al., 1987; Bevilacqua et al., 1989). Analysis of the regulatory sequences in the E-selectin gene suggests the NK-KB and AP-1 transcription factors are involved in the inducible expression of E-selectin. NF-KB and AP-1 binding sites are not unique to the E-selectin gene and cannot account for the exquisite tissue specificity of E-selectin expression (Montgomery et al., 1991; Whelen et al., 1991). E-selectin is only expressed by endothelial cells, and the characterization of the regulatory events that control this restriction is an intense area of study. Internalization of the antigen appears to account for the down-regulation of surface expression, but recent reports suggest that proteolytic cleavage and shedding of the molecule may also take place (Newman et al., 1993). This latter observation is similar to the regulation of L-selectin expression on leukocytes (see above). Effect of Anti-E-selectin Treatment on Inflammation As described for L-selectin, evaluation of the effect of E-selectin inhibitors in vivo is of considerable interest, though these studies will not be reviewed in depth here. Most of the previous limitations to the analysis of E-selectin were related to lack of reagents in appropriate animal models. This was addressed by the cloning studies of Saunders et al. (1992) and Weller et al. (1992) which resulted in the identification of cDNAs encoding both P-selectin and E-selectin in the mouse. These studies were followed by the development of function-blocking antibodies. Also gene knockout mice have been developed, but these studies were done after the writing of this chapter and their summary cannot be appropriately addressed at the proof stage. C. Platelet/Endothelial Cell Selectin (P-selectin) P-selectin is a 140 kD glycoprotein that was originally isolated from thrombin or histamine-activated platelets. The molecule is also found in the Weibel—Palade bodies of endothelial cells. As described with platelets, activation of endothelial cells with thrombin causes a rapid (within minutes) translocation of the intracellular antigen to the cell surface (Stenberg et al., 1985; Berman et al., 1986; Bonfati et al., 1989; McEver et al., 1989). Clues to the function of P-selectin came after the cloning of its cDNA (see below) and the determination of its relationship to L- and E-selectin. Following the cloning studies, anti-P-selectin antibodies were shown to block activated platelet binding to myeloid cells, such as neutrophils and monocytes (Larsen et al., 1989; Larsen et al., 1990). Neutrophil adhesion to thrombin-activated HUVECs is blocked by treatment of the endothelial cells with anti-P-selectin mAbs (Geng et al., 1990). Lorant et al. (1993) showed that P-selectin mediates a "tether-

196

MARKA.JUTILA

ing" interaction of neutrophils with endothehal cells, which may allow for activation of the leukocyte by platelet activating factors expressed on the surface of the endothelium. Finally, neutrophils in P-selectin gene knock-out mice exhibit defective leukocyte-endothelial cell rolling interactions and transendothelial migration (Mayadas et al, 1993). At the structural level, the main difference between P-selectin and E- and L-selectin is in the number of SCRs: P-selectin has 9, whereas E- and L-selectin have 6 and 2, respectively. P-selectin mediates adhesion by recognition of carbohydrates on the surface of leukocytes. In general, the P-selectin-carbohydrate interactions are similar to the other selectins (Berg et al., 1992; Foxall et al., 1992; Larken et al, 1992; Moore et al., 1992); however, P-selectin appears to bind a more wide array of carbohydrate structures than either L- or E-selectin. This latter result suggests that P-selectin may mediate a greater diversity of cell-cell interactions than the other selectins, though this hypothesis lacks the experimental evidence. Protein on the surface of neutrophils is important in the presentation of the carbohydrate ligands to P-selectin (Moore et al., 1992). Moore et al. (1992) identified a 250 kD (under non-reducing conditions) glycoprotein on neutrophils that is specifically bound by P-selectin. This molecule appears to be heavily glycosylated and is made up of two disulfide-linked chains of approximately 120 kD. Recently, a cDNA encoding the 250 kD (120 kD monomers) P-selectin ligand on neutrophils (PSGL-1) was isolated (Sako et al., 1993). Recently, it was shown that PSGL-1 supports leukocyte-dependent rolling under physiological flow (Norman et al., 1995). The cDNA sequence predicts regions in the glycoprotein that are heavily decorated with 0-linked carbohydrates—similar to the mucin-like domains GlyCAM-1 and CD34. These results suggest that a family of mucin-like molecules may represent the key ligands for all of the selectins. L-selectin also appears to interact with P-selectin (Picker et al., 1991a). Affinity purified L-selecfin supports adhesion of P-selectin cDNA transfectants. However, anti-L-selectin mAbs have little effect on the binding of purified P-selecfin to neutrophils (Moore et al., 1992). Obviously, additional investigation is required to fully define the nature of the glycoprotein ligands (mucin-like and L-selectin) for P-selectin and their role in mediating function in vivo. Though P-selectin is expressed by many types of vessels in a variety of tissues, there appears to be preferential expression in postcapillary venules versus arteries (McEver et al, 1989). The rapid expression (within minutes) of P-selectin on the surface of endothelial cells following thrombin stimulation in vitro, suggests a role for this protein during the earliest phases of the acute inflammatory process. Interestingly, Stoolman et al. (1993) have shown that P-selectin is expressed at very high levels on venules in chronically inflamed synovium. Expression appears to be on the surface of the endothelial cells. Furthermore, these authors have demonstrated that P-selectin on the inflamed venules supports monocyte adhesion, whereas intracellular antigen expressed in venules in other tissues, foreskin for example, does not support binding. We have obtained similar results in a goat lenti-virus-induced model of arthritis (Jutila et al., unpublished observations). What

The Select!n Family

197

these latter results suggest, is that P-selectin may be important in chronic as well as acute inflammation.

IV. MODEL OF SELECTIN FUNCTION/N V/VO As discussed for L-selectin, P- and E-selectin also support leukocyte adhesion under flow (Butcher, 1991; Lawrence and Springer, 1991; Kishimoto, 1991; Spertini, 1991a; Jutila, 1992; Lasky, 1992). These observations form the basis for models of leukocyte-endothelial cell interactions that were originally published (Butcher, 1991; Kishimoto, 1991; Jutila, 1992). The model described below concentrates on the role of selectins. Because of the unique regulation of selectin expression, it is likely that they mediate the initial interactions that take place between the leukocyte in circulation and the vascular endothelium and/or platelets. In inflammation, the initiating event is a change in the endothelium caused by the release of inflammatory mediators which cause expression of P-selectin within minutes and/or E-selectin within hours. The circulating leukocyte recognizes the altered endothelium and begins a reversible rolling interaction that is mediated by selectins (Ley et al, 1991; von Andrian et al, 1991; von Andrian et al, 1992). Eventually, the leukocyte stops, tightly adheres to the vascular lining, and eventually migrates into the underlying tissue. The tight adhesion and transendothelial migration steps are mediated by the leukocyte integrins (von Andrian et al, 1991). The transition of a selectin-dependent rolling interaction to the integrin-dependent events requires a signal to the leukocyte, which is likely a chemotactic factor, such as IL-8 or PAF, that is released by the inflamed tissues or is actually expressed on the surface of the endothelium (Zimmerman et al., 1992). The signaling event causes up-regulation of the integrins, but also rapid shedding of L-selectin from the leukocyte.The loss of L-selectin may allow the cell to release from the endothelium and migrate into the underlying tissue. Since L-selectin-negative neutrophils no longer retain the capacity to traffic via the vascular system (Jutila et al., 1989), the loss of L-selectin on activated neutrophils may also provide a protective mechanism that prevents the interaction of activated neutrophils, which inadvertently find themselves back in circulation, from interacting with healthy endothelium in other tissues. Cross-linked-induced shedding of L-selectin provides a mechanism for leukocytes that have initially arrested, but don't respond to the inflammatory signals being produced, such as eosinophils or monocytes at certain sites of acute inflammation, to release and re-enter the circulation. Lymphocyte interactions with HEV in peripheral lymph nodes during homing can also be incorporated in the model above. Unlike inflamed venules, selectin ligands are constitutively expressed on lymphoid tissue HEV. L-selectin on lymphocytes may mediate the initial interaction with the endothelium, which is followed by tight adhesion and transendothelial migration. Again, the lymphocyte integrins likely mediate the latter two events. In vivo mAb blocking studies support this model—both anti-L-selectin (Gallatin et al., 1983) and anti-integrin (Hamann et al., 1988) mAbs can block lymphocyte recirculation in vivo.

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An important area of future research is identification of the chemotactic factors that regulate the activation of the leukocyte once it has begun to roll on the endothelium. As mentioned above, chemotactic factors, such as PAF and IL-8, are involved in in vitro systems in increasing leukocyte adhesion to endothelial cells. These factors likely have important roles in inflammation. The chemotactic factors that regulate the entry of lymphocytes into lymphoid tissues are unknown; however, mononuclear cell-specific chemotactic factors have recently been defined (Oppenheim et al., 1991). The function of these factors in vivo remains to be determined.

V. CONCLUSION The study of selectins has contributed significant insight of the molecular basis for leukocyte extravasation. The control of selectin gene expression and the regulation of the expression of surface antigen are two novel aspects of these molecules. Areas of importance in future studies of selectins will be: (1) elucidation of the regulation of selectin gene expression, (2) characterization of the signaling events required for L-selectin shedding, (3) determination ofany role for protein-protein interactions between selectins and their ligands, and (4) generation of novel inhibitors of selectin function. This latter point is particularly relevant to the generation of new, anti-inflammatory therapeutics to treat diseases, such as arthritis, psoriasis, sepsis, and reperfusion injury. Preliminary studies have shown that blockers of L- and E-selectin function are very effective in inhibiting acute inflammatory events (Lewinsohn et al., 1987; Jutila et al., 1989; Jutila et al., 1990; Gundel et al, 1991; Mulligan et al, 1991; Watson et al., 1991a). Emphasis must be given to the development of new animal models to determine the full extent of the effectiveness of anti-selectin treatments in vivo.

ACKNOWLEDGMENTS The expert technical assistance of Sandy Kurk, Gayle Watts, and Kathryn Jutila was instrumental in the generation of much of the data reviewed here and is greatly appreciated, as was the contribution of former co-workers and collaborators: Rupert Kallmann, Frans Kroese, Ellen Berg, T. Kei Kishimoto, Louis Picker, and especially Eugene Butcher. The role of graduate students, Aiyappa Palecanda, Bruce Walcheck, and Rob Bargatze, in generating much of the new data summarized here is also acknowledged. The efforts of Dana Hoover in the preparation of the manuscript are greatly appreciated. Parts of these studies were funded by grants from the USDA (CRSR-90-01666), Pardee Research Foundation, American Cancer Society (ACS CD476), and the Montana Agricultural Experiment Station.

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Springer, T.A., & Lasky, L.A. (1991). Sticky sugars for selectins. Nature 349, 196-197. Stamper, H.B., & Woodruflf, J.J. (1976). Lymphocyte homing into lymph nodes: In vitro demonstration of the selective affinity of recirculating lymphocytes for high endothelial venules. J. Exp. Med. 144, 828-833. Steegmaier, M., Levinovitz, A., Isenmann, S., Borges, E., Lenter, M., Kocher, H.P., Kleuser, B., & Vestweber, D. (1995). The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor. Nature 373, 615. Stenberg, P.E., McEver, R.P., Shuman, M.A„ Jacques, Y.V., & Sainton, D.F. (1985). A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J. Cell Biol. 101,880-886. Streeter, P.R., Rouse, B.T.N., & Butcher, E.C. (1988). Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J. Cell Biol. 107,1853-1862. Stoolman, L., Grober, J., Bowen, B., Reddy, R, Shih, J., Thompson, C, Fox, D.A., & Ebling, H. (1993). Selectin-dependent monocyte adhesion in frozen sections of Rheumatoid synovitis. In: Structure, Function, and Regulation of Molecules Involved in Leukocyte Adhesion (Lipsky, P.E., Rothlein, R., Kishimoto, T.K., Faanes, R.B., Smith, C.W., Eds.). Springer-Verlag, New York, pp. 200-216. Tedder, T., Isaacs, C , Ernst, T., Demetri, G., Alder, G., & Disteche, C. (1989). Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1: Homology with the mouse lymphocyte homing receptor and other human adhesion molecules. J. Exp.Med. 170,123-133. Vestweber, D., Muhlhofif, J., & Levinovitz, A. (1993). Identification of a glycoprotein ligand for mouse E-selectin. J. Cell. Biochem. 17A: C314. von Andrian, U.H., Chambers, J.D., McEvoy, L., Bargatze, R.F., Arfors, K.-E., & Butcher, E.C. (1991). Two step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and leukocyte beta-2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88, 1538-1542. von Andrian, U.H., Hansell, R, Chambers, J.D., Berger, E.M., Filho, I.T., Butcher, E.C, & Arfors, K.-E. (1992). L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo. Am. J. Physiol. 263,1034-1044. Walcheck, B., White, M., Kurk, S., & Jutila, M.A. (1992a). Characterization of the bovine peripheral lymph node homing receptor: A lectin cell adhesion molecule. European J. Immunol. 22,469-476. Walcheck, B., Watts, G., Kurk, S., Bargatze, R., & Jutila, M.A. (1992b). L-selectin expression and function on gamma/delta T cells. FASEB J. A1089. Walcheck, B., Watts, G., & Jutila, M.A. (1993). Bovine gamma/delta T cells bind E-selectin via a novel glycoprotein receptor. First characterization of a lymphocyte/E-selectin interaction in an animal model. J. Exp. Med. 178, 853-865. Walz, G., Aruffo, A., Kolanus, W, Bevilacqua, M., & Seed, B. (1991). Recognition by ELAM-1 of the Sialyl-Lex determinant on myeloid and tumor cells. Science 250,1132-1135. Watson, S.R., Fennie, C , & Lasky, L.A. (1991a). Neutrophil influx into an inflammatory site inhibited by a soluble homing receptor-IgG chimera. Nature 349, 164-167. Watson, S.R., Imai, Y., Fennie, C, Geoffrey, J., Singer, M., Rosen, S.D., & Lasky, L.A. (1991b). The complement binding-like domains of the murine homing receptor facilitate lectin activity. J. Cell Biol. 115,235-344. Weller, A., Isenmann, S., & Vestweber, D. (1992). Cloning of the mouse endothelial selectins: expression of both E- and P-selectin is inducible by tumor necrosis factor. J. Biol. Chem. 287,15176-15183. Whelen, J., Ghersa, P., Hooft van Huijsduijnen, R., Gray, J., Chandra, G., Talabot, F., & Delamater, J.F. (1991). A NK kappa B-like factor is essential but not sufficient for cytokine induction of endothelial leukocyte adhesion molecule 1 (ELAM-1) gene transcription. Nucleic Acid Res. 19, 2645-2653. Zimmerman, G.A., Prescott, S.M., & Mclntyre, T.M. (1992). Endothelial cell interactions with granulocytes: Tethering and signaling molecules. Immunol. Today 13,93-99.

THE CD44 FAMILY OF CELL ADHESION MOLECULES: FUNCTIONAL ASPECTS

Charles B. Underhill

I. Introduction II. Structure of CD44 A. Functional Domains B. Multiple Isoforms of CD44 III. Lymphocyte Homing and Activation IV. Macrophages A. Distribution B. Hyaluronan Induced Aggregation C. Degradation of Hyaluronan V. Hemopoietic Tissue VI. Hair Follicle Development VII. Epithelial Cells A. Distribution B. CD44 is Associated with Proliferation C. Possible Functions of CD44 VIII. TumorCells A. Association with Metastatic Cancers B. Possible Mechanisms of Action IX. Conclusion Acknowledgments References Biomembranes Volume 3, pages 205-218. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-660-6.

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CHARLES B. UNDERHILL

I. INTRODUCTION The realization that the hyaluronan receptor was identical to CD44 came as a surprise to many of us who had been working with this molecule (Aruffo et al., 1990; Culty et al., 1990; Miyake et al., 1990a). Indeed, there was no obvious reason to suspect that these molecules might be related, since they had been investigated in completely different functional contexts. The hyaluronan receptor was originally examined with regard to its ability to bind hyaluronan, while CD44 was characterized for its role in lymphocyte homing. As it turns out, both of these distinct functions were carried out by CD44 and, more recently, this same molecule has been implicated in other phenomena, including morphogenesis, inflammation, lymphocyte maturation, cell migration, and tumor metastasis. In the present review, I will concentrate on the functional aspects of this molecule, with particular emphasis on its interaction with hyaluronan. At the same time, I will speculate on some of the mechanisms by which CD44 mediates these diverse functions.

IL STRUCTURE OF CD44 A. Functional Domains

As illustrated in Figure 1, CD44 is a highly elongated molecule which can be divided into three distinct regions; the N-terminal, middle, and C-terminal domains. The N-terminal domain is responsible for binding hyaluronan and is structurally similar to the link protein and core protein of cartilage, which can also bind hyaluronan (Culty et al, 1990; He et al, 1992). However, CD44 recognizes a six-sugar sequence of hyaluronan while the cartilage core protein recognizes a 10-sugar sequence (Hascall and Heinegard, 1974; Underbill and Toole, 1979; Underbill etal., 1983). The middle domain of CD44 is responsible for the binding of lymphocytes to high endothelial cells of mucosal lymphoid tissue, a phenomenon which is blocked by an antibody (termed Hermes-3) directed against this region (Jalkanen et al., 1987; Berg et al., 1989). This central domain is the site at which most of the structural variations occur between the different isoforms of CD44. Not surprisingly, this region has also undergone a higher rate of evolutionary change than either the N- or C-terminal domains (Goldstein and Butcher, 1990). The C-terminal domain contains the cytoplasmic region, which can be associated with actin filaments of the cytoskeleton through an ankyin-like molecule, as illustrated in Figure 1 (Lacy and Underbill 1987; Kalomiris and Bourguignon 1988). Furthermore, Bourguignon and her associates have shown that the interaction between CD44 and the ankyrin-like molecule is modified by the attachment of phosphate and fatty acid residues and by the binding of guanine nucleotides to the cytoplasmic domain of CD44 (Kalomiris and Bourguignon, 1989; Bourguignon

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The CD44 Family of Cell Adhesion Molecules

1 Membrane Ankyrin Actin Figure h Schematic view of CD44 and its interaction with hyaluronan and the cytoskeieton.

et al, 1991; Lokeshwar and Bourguignon, 1991, 1992). This interaction between CD44 and the cytoskeieton may be important in regulating the hyaluronan binding activity on the cell surface. If the cytoskeieton induces several molecules of CD44 to form clusters, then they vôuld be able to interact simultaneously with a single molecule of hyaluronan (see Figure 1). This cooperative binding would have a higher affinity for hyaluronan than that of a single molecule of CD44 interacting with hyaluronan (Underbill and Toole, 1980; Underbill, 1989). Thus, by controlling the distribution of CD44 on the cell surface, the cytoskeieton may indirectly regulate the binding affinity for hyaluronan on the cell surface. Consistent with this possibility is that cells transfected with forms of CD44 lacking the cytoplasmic domain have a greatly reduced binding affinity for hyaluronan (Hyman et al., 1991; Lesley etal, 1992). B. Multiple Isofornns of CD44

CD44 comes in a wide variety of different molecular weight forms, ranging from 80 to well over 200 kDa. Part of this variation is due to alterative splicing of the mRNA (Brown etal, 1991; Dougherty etal., 1991; Cooper etal, 1992). In humans, the single gene which codes for CD44 contains 19 exons which can give rise to 15

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or more different splicing variations (Goodfellow et al., 1982; Goldstein et al., 1989; Dougherty et al, 1991; He et al., 1992; Screaton et al., 1992). As indicated above, most of these variants result in changes in the middle domain of the molecule (for review see Haynes et al, 1991). The two most common isoforms are CD44H (approximately 85 kDa) which is associated with hemopoietic tissue and CD44E (approximately 150 kDa) which is associated with epithelial cells. The different isoforms differ both structurally and functionally. For example, cells transfected with the CD44H isoform can mediate the binding of lymphocytes to high endothelial cells, whereas cells transfected with the CD44E isoform lack this activity (Berg et al, 1989; Picker et al., 1989). Presumably, a structural change in the middle domain of CD44 alters its ability to interact with mucosal addressin (see following section). The various isoforms of CD44 may also differ with respect to their ability to bind hyaluronan, however, the studies examining this issue are not totally consistent. Stamenkovic and his associates (1991) have shown that cells transfected with a human CD44E isoform do not readily bind to a hyaluronan-coated substrate. On the other hand, He et al. (1992) found that cells transfected with most of the higher molecular weight isoforms of CD44 can bind soluble hyaluronan. Perhaps, this discrepancy is due to differences in the techniques used to measure hyaluronan binding. Clearly, the binding of hyaluronan by the different isoforms of CD44 is an area that deserves further investigation. Another source of structural variation in CD44 is due to differences in the degree of glycosylation. All of the different isoforms of CD44 are highly glycosylated with both N- and 0-linked carbohydrate side-chains which can account for more than half of the molecular mass of the molecule (Carter and Wayner, 1988; Jalkanen et al., 1988; Brown et al, 1991). In the case of macrophages, the extent of glycosylation of CD44 can vary depending upon the activation state of the cell (Camp et al., 1991). It is possible that these carbohydrates modify the ability of CD44 to bind hyaluronan or mediate lymphocyte homing. Heparan and chondroitin sulfate can also be attached to some isoforms of CD44 present in the epidermis and other tissues (Jalkanen et al, 1988; Brown et al, 1991; Kugelman et al, 1992). The chondroitin sulfate side-chains have been shown to be responsible for the binding of CD44 to Type I collagen and fibronectin (Carter and Wayner, 1988; Jalkanen and Jalkanen, 1992), and are required for the migration of melanocytes on collagen (Faassen et al, 1992).

III. LYMPHOCYTE HOMING AND ACTIVATION The connection between CD44 and lymphocyte homing was first suggested by the work of Dr. Eugene Butcher and his collaborators (Berg et al, 1989). These researchers were studying the mechanism of lymphocyte homing by examining the adhesion of lymphocytes to cryostat sections of lymphoid tissue. In the course of these studies, they found that a monoclonal antibody, directed against CD44


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(termed Hermes-3), could block the binding of lymphocytes to the high endothelial cells of mucosal lymphoid tissue (i.e., Peyer's patches), but not to endothelial cells of peripheral lymph nodes (Jalkanen et al., 1987; Streeter et al., 1988). Subsequent research revealed that the CD44 was interacting with a protein termed mucosal addressin, which was specifically present on the endothelial cells of mucosal lymphoid tissue. In addition to its involvement in lymphocyte homing, CD44 may also participate in the activation of lymphocytes. While antibodies against CD44 do not directly stimulate lymphocytes, they do enhance the stimulatory effect of other agents such as antibodies to CD2 and interleukin-2 (Demming et al, 1990; Chong et al, 1992; Conrad et al., 1992). Presumably, this activation occurs through the cross-linking of the cell surface proteins. The question still remains as to whether hyaluronan itself has a similar effect on lymphocytes as the antibodies to CD44. It is possible that hyaluronan helps to prime the lymphocytes so that they are more susceptible to activation with other agents. In this manner, lymphocytes in connective tissue, which is rich in hyaluronan, may be more readily activated than those in lymphoid tissue, which is relatively deficient in hyaluronan.

IV. MACROPHAGES A.

Distribution

While CD44 is particularly abundant on most mature macrophages, it is not uniformly expressed throughout the family of mononuclear phagocytes. In rodents, CD44 is very prominent on mature macrophages of the lungs, peritoneal cavity, and dermis, but is greatly reduced or absent from the Kuppfer cells of the liver and microglia of the brain (Green et al., 1988a). Likewise, peripheral blood monocytes, which give rise to the mononuclear phagocytic cells, generally express lower levels of CD44 than mature macrophages of the lungs or peritoneal cavity. B. Hyaluronan Induced Aggregation

The ability of pulmonary and peritoneal macrophages to interact with hyaluronan has been recognized for some time. More than a decade ago, Mervik and his associates showed that the addition of hyaluronan to a suspension of macrophages induces them to aggregate (Love et al., 1979). Subsequently, Green et al. (1988b) showed that this aggregation was inhibited by blocking antibodies against CD44. Presumably, individual molecules of hyaluronan interact with CD44 on the surfaces of adjacent cells, linking them together. Hyaluronan-induced aggregation of macrophages may occur in both the lungs and peritoneal cavity under certain conditions. In response to inflammatory cytokines, both the fibroblasts of the lungs and the mesothelial cells of the peritoneal cavity secrete hyaluronan which, in turn, causes the aggregation of macrophages (Love et al,

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1979; Sampson et al., 1992). This phenomenon is responsible for the "macrophage disappearance reaction," which is considered to be a form of delayed type hypersensitivity (Galindo et al., 1975; Shannon and Love, 1980; Shannon et al., 1980). This reaction occurs when mice are challenged with an immunogen and then the number of peritoneal macrophages is determined from peritoneal washes. In the case of naive mice, large numbers of macrophages are recovered, whereas in previously sensitized mice, the number of macrophages is greatly reduced. This phenomenon has been traced to a hyaluronan-induced aggregation of the macrophages, which prevents them from being removed during a peritoneal wash, resulting in their apparent disappearance. While the biological significance of this hyaluronan-induced aggregation is still an open question, one possibility is that aggregation of macrophages helps these cells to combat infection. Along these lines, hyaluronan has been shown to stimulate the rate of phagocytosis by monocytes (Ahlgren and Jarstrand, 1984). C. Degradation of Hyaluronan

Macrophages also play an important role in the degradation of hyaluronan, a process which depends upon CD44 (Culty et al., 1992). When fluorescein or isotopically tagged hyaluronan was added to cultures of alveolar macrophages, initially it was bound to the plasma membrane and then taken up inside the cells, where it appeared in small vesicles (Culty et al., 1992). Eventually this hyaluronan was degraded by acid hydrolases present in lysosomes. The process of hyaluronan binding, internalization and degradation was blocked by antibodies to CD44. This suggests that CD44 is required for the critical first step in the process of degradation, namely, the binding to the cell surface. The degradation of hyaluronan by macrophages appears to be an important factor during the morphogenesis of the lung tissue. During early stages of development, large amounts of hyaluronan are present in the interstitial spaces of the lungs, however, prior to birth most of this hyaluronan is lost and the interstitial space shrinks so that gas exchange can take place. The removal of the hyaluronan is accompanied by a general increase in the number of CD44 positive macrophages present in the lungs, many of which were found to contain hyaluronan in their cytoplasms (Underbill et al, 1993). Presumably, these cells had taken up hyaluronan from the lung tissue and were in the process of removing it, by either degrading it directly or by migrating out of the lungs, and taking the hyaluronan with them. Further evidence for the role of macrophages in removing hyaluronan from the lungs camefroman experiment in which newborn mice were injected with blocking antibodies to CD44. After a period of 10 days, the hyaluronan content of the lungs of the injected mice was approximately twice that of control mice (Underbill et al., 1993). Apparently, the antibody blocked the ability of the macrophages to degrade the hyaluronan. It is likely that macrophages play a similar role in the turnover of hyaluronan in other tissues, such as the skin and lamina propria of various tissues.


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V. HEMOPOIETIC TISSUE CD44 is present on a subset of the cells in both myeloid and lymphoid hemopoietic tissues. Indeed, the ability to interact with hyaluronan may be an important factor in the cell maturation process. Miyake et al. (1990b) have shown that antibodies directed against CD44 prevent the maturation of B cells when co-cultured with stromal cells from bone marrow. Presumably, the maturation process requires a hyaluronan-dependent adhesion between the lymphocytes and the stromal cells. Lymphoid cells expressing CD44 also play an important role in removing hyaluronan from the lymph so that the circulating levels are maintained at low levels. Fraser and coworkers (1988,1989) have shown that when [^H]-hyaluronan was injected either intravenously or into afferent lymphatic vessels, a large fraction of it was removed by the dendritic cells of the spleen and lymph nodes. Not surprisingly, dendritic cells also express relatively large amounts of CD44, which is presumably responsible for their ability to bind and degrade the hyaluronan.

VI. HAIR FOLLICLE DEVELOPMENT Histochemical staining of embryonic tissue has revealed an inverse correlation between the expression of CD44 and the presence of hyaluronan in a variety of tissues including the lungs, heart, liver, spleen, and other hemopoietic tissue. However, the most striking example of this occurs in the morphogenesis of the hair follicle, which involves an inductive interaction between the epidermal cells and a small cluster of mesenchymal cells termed the dermal condensation. The cells in this condensation specifically express CD44, while the surrounding mesenchymal cells do not (see Figure 2a). Furthermore, these mesenchymal cells expressed CD44 only during the inductive phases of follicle formation, and lost it once the mature hair follicle had formed. In contrast, hyaluronan was conspicuously absent from the region of the inductive mesenchymal cells as long as the receptor was expressed (see Figure 2b). However, in the mature hair follicle, when CD44 was absent, then hyaluronan reappeared. Thus, the presence of hyaluronan was inversely correlated with the expression of CD44 both temporally and spatially. This inverse correlation suggests, but does not prove, that the expression of CD44 allows the cells to degrade hyaluronan. The absence of hyaluronan from the tip of the developing hair follicle may account for the condensed nature of the inductive mesenchymal cells in this region. Indeed, several studies have demonstrated that treatment of embryonic tissue with hyaluronidase leads to a marked condensation of mesenchymal tissue (Solursh et al, 1979; Morriss-Kay et al., 1986). Apparently, hyaluronan present in these regions keeps the cells separated and its removal causes these tissues to collapse.


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a

Figure 2. Developing hair follicle from a hamster embryo. (A) The section is stained for CD44 and shows that it is present on the cells of the dermal condensation. (B) A similar section is stained for hyaluronan, which is present throughout most of the dermis except in the region of the dermal condensation.

Vll. EPITHELIAL CELLS A.

Distribution

Many, but not all, types of epithelia express CD44. For example, it was prominent on the basal cells of stratified squamous epithelia of the skin, tongue, and esophagus, and on the simple epitheUa lining the oviduct and large intestines. However, it was absent from most mesothelial cells, and was only occasionally observed on endothelial cells during the embryonic development of rodents. B. CD44 is Associated with Proliferation

One of the more unusual aspects of CD44 is that its expression appeared to be directly related to the rate of cell division in the epithelium (Alho and Underbill,


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1989). In the case of the epidermis, the actively dividing cells at the basal regions expressed CD44, while the non-dividing cells in the more superficial layers did not. Similarly, in the epithelium of the large intestines, the dividing cells at the base of the crypts of Lieberkuhn expressed CD44, while the non-proliferating cells further up the crypts and lining the surface did not. It should be noted that this unusual situation holds true only for epithelial cells and is not the case for other cell types (e.g., macrophages). C. Possible Functions of CD44

The biological significance of the CD44 on epithelial cells is still an open question. Earlier, we speculated that CD44 helps to bind the epithelial cells to the basement membrane, which was rich in hyaluronan (Alho and Underbill, 1989). However, we no longer favor this possibility, since closer examination revealed that CD44 was generally absent from the basal surface of the epithelial cells, but instead was present along the lateral surfaces of the cells. Thus, it was not in a position to interact with hyaluronan throughout most of the basement membrane. In the case of the epidermis and other stratified epithelia, CD44 may serve to maintain spaces between the cells so that nutrients can get to the more superficial layers. While hyaluronan often coincides with the presence of CD44 between these cells in the epidermis, it is not clear whether the isoforms of CD44 present on the keratinocytes can bind hyaluronan (Stamenkovic et al., 1991). However, this CD44 may serve as a core protein for the attachment of heparin and chondroitin sulfate side-chains (Carter and Wayner, 1988; Jalkanen and Jalkanen, 1992). These glycosaminoglycans may also function to maintain the spaces between the epithelial cells.

Vlll. TUMOR CELLS A. Association with Metastatic Cancers

Recently, several studies have suggested a connection between the expression of CD44 and the metastatic behavior of tumor cells. For example, high levels of CD44 have been found associated with several types of carcinomas, high grade gliomas, and many non-Hodgkin's lymphomas (Stamenkovic et al., 1989; Horst et al, 1990; Kuppner et al., 1992). Indeed, in the case of lymphomas, large amounts of this protein are correlated with the rapid dissemination and negative prognosis of these tumors (Horst et al., 1990). Thus, CD44 may be a useful marker of the diagnosis of some types of cancer (Matsumura and Tarin, 1992). More direct evidence that the expression of CD44 is related to the metastatic behavior of tumor cells comes from the work of Gunthert et al. (1991). They found that several highly metastatic cell lines from rats express a particular isoform of CD44, which was absent from their non-turmorigenic counterparts. This same

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isoform of CD44 is also expressed in human colorectal carcinomas and adenomatous polyps (Heider et al., 1993). More importantly, when non-metastatic cells were transfected with cDNA for this isoform of CD44, they were converted into a more metastatic phenotype suggesting that this isoform of CD44 is directly responsible for the metastatic behavior of these cells (Gunthert et al., 1991). Other isoforms of CD44 also appear to influence the metastatic behavior of cells. Sy et al. (1991) have shown that when human lymphoma cells were transfected with the hemopoietic isoform of CD44 (CD44H), but not the epithelial isoform (CD44E), there was a marked increase in tumor formation and metastatic behavior. In addition, the growth of these cells in nude mice was inhibited by a soluble fusion protein of CD44 and immunoglobulin, which may competitively inhibit the binding of hyaluronan by CD44 on the surfaces of tumor cells (Sy et al., 1992). However, they also noted that lymphoma cells lacking CD44 formed both primary and metastatic tumors, but at a lower rate. Based on these results, these researchers concluded that expression of CD44H promotes, but is not required for tumor growth and metastasis. B. Possible Mechanisms of Action

There are several possible ways in which CD44 could alter the metastatic behavior of cells. First, the expression of CD44 may allow these cells to bind and degrade hyaluronan in the extracellular matrix, similar to macrophages. Since hyaluronan is a major component of the extracellular matrix surrounding blood vessels, its breakdown may allow tumor cells to enter the circulation. A second possibility is that CD44 allows cells to migrate through the extracellular matrix and thereby establish metastases. Recently, Thomas and coworkers (1992) have shown that melanoma cells expressing the CD44H isoform have an enhanced rate of migration on hyaluronan coated substratum. In addition, this enhanced migration was blocked by treating the cells with blocking antibodies to CD44. In a similar fashion, Faassen et al. (1992) have also found that CD44 is important in the migration of melanoma cells on a substrate composed of Type I collagen. However, in this case, the effect appears to be mediated by chondroitin sulfate residues attached to the CD44 since it could be inhibited by treatment with chondroitinase. Thus, the presence of CD44 on the surfaces of tumor cells may enhance their ability to migrate away and form secondary tumors.

IX. CONCLUSION CD44 impacts upon a number of different areas of research including immunology, developmental biology, and tumor biology. From my perspective, one of the most important aspects of CD44 is its ability to interact with hyaluronan, which is responsible for cell adhesion, and for the ability of cells to take up and degrade hyaluronan. Hyaluronan is a major component of many extracellular matrices and


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is responsible for maintaining the space between cells. The removal of this hyaluronan by a CD44-dependent mechanism can lead to shrinkage of the extracellular space that could be a driving force in morphogenesis.

ACKNOWLEDGMENTS The author thanks Tony Shizari for his help with this manuscript. This work was supported by United States Health Service grants HD26758 and CA35592 and by USAMRMC grant N0.DAMD17-94-J-4284.

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Dougherty, G.J., Lansdrop, P.M., Cooper, D.L., & Humphries, R.K. (1991). Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte "homing" receptor expressed by hemopoietic cells. J. Exp. Med. 174, 1-5. Faassen, A.E., Schrager, J.A., Klein, D.J., Oegema, T.R., Couchman, J.R., & McCarthy, J.B. (1992). A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in Type I collagen-mediated melanoma cell motility and invasion. J. Cell Biol. 116, 521-531. Fraser, J.R.E., & Laurent, T.C. (1989). Turnover and metabolism of hyaluronan. In: The Biology of Hyaluronan CIBA Found. Symp. 143 (Laurent, T.C, Ed.). Wiley, Chinchester, pp. 41-59. Fraser, J.R.E., Kimpton, W.G., Laurent, T.C, Cahill, R.R, & Vakakis, N. (1988). Uptake and degradation of hyaluronan in lymphatic tissue. Biochem. J. 256,153-158. Galindo, B. Myrvik, Q.N., & Love, S.H. (1975). A macrophage agglutinating factor produced during a pulmonary delayed hypersensitivity reaction. J. Reticuloendothel. Soc. 18,295-304. Goldstein, L.A., & Butcher, E.C (1990). Identification of mRNA that encodes an alternative form of H-CAM (CD44) in lymphoid and nonlymphoid tissues. Immumogenetics 32, 389-397. Goldstein, L.A., Zhou, D.F.H., Picker, L.J., Minty, C.N., Bargatze, R.F., Ding, J.F., & Butcher, E.C. (1989). A human lymphocyte homing receptor, the Hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell 56,1063-1072. Goodfellow, P.N., Banting, G., Wiles, M.V., Tunnacliffe, A., Parker, M., Solomon, E., Dalchau, R., & Fabre, J.W. (1982). The gene, MIC4, which controls expression of the antigen defined by monoclonal antibody F 10.44.2, is on human chromosome 11. Eur. J. Immunol. 12, 659-663, Green, S.J., Tarone, G., & Underbill, CB. (1988a). Distribution of hyaluronate and hyaluronate receptors in the adult lung. J. Cell Sci. 89, 145-156. Green, S.J., Tarone, G., & Underbill, CB. (1988b). Aggregation of macrophages andfibroblastsis inhibited by a monoclonal antibody to the hyaluronate receptor. Exp. Cell Res. 178, 224-232. Gunthert, U., Hofmann, M., Rudy, W, Reber, S., Zoller, M., Hausmann, I., Matzku, S., Wenzel, A., Ponta, H., & Herrich, P. (1991). A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65, 13-24. Hascall, V.C, & Heinegard, D. (1974). Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction. J. Biol. Chem. 249,4242-4249. Haynes, B.F., Liao, H-X., & Patton, K.L. (1991). The transmembrane hyaluronate receptor (CD44); Multiple functions, multiple forms. Cancer Cells 3, 347-350. He, Q., Lesley, J., Hyman, R., Ishihara, K., & Kincade, P.W. (1992). Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition. J.CellBiol. 119,1711-1719. Heider, K.H., Hoftnann, M., Hors, E., van den Berg, F., & Ponta, H. (1993). A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J. Cell Biol. 120, 227-233. Horst, E., Meijer, C.J.L.M., Radaszkiewicz, T., Ossekoppele, G.J., Van Krieken, J.H.J.M., & Pals, S.T. (1990). Adhesion molecules in the prognosis of diffuse large-cell lymphoma: Expression of a lymphocyte homing receptor (CD44), LFA-1 (CDlla/18), and ICAM-1 (CD54). Leukemia 4, 595-599. Hyman, R., Lesley, J., & Schulte, R. (1991). Somatic cell mutants distinguish CD44 expression and hyaluronic acid binding. Immunogenetics 33, 392-395. Jalkanen, S., & Jalkanen, M. (1992). Lymphocyte CD44 binds the COOH-terminal heparin-binding domain offibronectin.J. Cell Biol. 116, 817-825. Jalkanen, S., Bargatze, R.F., Toyos, J.D.L., & Butcher, E.C. (1987). Lymphocyte recognition of high endothelium: Antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J. Cell Biol. 105, 983-990.


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Jalkanen, S., Jalkanen, M., Bargatze, R., Tammi, M., & Butcher, E.G. (1988). Biochemical properties of glycoproteins involved in lymphocyte recognition of high endothelial venules in man. J. Immunol 141, 1615-1623. Kalomiris, E.L., & Bourguignon, L.Y.W. (1988). Mouse T lymphoma cells contain a transmembrane glycoprotein (gp85) that binds ankyrin. J. Cell Biol. 106, 319-327. Kalomiris, E.L., & Bourguignon, L.Y.W. (1989). Lymphoma protein kinase C is associated with the transmembrane glycoprotein, GP85, and may function in GP85-ankyrin binding. J. Biol. Chem. 264,8113-8119. Kugelman, L.C., Ganguly, S., Haggerty, J.G., Weissman, S.M., & Milstone, L.M. (1992). The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44. J. Invest. Dermat. 99,381-385. Kuppner, M.C., Meir, E.V., Gauthier, T., Hamou, M-R, & De Tribolet, N. (1992). Differential expression of the CD44 molecule in human brain tumours. Int. J. Cancer 50, 572-577. Lacy, B.E., & Underbill, C.B. (1987). The hyaluronate receptor is associated with actin filaments. J. CellBiol. 105,1395-1404. Lesley, J., He, Q., Miyake, K., Hamann, A., Hyman, R., & Kincade, RW. (1992). Requirements of hyaluronic acid binding by CD44: A role for the cytoplasmic domain and activation by antibody. J. Exp. Med. 175,257-266. Lokeshwar, V.B., & Bourguignon, L.Y.W. (1992). The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction. J. Biol. Chem. 267, 22073-22078. Lokeshwar, V.B., & Bourguignon, L.Y.W. (1991). Post-translational protein modification and expression of ankyrin-binding site(s) in GP85 (Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis. J. Biol. Chem. 266,17983-17989. Love, S.W., Shannon, B.T., Myrvik, Q.N., & Lynn, W.S. (1979). Characterization of macrophage agglutinating factor as a hyaluronate acid-protein complex. J. Reticuloendothel. Soc. 25,269-282. Matsumura, Y, & Tarin, D. (1992). Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet 340, 1053-1058. Miyake, K, Underbill, C.B., Lesley, J., & Kincade, P.W. (1990a). Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J. Exp. Med. 172, 69-75. Miyake, K., Medina, K.L., Hayashi, S.I., Ono, S., Hamaoka, T., & Kincade, RW. (1990b). Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long term bone marrow cultures. J. Exp. Med. 171,477-488. Morriss-Kay, G.M., Tuckett, E, & Solursh, M. (1986). The effects of Streptomyces hyaluronidase on tissue organization and cell cycle time in rat embryos. J. Embryol. Exp. Morph. 98, 59-70. Picker, L., Nakache, M., & Butcher, E.C, (1989). Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types. J. Cell Biol. 109, 927-937. Sampson, P.M., Rochester, C.L., Freundlich, B., «fe Ellias, J.A. (1992). Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. J. Clin. Invest. 90,1492-1503. Screaton, G.R., Bell, M.V., Jackson, D.G., Gomelis, R.B., Gerth, U., & Bell, J.I. (1992). Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spHced exons. Proc. Natl. Acad. Sci. USA 89,12160-12164. Shannon, B.T., & Love, S.H. (1980). Additional evidence for the role of hyaluronic acid in the macrophage disappearance reaction. Immunol. Commun. 9, 735-746. Shannon, B.T., Love, S.H., & Myrvik, Q.N. (1980). Participation of hyaluronic acid in the macrophage disappearance reaction. Immunol. Commun. 9, 357—370. Solursh, M., Fisher, M., Meier, S., & Singley, C.T. (1979). The role of extracellular matrix in the formation of the scleratome. J. Embryol. Exp. Morphol. 54, 75-98. Stamenkovic, I., Amiot, M., Pesando, J.M., & Seed, B. (1989). A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell 56,1057-1062.

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Stamenkovic, I., Aruffo, A., Amist, M., & Seed, B. (1991). The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronan-bearing cells. EMBO J. 10,343-347. Streeter, P.R., Berg, E.L., Rouse, B.T.N., Bargatze, R.F,, & Butcher, E.G. (1988). A tissue-specific endothelial cell molecule involved in lymphocyte homing. Nature 311,41-46. Sy, M.M., Guo, Y.J., & Stamenkovic, I. (1991). Distinct effects of two CD44 isoforms on tumor growth in vivo. J. Exp. Med. 174, 859-866. Sy, M.S., Guo, Y.J., & Stamenkovic, I. (1992). Inhibition of tumor growth in vivo with a soluble CD44-immunoglobulin fusion protein. J. Exp. Med. 176,623-627. Thomas, L., Byers, R., Vink, J., & Stamenkovic, I. (1992). CD44H regulates tumor cell migration of hyaluronate-coated substrate. J. Cell Biol. 118,971-977. Underbill, C.B. (1989). The interaction of hyaluronate with the cell surface: The hyaluronate receptor and the core protein. In: The Biology ofHyaluronan, CIBA Found. Symp. 143 (Laurent, T.C., Ed.). Wiley, Chinchester, pp. 87-106. Underbill, C.B., & Toole, B.R (1979). The binding of hyaluronate to the surface of cultured cells. J. Cell Biol. 82,475-484. Underbill, C.B., & Toole, B.R (1980). Physical characteristics of hyaluronate binding to the surface of simian virus 40 transformed 3T3 cells. J. Biol. Chem. 255,4544-4549. Underbill, C.B., Chi-Rosso, G., & Toole, B.R (1983). Effects of detergent solubilization on the byaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells. J. Biol. Chem. 258,8086-8091. Underbill, C.B., Nguyen, H.A., Shizari, M., & Culty, M. (1993). CD44 positive macrophages take up hyaluronan during lung development. Dev. Biol. 155, 324—336.

MEMBRANE-ASSOCIATED MUCINS

Hans L. Vos, Jelle Wesseling, and John Milkens

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Structure of Episialin/MUCl Expression Pattern of Episialin/MUC 1 mRNA and Protein Effects ofEpisialin/MUCl on Cellular Adhesion Effects of Episialin on Interactions with the Immune System Episialin/MUCl as an Adhesion Molecule Anti-adhesive Effects of Other Mucin-like Proteins The Effects ofOther Mucin-like Molecules on Tumor Cells Conclusion: The Functions of Membrane-associated Mucins References

219 220 221 221 224 224 225 225 226 227

I. INTRODUCTION Mucins are characterized by the presence of a large number of 0-linked glycans (50-80% carbohydrate by weight) and by a large molecular mass, often exceeding 200 kDa. Serine and threonine residues, which function as the attachment sites for the glycans, are abundant in all mucins. Mucin molecules have an extended thread-like conformation as a result of steric hindrance between the 0-linked glycans and the presence of a large number of proline residues in their protein

Biomembranes Volume 3, pages 21^230. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-660-6. 219

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HANS L. VOS, JELLE WESSELING, and JOHN HILKENS

backbone (Jentoft, 1990). The mucins can be divided into the classical mucins that form the mucus layer in, for example, the gastro-intestinal system, and the membrane-associated mucins, that share many of the characteristics of the classical mucins, but are not involved in mucus formation. The mucins of mucus form an extracellular network via disulfide bonds and are often produced and secreted by specialized cells. They serve as lubricants and protect the underlying layer of epithelial cells. Several of these mucins have been characterized at the cDNA level, but in most cases, only partial amino acid sequences are available. A common characteristic apart from the anticipated presence of a large number of serine and threonine residues, is the presence of many proline residues that not only prevent a compact structure of the protein, but are also part of an as yet incompletely defined consensus sequence of 0-linked glycosylation. The mucin-like domain of a protein is often encoded by multiple tandem repeats, the number of which may vary in the genes that contain many nearly identical repeats. In addition to these classical mucins, several mucin-like proteins have been described which also contain an extended domain rich in 0-linked glycans that has all the characteristics of a genuine mucin. However, these proteins are monomeric transmembrane molecules and are, therefore, classified as membrane-associated mucins. Their 0-glycosylated domains may be relatively small, such as in the LDL receptor, in which case the mucin-like domain is mainly intended to lift the receptor domain above the glycocalyx, but it may also encompass most of the extracellular part of a glycoprotein. This is the case for episialin/MUC 1, one of the best characterized mucin-like proteins. It will serve as the prototypic membrane-associated mucin in this review. We will also discuss the properties of CD43, epiglycanin, and ascites sialoglycoprotein (ASGP).

11. STRUCTURE OF EPISIALIN/MUCl Episialin is encoded by the MUCl gene and is also known as the MUCl protein, polymorphic epithelial mucin (PEM), epithelial membrane antigen (EMA), the CA 15.3 antigen, and by various other names. The large mucin-like domain of episialin contains a large array of almost identical 20 amino acid repeats (Gendler et al., 1990; Lan et al, 1990; Lancaster et al., 1990; Ligtenberg et al., 1990; Tsarfaty et al., 1990; Wreschner et al., 1990). The number of repeats in the human gene ranges between about 30 and 90, but in the mouse gene (which contains only 16 less conserved repeats), no variability in the number of repeats has been observed (Spicer et al., 1991). Both the repeats and the immediate flanking regions are very rich in serine, threonine, and proline residues. Based on the low level of homology between the human and murine repeats, we assume that the only requirement for the mucin-like domain of the protein is to serve as a scaffold for 0-linked glycosylation and to give the protein its length. The structure of this domain has important implications for the possible fimctions of episialin and similarly structured molecules, as will be described below.

Membrane-Associated Mucins

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The cytoplasmic domain of episialin is highly conserved between various mammals (Spices et al., 1995a) reflecting its important functions. It is most likely involved in attachment to the actin skeleton (Parry et al., 1990), in intracellular sorting to the apical domain (Pemberton et al., 1996), and in the constitutive internalization of episialin/MUCl (Litvinov et al, 1993).

III. EXPRESSION PATTERN OF EPISIALIN/MUC1 mRNA AND PROTEIN Episialin/MUCl protein is mainly present at the apical surface of glandular epithelial cells of organs such as the mammary gland, pancreas, kidney, lung, and salivary gland, and in cells of the gastrointestinal tract. In carcinoma cells, the polarized expression pattern of episialin is often lost (Ellis et al, 1984; Hilkens et al., 1984; Cordell et al., 1985; Zotter et al., 1988). As a result, the proteins of the apical and basolateral membranes mix, whereas an increased expression level of episialin/MUCl ensures that the density of episialin molecules on the entire cell membrane is at least comparable to that on the apical membrane of normal epithelial cells. This over-expression is also observed at the level of the mRNA. Episialin mRNA levels are much higher in certain carcinomas relative to the adjacent normal epithelial tissue as observed on RNA dot blots of tumor tissue (Zaretsky et al., 1990) and by in situ hybridization of breast tumor sections (our unpublished results). The cause of this over-expression has not yet been established.

IV. EFFECTS OF EPISIALIN/MUC1 ON CELLULAR ADHESION Similar to the situation in mucus, a high density of membrane-associated mucins might act as a barrier to protect the cells against aggressive substances, such as those that occur in pancreatic and gall bladder ducts. However, the combination of over-production and a loss of polarization of episialin expression in breast tumor cells suggested to us that episialin might serve an additional function in these cells. We hypothesized that episialin, on the basis of its extended and relatively rigid structure, might interfere with the function of the adhesion molecules in the non-polarized carcinoma cells. The aggregation capabilities of episialin-expressing transfectants of both a normal mammary cell line (HBLIOO) and a melanoma cell line (A375) were therefore determined, using episialin-negative revertants as a control (Ligtenberg et al., 1992). The episialin levels on the cell membranes of the transfectants were similar to those on carcinoma cell lines. These levels of episialin inhibited cellular interactions between the transfectants to a large extent. In fact, the presence of episialin on only one of a pair of aggregating cells was already sufficient to block cellular adhesion (Ligtenberg et al, 1992). Interestingly, an in-frame deletion mutant of episialin that only carries three repeats (instead of 35 as in the other transfectants) is no longer able to prevent adhesion between mouse


222

60i

Col I ACA25-

Col IV

i ^ ACA25 +

Figure 1. The effect of episialin expression on the adhesion of A375 transfectants to extracellular matrix components. The percentage of episialin-negative (ACA25-; black bars) and episialin-positive (ACA25+; hatched bars) A375 melanoma cells binding to extracellular matrix components is shown. The extracellular matrix components used were: fibronectin (FN), laminin (LM), collagen I (Col I), and collagen IV (Col IV).

L-cells, that is mediated by an E-cadherin transgene, whereas a similar level of the full-length molecule completely inhibited cellular aggregation in this system (Wesseling et al, 1996). These results demonstrate that the length of the episialin molecule is responsible for its anti-adhesive effect. The transfected cell lines also showed a reduced adherence to various extracellular matrix components (Figure 1; Wesseling et al., 1995). Aproportion of the cells of some lines were even growing in suspension (Ligtenberg et al., 1992). Adhesion of these cells to the extracellular matrix is integrin-mediated, showing that episialin is also able to interfere with the action of these types of adhesion molecules, in addition to the cadherins. The combined results strongly suggest that episialin mediates an anti-adhesive effect and we postulate that this occurs by masking the adhesion molecules. We predict that episialin is towering at least 200 nm above the plasma membrane (Calafat et al., unpublished results), whereas other transmembrane proteins reach at most 30 nm above the plasma membrane. A schematic representation of episialin on the cell membrane of non-polarized gels is shown in Figure 2. Indeed, electron microscopy studies on breast carcinoma cell lines have shown that in the regions where the molecule is abundantly present, the adjacent membranes do not directly contact each other (Milkens et al., 1984; Calafat et al., unpublished results). Clearly, adhesion can only be affected if episialin is co-expressed on the same membrane-


223 100 nm

outside I inside

db dp VVVWrViVtVrVVYVrVV^^ VVVVrVrVrVVVrVrVrVry^^

VVVAVrVtVrVrVrVrVrVr^^

Figure 2. A model of the episialin molecules on the cell membrane of a non-polarized cell. The episialin molecules are depicted as the "bottle-brush" type of molecules, whereas all the other molecules are shown as globular proteins. The exact shapes of all proteins are arbitrary, but their respective sizes are within the range of average transmembrane and episialin molecules. The episialin molecules shown contain a relatively low number of repeats. The density of the molecules on the cell membrane is, again, arbitrary.

domain as the adhesion molecules and is present at a sufficient density. This is actually the case for many adenocarcinoma cells. This implies that the presence of episialin is likely to destabilize cell-cell interactions in vivo and may therefore be an important cofactor in metastasis. Interestingly, Muc-1 null mice show a delayed progression of mammary tumors (Spicer et al., 1995b). In polarized normal cells, episialin/MUCl might prevent (nonspecific) interactions between opposing apical membranes. The strong negative charge of the protein that is the result of the high number of sialic acid residues attached to the 0-linked glycans, might contribute to this effect. In this way, episialin might facilitate the formation and maintenance of the ducts during development (Hilkens

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et al., 1992). An identical model has also been suggested by Braga et al. (1992), who showed that the start of murine episialin/MUC-1 expression indeed coincides with epithelial differentiation during the development of the mouse embryo,

V. EFFECTS OF EPISIALIN ON INTERACTIONS WITH THE IMMUNE SYSTEM The function of episialin in tumor progression may not only be restricted to detachment of the cells from their normal surroundings—^the first step in metastasis. During the spread through the body via lymphatic or blood vessels, the expression of episialin may well be critical to the survival of cells. The same transfectants that were used in the adhesion assays are to some extent protected against in vitro immunogenic attack by cytotoxic T-lymphocytes (CTLs) as a result of the presence of episialin on their cell membranes (Van de Wiel-van Kemenade et al., 1993). Adhesive interactions of the CTLs with the transfectants are impaired, leading to a significantly delayed lysis of the cells. On the other hand, tumor-reacting CTL and cytotoxic T cell clones from tumor-draining lymph nodes of patients with breast and pancreatic cancer have been shown to specifically react with episialin (Bamd et al., 1989; Jerome et al, 1991,1993). However, we consider this to be outside the scope of this review as it is not clear if this is a general property of membrane-associated mucins in general.

Vl. EPISIALIN/MUC1 AS AN ADHESION MOLECULE Despite the role of episialin as an anti-adhesion molecule, it might also contribute to adhesive interactions in some situations. The extreme length of the molecule makes it perfectly suited to present its carbohydrate side chains to the cellular surroundings. In fact, it has recently been shown that episialin on colon carcinoma cells is one of the carriers of the sialyl-Lewis X and sialyl-Lewis A antigens (Baeckstrom et al., 1991, 1993; Hanski et al., 1993), that can function as ligands for the selectins. Colon carcinoma cells are known to adhere to activated endothelial cells and platelets that carry selectins, and this process is dependent on the presence of sialyl-Lewis A or X on the carcinoma cells (Lauri et al., 1991; Takada et al, 1991; Kojima et al., 1992; Majuri et al., 1992). Proteins that interact with selectins in vivo, such as GlyCAM-1 and MadCAM, have mucin-like domains (Lasky et al., 1992; Briskin et al., 1993) and these proteins may, therefore, be classified as membraneassociated mucins. In this case, the mucin-like domain has a function in the presentation of the carbohydrate ligands to the selectins, while it may at the same time act to lift other domains of GlyCAM-1 and MadCAM above the cell surface. The exposed nature of the carbohydrates on episialin, and the fact that they are present within a mucin-like domain, make it likely that the sialyl-Lewis structures on episialin are bona fide ligands for selectins.


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VII. ANTI-ADHESIVE EFFECTS OF OTHER MUCIN-LIKE PROTEINS Anti-adhesive properties similar to those of episialin/MUCl have also been observed for other mucin-like molecules, showing that this is a more general characteristic of these molecules. The best characterized example is the CD43 molecule, also known as leukosialin or sialophorin. The extracellular domain of this protein is almost entirely composed of a mucin-like domain consisting of semi-repetitive sequences that are heavily glycosylated and have an overall length of about 45 nm (Cyster et al., 1991). This is much smaller than the observed length of episialin/MUCl, but the CD43 molecule is still larger than most adhesion molecules. CD43/leukosialin is present on hematopoietic cells such as T cells and granulocytes, and it is shed from these cells during activation in vitro at the same time that integrins are upregulated (Rieu et al., 1992; Bazil and Strominger, 1993; Nathan et al., 1993). This suggests that CD43, like episialin, is counteracting interactions mediated by integrins or other adhesion molecules when the cells are in a resting state. This view is reinforced by the observation that T cells not expressing CD43 show spontaneous aggregation both in vivo and in vitro (Manjunath et al., 1993, 1995), whereas HeLa transfectants expressing CD43 show a decreased adherence to T-lymphocytes (Ardman et al., 1992), and are protected against lysis by alio specific T cells (McFarland et al., 1995). This is exactly the phenotype we have observed for episialin/MUCl transfectants. In addition, it has been shown that CD43 accumulates in the cleavage fiirrow during cell division (Yonemura et al., 1993). This might be interpreted as another anti-adhesive function in view of the data cited above. The presence of CD43 would prevent adhesive interactions in the cleavage furrow, thus ensuring the complete separation of the cells following division. The CD43 molecules might be envisioned to act as a kind of "negative zipper." Another mucin-like molecule, CD34, is also conversely regulated with respect to integrins on endothelial cells (Delia et al., 1993), once more suggesting that mucin-like molecules are able to prevent integrin action. Recently, it has been shown that CD34 on endothelial cells also presents ligands to selectins (Baumhueteretal., 1993).

VIII. THE EFFECTS OF OTHER MUCIN-LIKE MOLECULES ON TUMOR CELLS Two additional membrane-associated mucin-like molecules, epiglycanin and ASGP-1, have been shown to affect cellular interactions of rodent carcinoma cells. Epiglycanin is produced by TA3Ha mouse mammary carcinoma cells as one of the major components of their cellular surface. These cells grow readily in suspension, again suggesting an anti-adhesive effect. Epiglycanin, a long rod-like molecule, has been implicated in the masking of the histocompatibility antigens, thereby render-

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ing the cells allotransplantable (Codington et al., 1973, 1978; Miller et al, 1982), and in the prevention of cellular adhesion to the extracellular matrix (Kemperman et al, 1994). Fung and Longenecker (1991) observed immunosuppressive properties of this protein. ASGP-1 (ascites sialoglycoprotein) has many properties in common with episialin with respect to biosynthesis and processing (Carraway et al., 1992). It is made from a precursor that is later split into a mucin-like domain (ASGP-1) and a cell-anchoring domain (ASGP-2). It is abundantly present on the surface of the rat mammary carcinoma cell line 13762. Resistance of these cells to lysis by spleen lymphocytes and NK cells was shown to correlate with the expression level of this sialomucin (Sherblom and Moody, 1986; Bharathan et al., 1990; Moriarty et al., 1990). Moreover, studies on different metastatic clones of this rat mammary tumor cell line have established a positive correlation between their metastatic potential and the expression level of ASGP-1 (Steck and Nicolson, 1983).

IX. CONCLUSION: THE FUNCTIONS OF MEMBRANE-ASSOCIATED MUCINS A clear picture is emerging from the data presented above regarding the role of membrane-associated mucins in normal and carcinoma cells. All properties of mucin-like domains are based on the extreme length and relative rigidity of such domains. Thus in normal cells, this may enable the cell to present certain ligands (such as sialyl Lewis X or A) or receptor domains (such as the LDL receptor) to the outside world, or the mucin-like domain may assist in duct formation by counteracting cellular interactions at the apical membrane. In carcinoma cells, the same anti-adhesive effects are employed to isolate the carcinoma cells from its surroundings, thus enhancing their ability to metastasize and survive in a system that aims for their destruction. The anti-adhesive function is effective only when the mucinlike protein is over-expressed on the entire cell membrane, so that most adhesion molecules are masked. Since present evidence indicates that episialin, epiglycanin, and ASGP are different proteins, the exact nature of the transmembrane mucin that should provide the anti-adhesion function to the tumor cell seems to be of little importance if the molecule is sufficiently large. It should be emphasized that over-expressing a mucin-like molecule is by no means the only way to obtain a less adherent cell. For instance, a large body of evidence shows that lowering the effective amount of the adhesion molecule E-cadherin at the cell surface increases the invasive potential of the cells (Behrens et al., 1989; Frixen et al., 1991; Navarro et al., 1991; Vleminckx et al, 1991). Over-production of a membrane-associated mucin, such as episialin, might be considered as an alternative mechanism to lower the effectiveness of cadherin-mediated cellular adhesion, and, in this way, episialin might enhance invasion. An important remaining question regarding the role of membrane-associated mucins in metastasis is how disseminated cells, expressing high levels of an


227

anti-adhesion molecule (or low levels of an adhesion molecule), will re-adhere to form a distant metastasis. We speculate that the loss of adhesive properties reflects only a relatively small shift in the adhesional balance that might be reversed in several ways. Down-regulation of the expression of the tumor-associated membrane-associated mucins by lowering transcription or by active shedding of the protein (as occurs for CD43) is one obvious mechanism, but the levels of these mucins on metastatic cells are still high. Therefore, cells should be able to increase their capability to adhere. This situation can be reached in several ways. The adhesion molecules may be activated, or their expression may be upregulated, or the cells may undergo a kind of pseudo-polarization that liberates one part of the membrane from obscuring mucin-like molecules. These mechanisms should be able to tip the balance of adhesion once more, now in the direction of adhesion. Despite the fact that we now can model some of the functions of mucin-like proteins, it is clear that our knowledge of the role of these proteins in development and disease is still far from complete. Because they participate in interactions between several different cell types, progress will mainly depend on the availability of in vivo systems, such as transgenic mice and gene replacement mice, to study the roles of these interesting and clinically important proteins. REFERENCES Ardman, B., Sikorski, M.A., & Staunton, D.E. (1992). CD43 interferes with T-lymphocyte adhesion. Proc. Natl. Acad. Sci. USA 89, 5001-5005. Baeckstrom, D., Hansson, G.C., Nilsson, O., Johansson, C , Gendler, S.J., & Lindholm, L. (1991). Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Le- epitope on distinct core proteins. J. Biol. Chem. 266, 21537-21547. Baeckstrom, D., Nilsson, O., Price, M.R., Lindholm, L., & Hansson, G.C. (1993). Discrimination of MUCl mucins from other sialyl-Le--carrying glycoproteins produced by colon carcinoma cells using a novel monoclonal antibody. Cancer Res. 53, 755—761. Bamd, D.L., Lan, M.S., Metzgar, R.S., & Finn, O.J. (1989). MHC-unrestricted recognition of tumorassociated mucins by human cytotoxic T cells. Proc. Natl. Acad. Sci. USA 86, 7159-7163. Baumhueter, S., Singer, M.S., Henzel, W., Hemmerich, S., Renz, M., Rosen, S.D., & Lasky, L.A. (1993). Binding of L-selectin to the vascular sialomucin CD34. Science 262,436-438. Bazil, v., & Strominger, J.L. (1993). CD43, the major sialoglycoprotein of human leukocytes, is proteolytically cleaved from the surface of stimulated lymphocytes and granulocytes. Proc. Natl. Acad. Sci. USA 90, 3792-3796. Behrens, J., Mareel, M.M., VanRoy, P.M., & Birchmeier, W. (1989). Dissecting tumor cell invasion: Epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol. 108,2435-2447. Bharathan, S., Moriarty, J., Moody, C.E., & Sherblom, A.P. (1990). Effect of tunicamycin on sialomucin and natural killer susceptibility of rat mammary tumor ascites cells. Cancer Res. 50, 5250-5256. Braga, V.M.M., Pemberton, L.F., Duliig, T., & Gendler, S.J. (1992). Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development. Development 115,427-437. Briskin, M.J., McEvoy, L.M., & Butcher, E.C. (1993). MadCAM-1 has homology to immunoglobulin and mucin-like adhesion receptors and to IgAl. Nature 363,461^64.

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Kojima N., Handa, K., Newman, W., & Hakomori, S. (1992). Inhibition of selectin-dependent tumor cell adhesion to endothelial cells and platelets by blocking 0-glycosylation of these cells. Biochem. Biophys. Res. Commun. 182, 1288-1295. Lan, M.S., Batra, S.K., Qi, W.-N., Metzgar, R.S., & Hollingsworth, M.A. (1990). Cloning and sequencing of a human pancreatic tumor mucin cDNA. J. Biol. Chem. 265, 15294-15299. Lancaster, C.A., Peat, N., Duhig, T., Wilson, D., Taylor-Papadimitriou, J., & Gendler, S.J. (1990). Structure and expression of the human polymorphic epithelial mucin gene: An expressed VNTR unit. Biochem. Biophys. Res. Commun. 173, 101^1029. Lasky, L.A., Singer, M.S., Dowbenko, D., Imai, Y., Henzel, W.J., Grimley, C , Fennie, C , Gillett, N., Watson, S., & Rosen, S.D. (1992). An endothelial ligand for L-selectin is a novel mucin-like molecule. Cell 69,927-938. Lauri, D., Needham, L., Martin-Padura, I., & Dejana, E. (1991). Tumor cell adhesion to endothelial cells: Endothelial leukocyte adhesion molecule-1 as an inducible adhesive receptor specific for colon carcinoma cells. J. Natl. Cancer Inst. 83, 1321—1324. Ligtenberg, M.J.L., Vos, H.L., Gennissen, A.M.C., & Hilkens, J. (1990). Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J. Biol. Chem. 265,5573-5578. Ligtenberg, M.J.L., Buijs, R, Vos, H.L., & Hilkens, J. (1992). Suppression of cellular aggregation by high levels of episialin. Cancer Res. 52,2318-2324. Litvinov, S.V., & Hilkens, J. (1993). The epithelial sialomucin, episialin, is sialylated during recycHng. J. Biol. Chem. 268, 21364-21371. Majuri, M.-L., Mattila, P., & Renkonen, R. (1992). Recombinant E-selectin-protein mediates tumor cell adhesion via sialyl-Lea and sialyl-Lex. Biochem. Biophys. Res. Commun. 182,1376-1382. Manjunath, N., Johnson, R.S., Staunton, D.E., Pasqualini, R., & Ardman, B. (1993). Targeted disruption of CD43 gene enhances T lymphocyte adhesion. J. Immunol. 151, 1528-1534. Manjunath, N., Correa, M., Ardman, M., & Ardman, B. (1995). Negative regulation of T-cell adhesion and activation by CD43. Nature 377, 535-538. McFarland, T.A., Ardman, B., Manjunath, N., Fabry, J.A., & Lieberman, J. (1995). CD43 diminishes susceptibility to T-lymphocyte mediated cytolysis. J. Immunol. 154, 1097-1104. Miller, S.C., Codington, J.F., & Klein, G. (1982). Further studies on the relationship between allotransplantability and the presence of cell surface glycoprotein epiglycanin in the TA3-MM mouse mammary carcinoma ascites cell. J. Natl. Cancer Inst. 68, 981-988. Moriarty, J., Skelly, CM., Bharathan, S., Moody, C.E., & Sherblom, A.R (1990). Sialomucin and lytic susceptibility of rat mammary tumor ascites cells. Cancer Res. 50, 6800-6805. Nathan, C , Xie, Q.-W, Halbwachs-Mecarelli, L., & Jin, WW. (1993). Albumin inhibits neutrophil spreading and hydrogen peroxidase release by blocking the shedding of CD43 (sialophorin, leukosiaHn). J. Cell Biol. 122, 243-256. Navarro, P., Gomez, M., Pizarro, A., Gamallo, C , Quintanilla, M., & Cano, A. (1991). A role for the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis. J. Cell Biol. 115, 517-533. Parry, G., Beck, J.C, Moss, L., Bartley, J., & Ojakian, G.K. (1990). Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membranecytoskeleton interactions. Exp. Cell Res. 188, 302-311. Pemberton, L.F., Rughetti, A., Taylor-Papadimitriou, J., & Gendler, S.J. (1996). The epithelial mucin MUCl contains at least two discrete signals specifying membrane localization in cells. J. Biol. Chem. 271,2332-2340. Rieu, P., Porteu, F., Bessou, G., Lesavre, P., & Halbwachs-Mecarelli, L. (1992). Human neutrophils release their major membrane sialoprotein, leukosialin (CD43), during cell activation. European J. Immunol. 22, 3021-3026. Sherblom, A.P., & Moody, C.E. (1986). Cell surface sialomucin and resistance of natural cell-mediated cytotoxicity of rat mammary tumor ascites cells. Cancer Res. 46,4543-4546.

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Spicer, A.R, Parry, G., Patton, S., & Gendler, SJ. (1991). Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUCl, reveals conservation of potential 0-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism. J. Biol. Chem. 266, 15099-15109. Spicer, A.R, Duhig, T., Chilton, B.S., & Gendler, S.J. (1995a). Analysis of mammalian MUCl genes reveals potential functionally important domains. Mamm. Genome 6,885-888. Spicer, A.P., Rowse, G.J., Lidner, T.K., & Gendler, S.J. (1995b). Delayed mammary tumor progression in Muc-1 null mice. J. Biol. Chem. 270,30093-30101. Steck, P.A., & Nicolson, G.L. (1983). Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials. Exp. Cell Res. 147,255-267, Takada, A., Ohmori, K., Takahashi, N., Tsuyuoka, K., Yago, A., Zenita, K., Hasegawa, A., & Kannagi, R. (1991). Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. Biochem. Biophys. Res. Commun. 179, 713-719. Tsarfaty, I., Hareuveni, M., Horev, J., Zaretsky, J., Weiss, M., Jeltsch, J.M., Gamier, J.M., Lathe, R., Keydar, I., & Wreschner, D.H. (1990). Isolation and characterization of an expressed hypervariable gene coding for a breast-cancer-associated antigen. Gene 93, 313-318. Van de Wiel-van Kemenade, E., Ligtenberg, M.J.L., de Boer, A.J., Buijs, R, Vos, H.L., Melief, C.J.M., Hilkens, J., & Figdor, C.G. (1993). Episialin (MUCl) inhibits cytotoxic lymphocyte-target cell interaction. J. Immunol. 151, 767—776. Vleminckx, K., Vakaet, L., Mareel, M., Fiers, W., & Van Roy, F. (1991). Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 66, 107-119. Wesseling, J., van der Valk, S.W., Vos, H.L., Sonnenberg, A., & Hilkens, J. (1995). Episialin (MUCl) overexpression inhibits integrin-mediated cell-adhesion to extracellular matrix components. J. Cell Biol. 129,255-265. Wesseling, J., van der Valk, S.W, & Hilkens, J. (1996). A mechanism for inhibition of E-cadherinmediated cell-cell adhesion by the membrane-associated mucin episialin/MUCl. Mol. Biol. Cell 7, 565-577. Wreschner, D.H., Hareuveni, M., Tsarfaty, I., Smorodinsky, N., Horev, J., Zaretsky, J., Kotkes, P., Weiss, M., Lathe, R., Dion, A., & Keydar, I. (1990). Human epithelial tumor antigen cDNA sequences. Differential splicing may generate multiple protein forms. Eur. J. Biochem. 189,463-^73. Yonemura, S., Nagafuchi, A., Sato, N., & Tsukita, S. (1993). Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons. J. Cell Biol. 120,437-449. Zaretsky, J.Z., Weiss, M., Tsarfaty, I., Hareuveni, M., Wreschner, D.H., & Keydar, I. (1990). Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen. A comparative analysis in breast cancer. FEBS Letters 265,46-50. Zotter, S., Hageman, Ph.C, Lossnitzer, A., van den Tweel, J., Hilkens, J., Mooi, W.J., & Hilgers, J. (1988). Monoclonal antibodies to epithelial sialomucins recognize epitopes on different cellular sides in adenolymphomas of the parotid gland. Int. J. Cancer Supplement 3,45-49.

PLATELET MEMBRANE GLYCOPROTEINS

Kenneth J. Clemetson

I. Introduction II. Platelet Surface Glycoproteins III. Platelet Integrins A. GPIIb-IIIa (allbp3) B. Vitronectin Receptor (avp3) C. GPIa-IIa(a2pl) D. GPIc-IIa(a5pl) E. GPIc'-IIa(a6pl) IV. Glycoprotein lb-V-IX (Leucine-rich Repeat Glycoproteins) A. Glycoprotein Iba B. Glycoprotein Ibp C. Glycoprotein IX D. Glycoprotein V E. Polymorphism within GPIba V. Function oftheGPIb-V-IX Complex A. Bleeding Disorders B. Biochemical Evidence for Function C. Non-physiological Activators of the GPIb/vWf Axis D. The Role oftheGPIb-V-IX Complex in Thrombin Activation of Platelets . .


232 235 236 237 240 241 241 241 241 243 244 245 245 246 246 246 248 249 249

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VI.

VII. VIII.

X.

IX.

KENNETH J. CLEMETSON

E. Expression of GPIb-V-IX F. Platelet Activation and Signal Transduction via the GPIb/vWf Axis . . . Other Major Glycoproteins A. CD36 (GPIIIb or GPIV) B. PECAM (Platelet Endothelial Cell Adhesion Molecule, CD31) C. Glycoprotein VI D. CD9 Immunologically Relevant Molecules A. HLA(MHC Class I) B. Fc Receptors, FcyRII (CD32) Complement Protection Molecules A. Decay Accelerating Factor (DAF, CD55) B. CD59 C. C8 Binding Protein (C8bp) D. Membrane Cofactor Protein (MCP, CD46) Granule Membrane Glycoproteins A. P-selectin(GMP-140,PADGEM) B. GMP-33 C. Lysosome-associated Membrane Protein-1 (LAMP-1) and -2 (LAMP-2) D. CD63 (LIMP) E. Granulophysin Conclusions Acknowledgments References

250 .250 251 251 252 252 253 254 254 254 254 255 255 255 255 255 256 256 . 256 256 257 257 . 257 257

1. INTRODUCTION The bleeding problems associated with severe thrombocytopenia clearly illustrate the vital role that platelets have in haemostasis—the prevention of leakage from damaged blood vessels. Although platelets contain many components with a role in these fiinctions, surface glycoproteins are critical for at least two processes—âdhesion and aggregation. In platelets, adhesion has a restricted definition, referring to the attachment of platelets to subendothelium or to other cells, while platelet-i)latelet "adhesion" is referred to as aggregation to differentiate clearly these processes (Figures 1 and 2). Primary adhesion comprises the binding of unactivated, resting platelets to subendothelium (Figure 1) and secondary adhesion comprises the binding of activated platelets (via incomplete primary adhesion or temporary association with a thrombus) to subendothelium (Figure 2). When a vessel wall is damaged and endothelial cells are removed or cell-cell junctions are interrupted (exposing the extracellular matrix of the subendothelium), platelets adhere, are activated, spread, release storage granule contents, bind further platelets, and eventually, also bind some leukocytes and monocytes. A fiirther important step in thrombus formation is aggregation, defined as the sticking together of platelets, in which at least one of the two platelets in contact is activated. GPIIb-IIIa (integrin

Figure 1. Diagram of the processes involved in primary platelet adhesion after damage to the endothelial layer of a vessel wall. Initial attachment of resting platelets via GPlb/V/IX to von Willebrand factor bound to components of the subendothelium stops the platelets, brings them into closer contact with the subendothelium, and starts the process of activation. Other receptors, such as GPla/lla, interact with their ligands, such as collagen, thereby increasing activation and involving the cytoskeleton. GPllb/llla is also involved in the spreading process by forming focal contacts and linking the cytoskeleton more tightly. This is necessary for the spreading to occur over the subendothelial surface.

W Fibrinogen

Figure 2. Diagram of some of the changes occurring during the later stages of activation and thrombus formation. Fibrinogenforms links between GPllb/llla molecules on activated platelets and changes occur in glycoprotein and cytoskeleton distribution. These changes include the transfer of molecules from granules to the platelet surface (e.g., GPllb-llla and granule proteins such as CD62), removal of others to the surface-connectedcanalicular system (such as GPlb-IX), and changes in conformation and in spatial distribution (GPllb-llla).

Platelet Membrane Glycoproteins

235

allbp3) is implicated particularly in aggregation and in spreading. Aggregation involves conformational changes in GPIIb-IIIa exposing a fibrinogen binding site, followed by activation of additional signaling steps that induce GPIIb-IIIa association with the cytoskeleton. This association leads to clustering, which is necessary for the aggregation of platelets held together by fibrinogen bridging. After the initial bridging, additional glycoprotein receptors probably strengthen and stabilize the interaction between the platelets. Resting platelets bind to, and incorporate into, a growing thrombus if the platelets they contact are activated and have fibrinogen/GPIIb-IIIa clusters on their surface. Later cytoskeleton reorganization leads to clot retraction, drawing together the sides of the wound to assist repair. GPIIb-IIIa is required as the link between the subendothelium and the cytoskeleton of the individual platelets. The platelet release reaction, equivalent to activated secretion in other cells, involves the exocytosis of the content of storage granules and is also essential for platelet function. During this process, glycoproteins present on the granule membrane and not expressed on the surface of the resting platelet are exposed, changing the platelet surface properties. Platelets, like other cells, also have a wide range of other glycoproteins, such as agonist receptors. These modulate platelet activity during adhesion and aggregation by either increasing its sensitivity and response through feed-back reactions, or by dampening the response when this might be harmful to the organism. Platelets also contain glycoproteins that have an immunological function such as MHC Class I, Fc receptors and, as other blood cells, molecules involved in protection against complement.

IL PLATELET SURFACE GLYCOPROTEINS Platelet glycoprotein nomenclature was originally based on migration in various gel electrophoresis systems and detection by several surface-labeling methods.

Table 1. Platelet Surface Glycoproteins Platelet Nomenclature GPIa lb Ic Ic' lia lib Ilia lllb(IV) V VI VII VIII IX

Integrin

CD

a1

CDw49b CD42b/c CDw49e CDw49f CD29 CD41 CD61 CD36 CD42d

a5 a6

pi allb

p3

CD42a

236


7

6

1

1

Pl

5

1

4 1

kOa - 150 - 100

-•VI

-

IX*

50

_ 20

Figure 3. Fluorogram of a two-dimensional isoelectric focusing/SDS-polyacrylamide gel of resting platelets, surface-labeled using periodate/[^H]-NaBH4 to show the principle glycoproteins involved as adhesion receptors. P-Selectin is only seen when activated platelets are labeled.

Separations obtained using ^H-surface-labeling with two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis (Figure 3) are shown together with the traditional nomenclature. More general systems of nomenclature have been developed based on the integrin (ap) and cluster of differentiation (CD) systems so that the recent tendency has been toward rationalization of nomenclature. These names are indicated in Table 1.

III. PLATELET INTEGRINS Integrins are glycoprotein complexes, consisting of an a- and a (J-subunit that act as receptors for adhesive proteins present in plasma or on extracellular matrix and link these to the cell cytoskeleton (Hynes, 1987; see also Chapter 6). A common


237

property of integrins is that part of the site they recognize in the adhesive protein is an RGD sequence and peptides related to this sequence can be used to inhibit binding (Plow et al., 1985). Specificity of binding to individual adhesive proteins is assured by the conformation of the RGD sequence and by flanking sequences. Platelets are known to contain two integrins of the P3 family, allbpB (GPIIb-IIIa) and avP3 (vitronectin receptor), and three of the pi family, a2pl (GPIa-IIa), aSpi (GPIc-IIa), and a6pi (GPIc'-IIa) (Sonnenberg et al, 1991 and references therein). A. GPIIb-IIIa (allbpB) GPIIb-IIIa are major glycoproteins on the platelet surface with about 50,000 copies/platelet, constituting about 1—2 % of the total platelet protein. A diagram of the general structure of GPIIb-IIIa is shown in Figure 4. GPIIb-IIIa can act as a receptor for a number of adhesive proteins including fibrinogen (Marguerie et al., 1979), fibronectin (Plow et al, 1985; Parise and Phillips, 1986), von Willebrand factor (Plow et al., 1985), and possibly thrombospondin (Karczewski et al, 1989) and vitronectin (Mohri and Ohkubo, 1991). GPIIb-IIIa is normally cryptic in resting platelets and becomes activated and able to bind fibrinogen in activated platelets (Figure 5). A separate pool of GPIIb-IIIa is present on the membranes of platelet a-granules which fuse to the plasma membranes during exocytosis. Binding of

-binding sites Disulphide linked loops

Phosphorylation site(s) P04\

Cytoplasmic domains

Figure 4. Diagram of the structure of GPIIb-IIIa (allbp3). The cytoplasmic domain of GPIIIa can be phosphorylated on threonine or tyrosine, but the physiological significance of this modification remains unclear.


238

Fibrinogen ^ bound

Aggregation

GPIIIa GPIIb

Signal ^ Resting state

Activated Changes in receptor

•skeleton alin, vinculin) Changes in receptor and fibrinogen

Clustering ther cytoskeleton Calcium channels Kinase and phosphatase activation Clot retraction

figure 5. Diagram of the various stages in activation and occupation of GPIIb-IIla during platelet activation. Many of these stages involve subtle conformational changes that can be detected and quantitated with monoclonal antibodies using flow cytometry.

fibrinogen induces changes in GPIIb-IIIa including formation of calcium channels, linkage to cytoskeleton, and activation of tyrosine kinases (Figure 5). Changes in fibrinogen, or simply clustering induced by binding either to GPIIb-IIIa or to surfaces, allow it to interact with GPIIb-IIIa on unactivated platelets, activating them in turn. These are all complex processes and are incompletely understood. Many studies use monoclonal antibodies that recognize particular epitopes in the GPIIb-IIIa complex. These may be epitopes that are only exposed on GPIIb-IIIa after activation, only exposed after the binding of fibrinogen (Ligand Induced Binding Sites, LIBS) (Du et al., 1991), or only exposed on fibrinogen after binding to GPIIb-IIIa (Receptor Induced Binding Sites, RIBS) (Zamarron et al, 1991), representing particular functional states. Studies on the kinetics of these changes provide vital information on how platelets react. With the development of antiplatelet reagents, such as snake peptides that act by binding to the RGD binding site and blocking fibrinogen binding, these techniques can be used to follow the changes in conformation in GPIIb-IIIa. Normally, if fibrinogen or other adhesive proteins are absent, that is, in washed platelets, the activation of GPIIb-IIIa is transient. This is also seen in the reversible aggregation that occurs with ADP-activated platelets where there is no release. The mechanism of activation of GPIIb-IIIa is still not known, but investigation of the effects of various reagents on the kinetics of "opening" by


239

measurement of fibrinogen or specific antibody binding may cast light on factors influencing this process. Glanzmann's thrombasthenia is a bleeding disorder caused by the absence, deficiency, or malfunction of Gllb-IIIa (George et al, 1990) and its study has provided much of the information about the function of this complex. In the classic disorder, deletions or mutations in either chain prevent or reduce expression of the complex (Newman et al., 1991). In some of the rarer patients with more or less normal amounts of GPIIb-IIIa, but otherwise typical symptoms, mutations have been found affecting the association of the complex (Nurden et al., 1987), fibrinogen binding (Lanza et al, 1992), or signal transduction by the complex (Chen et al., 1992). The use of conformation specific antibodies and flow cytometry has often simplified detection of the functional step that is affected. The synthesis of GPIIb-IIIa has been studied by several groups in both megakaryocytes and megakaryocytic cell lines. Separate precursors for GPIIb and Ilia were detected using [^^S]-methionine-labeling and immunoprecipitation. The large- and small-chain of GPIIb are made as a common precursor. Pulse-chase experiments showed that early assembly of the precursor forms of GPIIb and Illb is necessary for the maturation of GPIIb and the transport of the complex to the cell surface (Duperray et al., 1991). This provides a molecular basis for Glanzmann's thrombasthenia by explaining how defects in either GPIIb or Ilia result in the lack of expression of the other protein as well. GPIIb is one of the rare proteins that is completely specific for megakaryocytes and platelets. GPIIIa is also expressed on a wide range of other cells (such as endothelial cells and monocytes) in the vitronectin receptor (see below). Studies on the promoter region of the GPIIb gene have started to cast some light on the mechanisms of gene control within the megakaryocyte/erythrocyte lineage and its temporal regulation (Martin et al., 1993), While the major role of the integrin GPIIb-IIIa (allbp3) is clearly in plateletplatelet aggregation, there is considerable evidence that it also plays an important role in adhesion. Thus, Glanzmann's thrombasthenia platelets which are deficient in GPIIb-IIIa show a reduced adhesion to subendothelium, though not as dramatic as in Bemard-Soulier Syndrome (BSS) where GPIb is missing. Studies with various antibodies point to two effects here. One is that platelets, which have adhered and have been wrenched from the subendothelium by shear, are activated and GPIIb-IIIa can bind to vWf-coated exposed subendothelium downstream of the initial adhesion site (Ruggeri et al., 1983). The second is that GPIIb-IIIa is apparently necessary for the spreading of the adhering platelets on subendothelium and Glanzmann's thrombasthenia platelets show a much reduced contact area compared with normal (Lawrence and Gralnick, 1987). This role can be explained by activated GPIIb-IIIa binding to vWf and other adhesive proteins on the vessel wall (Savage et al, 1992) and thus, increasing the association between the platelet cytoskeleton and the subendothelium, but also by the important function of GPIIbIIIa in signal transduction via tyrosine kinases and phosphatases and in activation

240


of pathways involved in cytoskeletal rearrangement (Ferrell and Martin, 1989; Golden et al., 1990; Kieffer et al., 1992; Haimovich et al., 1993). In the absence of GPIIb-IIIa or if it is blocked, these cytoskeletal changes are prevented or severely reduced, thus preventing spreading from occurring efficiently. Recent studies have also shown that unactivated GPIIb-IIIa is also the receptor for surface-bound fibrinogen (Zamarron et al, 1991). Whether this may also mediate interactions between platelets and subendothelium appears unlikely, but cannot be completely excluded. GPIIb-IIIa has been expressed on the surface of cultured cells, but the physiological activation mechanism is lacking, thus, so far it has been less useful to study structure/function relationships than had been hoped. Several polymorphisms in GPIIb and Ilia have been reported that are clinically important because of possible antibody production after transfiision that could lead to problems with later transfusions. On GPIIIa, these include PL"^^ and PL"^^, localized to Leu33/Pro33 (Newman et al., 1989), Pen^ and Pen^ localized to Argl43/Glnl43 (Wang et al., 1992), and on GPIIb Bak' and Bak^ localized to Ile843/Ser843 (Lyman et al., 1990). We have seen that GPIIb-IIIa is critical for platelet function and is thus a very important target for the development of inhibitors in the search for "a better aspirin". Several of these are now in clinical trials. Since, at least theoretically, inhibition of GPIIb-IIIa produces a thrombasthenia-like state in the patient, there have been considerable worries about possible bleeding complications. It seems, however, that appropriate molecules and doses are capable of inhibiting platelet aggregation with only minor effects on bleeding, perhaps because mechanisms involved in adhesion are retained. Thus, these inhibitors will be of great interest in modulating platelet ftinction. B. Vitronectin Receptor (avjiS)

Integrins related to GPIIb-IIIa were detected by immunological methods on a wide variety of cells and were identified as the vitronectin receptor, avp3, with an identical P-chain and different a-chain (125 kDa + 20 kDa). The sequence of the a-subunit, av, (Fitzgerald et al, 1987; Suzuki et al., 1987) shows 45% similarity with a5, the subunit of the fibronectin receptor (Argraves et al., 1986; Suzuki et al., 1986) but only 36% with allb of the related platelet p3 integrin (Fitzgerald et al., 1987). Although the P-subunit has an identical amino acid sequence in different cells, there may be differences in post-translational processing, such as glycosylation (Thiagarajan et al., 1985). In contrast to GPIIb-IIIa, avP3 is expressed only in low amounts at the platelet surface—âbout 1,500 copies/platelet based on the binding of an av specific monoclonal antibody (Lawler and Hynes, 1989). In Glanzmann's thrombasthenia (caused by a defect in the gene for GPIIIa (P3)) both GPIIb-IIIa and avp3 expression are affected, whereas in cases caused by a defect in the GPIIb (allb) gene, the levels of avp3 are often elevated as if in compensation. No clear clinical differences could be seen, however. The vitronectin receptor shows a


241

variety of differences from GPIIb-IIIa both in cation requirements and in the question of activation that are still unclear. The receptor may have a role in the initial phases of adhesion since vitronectin is a subendothelial component and may also be involved in clot stabilization involving vitronectin released from a-granules, since anti-vitronectin antibodies inhibit the second wave of platelet aggregation (Asch and Podack, 1990). C. GPIa-lla(a2p1) This integrin is also present in much smaller amounts than GPIIb/IIIa (2,000 copies) on the platelet surface, which is probably related to its role in adhesion and activation instead of aggregation. First indications of its function as a collagen receptor came from patients with platelets deficient in GPIa-IIa who had mild bleeding disorders (Nieuwenhuis et al., 1985). The a- and p-chains are 160- and 130-kDa respectively and binding to collagen is Mg^^ dependent (Staatz et al, 1989). The a-subunit is a single chain. In contrast to GPIIb-IIIa, a tetrapeptide, DGEA, was found to be the main recognition site in collagen sequences (Staatz et al., 1991). Studies on collagen receptors on platelets are complicated by the fact that several types are present. GPIa-IIa binds collagen constitutively (Coller et al, 1989) which activates platelets (tyrosine phosphorylation of P125^'^^) giving clear evidence for outside-in signaling. D. GPIc-lla(a5pi) This is the fibronectin-binding integrin on platelets (Giancotti et al., 1987), present in fairly low amounts (1,000 copies per platelet). The a-chain is cleaved post-translationally into a small and large subunit as with GPIIb. The detailed mechanisms involved in the activation induced via this integrin have not yet been studied. E. GPIc'-lla(a6pi) This is one of the laminin receptors on platelets (Sonnenberg et al., 1988), the other is a 67 kDa non-integrin (Tandon et al., 1991). It is also present in fairly low amounts (1,000 copies per platelet). No detailed studies have yet been done to investigate the mechanisms of activation via this integrin. With both GPIc-IIa and GPIc'-IIa, RGD sequences are thought to be involved in the recognition site.

IV. GLYCOPROTEIN Ib-V-IX (LEUCINE-RICH REPEAT GLYCOPROTEINS) The glycoproteins that make up this complex (and family) have quite different structures from the integrins (Clemetson and Ltischer, 1988; Roth, 1991; Ruggeri, 1991; Wicki et al., 1992). The glycoprotein (GP) Ib-V-IX (CD49a, b, c, and d)


242

Leucine-rich domain 8.9 nm

vWf Thrombin ^^ Negatively

Highly 0-glycosylated segment 34 nm

Leucine-rich domain

15.9 nm

Actin Filaments Figure 6, Diagram of the GPIb-V-IX complex indicating the major domains of the four subunits, known binding sites, phosphorylation sites, and sites known to be involved in interactions with the cytoskeleton. The double loop region of GPIba contains the binding sites for von Willebrand factor and thrombin. The thrombin cleavage site on GPV is just under the double loop region.

complex consists of four chains each coded by separate genes present on different chromosomes. GPIb (CD49b and c) contains GPIba (150 kDa; CD49b, gene on chromosome 17pl2-ter, Wenger et al, 1989) and GPIbp (27 kDa; CD49c, chromosome 22; Bennett, 1990) linked by a disulphide bond (Phillips and Poh Agin, 1977) while GPIX (CD49a, 22 kDa, gene on chromosome 3, Hickey et al, 1990) is strongly non-covalently associated in a 1:1 ratio (Du et al., 1987) and GPV (CD49d, 82 kDa) weakly non-covalently associated with the complex in a 1:2 ratio (GPViGPIb; Modderman et al, 1992; Figure 6). There are around 25,000 copies of GPIb-IX per platelet (Bemdt et al, 1985).


243

A. Glycoprotein Iba GPIba has several distinct domains (Lopez et al., 1987). The N-terminal region, which was also directly sequenced as protein (Titani et al., 1987), consists of a loop formed by a disulphide bond followed by a domain consisting of 7 repeats of a 24 amino acid, leucine-rich sequence very similar to that found in an increasing number of proteins (for review see Roth, 1991). All of the GPIb-V-IX complex proteins contain this motif The first such protein to be described was the leucinerich glycoprotein found in plasma, containing nine such domains (Takahashi et al., 1985), the function of which is still unknown. Recently, the X-ray crystal structure of porcine ribonuclease inhibitor, which contains 16 such repeats, was determined and they were shown to form a novel a/p protein fold arranged in a horse shoe shaped structure (Kobe and Deisenhofer, 1993). Table 2 lists some typical proteins containing these repeats. This domain also contains a single free cysteine residue. The apparent low reactivity of the thiol group may be due to it being buried in the middle of the leucine-rich sequences, but there is some recent evidence for a population of dimers possibly linked by this residue (Clemetson and Hiigli, 1994). Following this region, two disulphide bonds form an overlapping double loop (Hess et al., 1991). This domain is important for the binding sites of the molecule (Figure 7) together with a sequence rich in negatively charged amino acids that then switches abruptly to positively charged amino acids. A domain follows with five repeats of nine amino acid motifs, rich in theonine and serine residues that are 0-glycosylated. The structure of this region strongly resembles that of the mucins

Table 2. Some Proteins Containing Leucine-rich Repeats^ Repeats

Protein

Species

Leucine-rich a2glycoprotein Platelet GPIba Platelet GPIbp Platelet GPIX Platelet GPV

Human

8

Human Human Human Human

6 1 1 15

Drosophila Toll Drosophila Chaoptin Drosophila Slit RNAse inhibitor Human Human Carboxypeptidase N

15 41 22 7 12

Consensus Sequence QLR-LDLSGN-LESLPPGLLQGLP

Reference Takahashi etal., 1985

-L-L-LS-N-LTTLP-GLL~LP -L-L-LS-N-LTTLP-GLL~LP -L-L-LS-N-LTTLP-GLL~LP -L~L-L~N-L~LP-LF~L-

Lopez etal., 1987 Lopez etal., 1988 Hickeyetal., 1989 Hickeyetal., 1993 Lanza etal., 1993 NL-L-L~N-L~LP-LF-H~ Hashimoto et al., 1988 -L-LDLS-N-L~IP—F-LReinkeetal., 1988 -L-L-L-N-I~L--F-LRothbergetal., 1990 SL-EL-LS-N-LGDAG-LC-S-LR-K- Schneider et al., 1988 .L-L-L-N-L~LP~F~LTanetaL, 1990

Note: Â large number of proteins containing leucine-rich domains are now known. Those shown here have the highest similarity to the GPIb complex. In addition, there are flanking regions to many of these domains which also show high degrees of similarity.

244


R

0.?"!]!

Cathepsin G 2«°

\rN^''' (^li

Elastase

—--^

^'^^v

rPcC NrQ-PTLGDEGDTGLYDYYPEEDTEGDKVRATRTWKFP QOONCE,

i

W 210 *-Yr- ^70

S

SS

040434

A

290

Figure 7. Detailed diagram of the double-loop region of GPIba, showing cathepsin G and elastase primary cleavage sites and sequences implicated in thrombin and von Willebrand factor binding as determined from results of studies with peptides and antibodies. The sequence of the 40 amino acid loop from Phe216-Thr240 has been implicated as a thrombin binding site, while the region from Asp235-Lys262 has been implicated in von Willebrand factor binding. The segment from Asp269-Asp287 is highly charged, including three sulphated tyrosine residues and shows some similarity to the C-terminal peptide of hirudin which is involved in binding to the exosite of thrombin.

with a high substitution by short O-Hnked oligosaccharides. Before reaching the membrane, there is an unglycosylated region and just above the membrane is the cysteine(s) forming the Hnk to GPIbp. The transmembrane region (29 amino acids) is followed by a 96 amino acid cytoplasmic domain that can associate with actin-binding protein (Andrews and Fox, 1991), and hence with the membrane-associated cytoskeleton. GPIba glycosylation has been intensively investigated. There are four putative N-glycosylation sites and biantennary, triantennary (Tsuji and Osawa, 1987), and tetraantennary monofucosylated chains (Korrel et al., 1988) have been described. There are many 0-glycosylation sites and the bulk of these are occupied by a biantennary hexasaccharide structure (Tsuji et al, 1983; Korrel et al., 1984). Minor amounts of penta- (Korrel et al., 1985), tetra-, and tri-0-linked saccharides were also detected by these authors. B. Glycoprotein Ibp

Although much smaller than the a-chain, the p-chain has several similar features (Lopez et al., 1988). The N-terminal region contains two disulphide loops followed


245

by a single 24 amino acid leucine-rich repeat, followed further by two disulphide loops with the single cysteine just above the membrane forming the link to the a-chain, the transmembrane region, and a 34 amino acid cytoplasmic domain. Just below the membrane lies a cysteine that can be palmitylated (Muszbek and Laposata, 1989) and in the middle of the cytoplasmic domain lies serine 166 which can be phosphorylated (Wyler et al., 1986) by cAMP dependent kinase (Wardell et al., 1989). GPIbp has a single N-glycosylation site with a lactosamine biantennary oligosaccharide within the leucine-rich domain (Wicki and Clemetson, 1987). There is no 0-glycosylation. C. Glycoprotein IX The overall structure of GPIX is very similar to that of GPIbp (Hickey et al., 1989, 1990). The N-terminal region contains two disulphide loops followed in succession by a single 24 amino acid leucine-rich repeat, two disulphide loops, a short sequence before the transmembrane region, and a six amino acid cytoplasmic domain. Just within the membrane from the cytoplasmic surface lies a cysteine that can be palmitylated (Muszbek and Laposata, 1989). As GPIbjJ, it has a single N-glycosylation site with a lactosamine biantennary oligosaccharide within the leucine-rich domain (Wicki and Clemetson, 1987). There is also no 0-glycosylation. D. Glycoprotein V GPV has recently been cloned by two groups (Hickey et al., 1993; Lanza et al., 1993) so that the complete primary structure is now known. A considerable part of the sequence was known from peptide sequencing (Shimomura et al., 1990). It shares many general structural features with the other members of the complex, in particular GPIba. Thus, from the N-terminus, it also contains two disulphide loops followed by 15 leucine-rich repeats, then two disulphide loops followed by the thrombin cleavage site (but no hirudin-like anionic site). After the cleavage site there is a sequence containing one N-glycosylation site and two 0-glycosylation sites, but no mucin-like repeats. This is followed by the transmembrane region and a 16 amino acid cytoplasmic domain with no phosphorylation sites (no serine, threonine, or tyrosine residues). Overall, there are eight N-glycosylation sites, with six of these in the leucine-rich repeat region. There are no palmitylation sites, supporting an earlier conclusion based on labeling studies (Muszbek and Laposata, 1989). The thrombin cleavage site does not appear to have a direct or indirect role in platelet activation by thrombin (Bienz et al, 1986), unlike the binding site on GPIba. A possible role in modulating GPIb-V-IX function was suggested by the inhibitory effect of alloantibodies from a Bernard-Soulier syndrome patient on ristocetin-induced aggregation of normal platelets (Drouin et al., 1989).

246

KENNETH J. CLEMETSON E. Polymorphism within GPlba

No polymorphisms have yet been reported for the other members of the complex (though there is a preliminary report for GPIbP; Wright et al, 1993), but several are known within GPIba. One of these, thought to be the Sib^-Sib^ system (Ishida et al., 1991) responsible for alloantibody induction, is a Thrl45 (89%)/Metl45 (11 %) (Murata et al., 1992) polymorphism. Another, with no apparent immunological consequences, involves the duplication or tripling of a 13 amino acid sequence between Ser399 and Thr411 (Lopez et al., 1992a). Originally, these size polymorphisms of GPIba were found in the Japanese population and described as A, B, C, and D from the highest to the lowest mass, with about 2,000 Da between each (Moroi et al., 1984). They were later reported from other populations as well (Jung et al., 1986). From molecular biology studies, D was shown to be the molecule with the single sequence from Ser399 to Thr411, while C was the duplicate and B is the quadruple form (Ishida et al, 1995). The A form, which is much rarer in Caucasian populations, has not yet been satisfactorily explained. The size differences found can be explained on the basis of the 13 amino acid segment which contains five threonine and serine residues that can be 0-glycosylated (see preceding). Since each hexasaccharide has a mass of about 1,200 Da, the observed mass difference of more than 2,000 Da per additional segment would predict glycosylation on two sites on average.

V. FUNCTION OF THE GPIb-V-IX COMPLEX A. Bleeding Disorders Much of what is known about GPIb-V-IX ftmction comes from studies of inherited bleeding disorders where expression of this complex on the platelets is defective. Bernard-Sou Her Syndrome In the Bernard-Soulier syndrome (BSS) GPIb-V-IX is either absent, severely depleted, or non-ftinctional (George et al., 1984; Clemetson and Luscher, 1988). This disorder provided most of the first evidence for the role of the GPIb complex (Nurdenand Caen, 1975; Jenkins et al, 1976) and for the association of GPIb with GPIX and GPV (Clemetson et al, 1982; Berndt et al., 1983). BSS platelets adhere poorly, if at all, to subendothelium at all shear rates (Weiss et al., 1974, 1978) emphasizing the importance of the GPIb-V-IX complex in this primary phase of haemostasis. BSS is a rare, autosomal, recessive genetic disorder. The long bleeding time, thrombocytopenia, and morphologically abnormal, unusually large ("giant") platelets are characteristic. Resting BSS platelets are incapable of interacting with vWf (Bithell et al, 1972; Howard et al., 1973) and therefore show dramatically decreased adhesion to subendothelium of damaged vascular wall. Aggregation to


247

other agonists except thrombin (see following) is normal (Bithell et al, 1972). In the classic form of BSS, all four chains are virtually completely absent (i.e., less than 1% can be detected) and the platelets are giant (up to the size of leukocytes) and have a more fluid membrane than normal. In vitro they do not aggregate to either ristocetin or botrocetin in the presence of vWf (Zucker et al, 1977), nor directly to asialo-vWf (De Marco and Shapiro, 1981) or animal vWf, such as bovine or porcine. Another characteristic difference between BSS and normal platelets is their reduced response to and binding of, thrombin (Jamieson and Okumura, 1978; Takamatsu et al., 1986). The thrombin receptor on BSS platelets is most probably normal and it is the absence of GPIb which causes this effect. GPIba contains a thrombin-binding site (Harmon and Jamieson, 1986; Okumura et al., 1978) which, when blocked by antibodies (Jenkins et al., 1983; Mazurov et al., 1991) or removed by proteolytic cleavage (Tam et al, 1980) reduces the response of normal platelets to a-thrombin. The cleaved form, y-thrombin, activates both normal and BSS platelets with similar kinetics, but does not bind to GPIb (Jandrot-Perrus et al., 1988, 1990). BSS platelets also show differences in coagulant activity from nornial (Walsh et al., 1975). Prothrombin consumption was reported lower than normal, whereas platelet factor 3 (= surface exposure of negatively charged lipids) activity was raised (Perret et al., 1983). The latter may be due either to the increased size of the platelets, although other giant platelet syndromes do not show the same effect, or to changes in the distribution of the lipid bilayer (Bevers et al., 1986). BSS platelets were also shown to have more easily deformable membranes than normal (White et al, 1984). Both these phenomena may also be associated with the absence of the GPIb-V-IX complex which, in resting platelets, is linked to actin-binding protein (Solum and Olsen, 1984; Okita et al., 1985) and hence to the membraneassociated cytoskeleton (Solum and Olsen, 1984; Fox, 1985). Several cases of BSS were shown to have mutations in or near the leucine-rich repeats of GPIba or GPIX indicating that these domains are critical for the complex function (Miller et al., 1992; Ware etal., 1993). Platelet-type von Willebrand's Disease Further evidence for the role of GPIb-V-IX and its functions comes from a another bleeding disorder, platelet-type or pseudo von Willebrand's disease where the platelets show an abnormal affinity for normal von Willebrand factor and, as a resuh, are aggregated and removed from the circulation (Miller et al., 1983). Such patients therefore develop thrombocytopenia and consequently, bleeding problems. The molecular defect in platelet-type von Willebrand disease has been established in two families. In one family, there is a G to T point mutation in the gene for GPIba causing a valine for glycine substitution at position 233 of the protein sequence (Miller et al., 1991). This substitution may lead to a different local peptide conformation and hence to the spontaneous binding of vWf to GPIb that is

248


characteristic of this syndrome. In the other family, an A to G mutation leads to a valine for methionine substitution in position 239 (Russell and Roth, 1993). These close mutations lie in a 40 amino acid, disulphide-bridged loop thought to form part of the vWf binding site (Hess et al., 1991). Figure 7 shows a schematic drawing of the double loop region of GPIba with the platelet-type von Willebrand disease mutation sites indicated. The equivalent disorder caused by mutations in vWf is Type IIB von Willebrand's disease (De Marco et al., 1985). Such mutations have been localized to the region of vWf identified as the GPIb binding site (Cooney et al., 1991; Randi et al., 1991). Thus, changes in the conformation of either the vWf binding site on GPIb or the GPIb binding site on vWf can lead to binding of one to the other, pointing to a role for such changes in the physiological functioning of the GPIb/vWfaxis. B. Biochemical Evidence for Function Both polyclonal and monoclonal antibodies have been described that block vWf-related platelet function and bind to GPIb (Ali-Briggs et al., 1981; Ruan et al., 1981; Coller et al., 1983). The epitopes lie on the N-terminal 45 kDa domain of GPIb. Treatment of platelets with proteases, such as elastase (Brower et al., 1985; Wicki and Clemetson, 1985), selectively removes the 45 kDa N-terminal domain and results in platelets that no longer bind vWf, also supporting the localization of the vWf-binding domain in this region. Further, peptides derived from the GPIb sequence are competitive inhibitors of the platelet/vWf interaction, thus defining the binding site more precisely (Katagiri et al., 1990; Vicente et al, 1990). Since vWf does not normally interact directly with GPIb, it was necessary to induce binding using either ristocetin, botrocetin, or asialo-vWf. It was first established that the disulphide bonds of the 45 kDa domain were necessary for optimal interactions, with the exception of that induced by ristocetin. The sequence Ser251 -Tyr279 was identified as inhibiting ristocetin/vWfinduced platelet agglutination, but also inhibited botrocetin-induced platelet agglutination at higher concentrations (Vicente et al, 1990). In both studies, very high amounts of peptide (about 0.5mM) were necessary. This peptide contains most of the smaller double loop (in linear form) plus the anionic region in the C-terminal direction and it contains a PG sequence (see below). The other study reported the peptide Asp235-Lys262 (which still contains the PG sequence) capable of inhibiting ristocetin/vWf-induced platelet aggregation at lljim, whereas the peptide Asp249-Asp274 (which also contains the PG) required 300|im for a comparable inhibition (Katagiri et al., 1990). Conversion of aspartic acid and glutamic acid residues to asparagine and glutamine, respectfully, by site-directed mutagenesis in the region between 251 and 279, eliminated binding to vWf in the presence of either ristocetin or botrocetin, whereas substitutions between 280 and 302 only affected botrocetin (Murata et al., 1991).


249

C. Non-physiological Activators of the CPIb/vWf Axis Ristocetin has been known for a number of years (Howard and Firkin, 1971) and has been useful for diagnosis of bleeding disorders related to the GPIb/vWf axis (Zucker et al., 1977). It is a glycopeptide antibiotic thought to act by binding D-ala containing peptides thereby preventing cross-linking within the peptidoglycans forming the growing cell wall of bacteria. How it induces the vWf/GPIb interaction is still controversial, but it is thought to bind to both vWf and GPIb (Sixma et al., 1991). A possible explanation that extends earlier ideas (Jenkins et al., 1979) suggests that dimeric ristocetin can link the two molecules and indicates that XPGX sequences in both are important for binding (Scott et al., 1991). Others have pointed to XPPX sequences in vWf as being important (Azuma et al., 1993). Botrocetin is a peptide mixture from the venom of the snake Bothoxjararaca (Read et al, 1978; Andrews et al., 1989) that interacts with von Willebrand factor and induces it to bind to GPIb on platelets. Human vWf treated with neuraminidase to remove sialic acid (asialo-vWf) also binds spontaneously to platelet GPIb. Further treatment to remove galactose residues makes the vWf again unresponsive arguing for an important role of oligosaccharides in the conformational changes in vWf. Recently, some other snake peptides have been discovered that affect the vWf/GPIb axis. Alboaggregin-B from Trimeresus albolabris has been shown to induce platelet agglutination to vWf by binding to GPIb (Peng et al., 1991), whereas echicetin from Echis carinatus blocks platelet agglutination by attachment to the same site (Peng et al., 1993). A consideration of all these various examples allows the construction of a model for the physiological process. Although it is not yet possible to test all the parameters involved, there is some evidence for most of them. Thus, the presence of vWf is necessary on the subendothelium where it undergoes a conformational change so that it can bind to GPIb on the platelets. The domain of vWf containing the GPIb binding site also has a collagen binding site in close proximity (Girma et al., 1986; Sixma et al., 1991), which could well be involved in the conformational change. The interaction of GPIb and vWf, together with the forces exerted on the platelet by shear, may start the activation process. D. The Role of the GPIb-V-IX Complex in Thrombin Activation of Platelets The GPIb-V-IX complex contains two thrombin interactive sites, one on GPIba and the other on GPV. The site on GPIba was shown to bind thrombin at an early stage in the characterization of glycocalicin (Okumura et al., 1978). In BSS, the platelets show a reduced response to thrombin which can be reproduced by treating platelets with enzymes that selectively remove the outer domains of GPIba, or by antibodies (polyclonal or monoclonal) that recognize the thrombin binding site on GPIba (Jenkins etal., 1983; De Marco etal, l991;Mazurovetal., 1991). This site only affects the platelet response to low doses of thrombin (Wicki and Clemetson, 1985; McGowan and Detwiler, 1986) and has little affect at high doses. y-Thrombin

250


does not bind to GPIb (Jandrot-Perrus et al., 1988) and showed the same slow kinetics with platelets whether or not GPIb was present (Jandrot-Perrus et al, 1990). The thrombin receptor is now known to belong to the seven-transmembrane, G-protein-coupled receptor family, and is the first known representative of a mechanism where proteolytic cleavage of the N-terminus of the receptor reveals a new N-terminus which acts as a "tethered ligand" capable of interacting with other extracellular loops to activate the receptor (Vu et al., 1991). The thrombin receptor N-terminus contains a highly charged domain similar to the C-terminal region of hirudin that is capable of binding to the anion-binding region of thrombin, thus facilitating the interaction between thrombin and the receptor (Liu et al., 1991). GPIb contains a region just on the C-terminal side of the double-loop which shows some similarity to this domain (Jandrot-Perrus et al., 1992a, 1992b). The thrombin receptor may be in close proximity to GPIb on the platelet surface so that the binding site on GPIb first alters the conformation of thrombin to make the binding site more accessible, and also aids in docking and directing the active site of the thrombin to the cleavage site of the receptor. Indeed, it is known that the active site of thrombin is not blocked by binding to GPIb. An open question is whether the hirudin-like sites on GPIb and the thrombin receptor are competitive or not. In order to obtain a synergistic effect, it would be necessary to have simultaneous binding of thrombin to both GPIb and the receptor. Another problem with this theory is the large number of GPIb molecules (25,000) in comparison with the thrombin receptor (1,200). Of course, a close proximity of a small number of the two sites might account for the "high avidity binding site" (Greco and Jamieson, 1991) by increasing the avidity for thrombin through simultaneous binding. An alternative explanation for the role of GPIb in thrombin-induced platelet activation might be related to signal transduction via GPIb to the platelet interior. While there is no direct evidence for such a signal, the phenomenon of priming is known from other cells. E. Expression of GPIb-V-IX Since very little is known about how the various chains of the complex interact with each other and with other molecules (such as in the cytoskeleton), it is of considerable interest to be able to express these chains in various combinations on non-megakaryocytic cells. Two studies have appeared, one reporting that expression of all three (Iba, Ibp, and IX) chains is necessary for the efficient overall expression of any single chain (Lopez et al., 1992b), while the other found that Iba by itself, although extensively degraded intracellularly, could be expressed intact in small amounts (Meyer et al., 1993). F. Platelet Activation and Signal Transduction via the GPIb/vWf Axis Does binding of GPIb to vWf induce transduction of signals? Recent results exposing platelets to shear-forces comparable to those encountered under more rigorous physiological conditions indicate that the GPIb/vWf axis is indeed capable


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of causing a weak signal, seen as a rise in calcium in the platelets (Kroll et al., 1991; Chow et al., 1992; Ikeda et al, 1993; Kroll et al, 1993). How such signals are induced and how they cause further platelet activation remains unclear, but it has been suggested that it is the force on the GPIb-V-IX complex, transmitted to the cytoskeleton, which is important for opening Ca^"^ channels. In the absence of shear, the primary adhesion process brings other receptors into action such as the collagen receptor(s). Other studies have shown that GPIb is removed from the surface, possibly into the surface-connected canalicular system, when platelets are activated with thrombin. This implies active modulation of the platelet surface-properties.

VI. OTHER MAJOR GLYCOPROTEINS A. CD36(GPIIIborGPIV) CD36 is a single chain molecule of 100 kDa found in the membranes of a wide variety of cells including platelets, monocytes, epithelial, and endothelial cells (Oquendo et al., 1989). This molecule has been the object of considerable activity over the last few years (Figure 8). CD36 is thought to act both as a thrombospondin and a collagen receptor on platelets, though the situation is probably more complex as both thrombospondin and collagen appear to have several different receptors. Even so, CD36 may be particularly important as it is able to transduce signals and several anti-CD36 monoclonal antibodies are able to activate platelets and monocytes by cross-linking CD36 and the Fc receptor (FcyRII in platelets). CD36 is a Cysteine-rich domains Exophosphorylation site Thrombospondin ^ ' or collagen binding

Proline-rich collagen-binding domains

NH2-

Membrane Cytoplasm OOH ppBOsrc-related kinases Fyn, Yes, Lyn, Hck

V Potential N-glycosylation sites

CXCBXBXXK sequence B = basic amino acid

Figure 8. Diagram of CD36 (GPIIIb or IV) showing putative domains and interaction sites with cytoplasmic tyrosine kinases of the src family.

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highly compact and hydrophobic molecule, and in the membrane it is resistant to proteolytic degradation. Part of this protease resistance undoubtedly comes from the fact that the extracellular domains are both N- and 0-glycosylated and contain six cysteines participating in three disulphide bonds. The C-terminal cytoplasmic domain of CD36 contains the sequence CACRSKTIK, which is homologous to the domains on CD4 and CDS involved in binding of src tyrosine kinases. Indeed, it has been shown that the src kinases Fyn, Lyn, and Yes co-precipitate with CDS 6 in immunoprecipitation studies (Huang et al, 1991). This provides a possible mechanism by which CD36 could operate as a signal transducing receptor and antibodies activating platelets have been shown to cause tyrosine phosphorylation of FcyRII. It remains unclear whether conformational changes occur during platelet activation by other agonists, making it an activatable receptor like GPIIb-IIIa and whether it associates with other receptors such as GPIIb-IIIa on activation. Recent studies using synthetic peptides have localized two domains, 93-110 and 139-155, involved in the receptor function for thrombospondin (Leung et al., 1992). These results also indicated that the 139—155 sequence induced a conformational change in thrombospondin (RIBS) allowing it to bind the 93-110 sequence further indicating that not only CD36, but also thrombospondin is affected by the binding. As well as thrombospondin and collagen, CD36 is also a receptor for oxidized LDL (Endemann et al., 1993) and for malaria-infected erythrocytes (Ockenhouse et al., 1989) which bind via the circumsporozite protein, sequestrin (Ockenhouse et al., 1991). B. PECAM (Platelet Endothelial Cell Adhesion Molecule, CD31)

PEC AM is a cell adhesion molecule of the immunoglobulin gene superfamily (Newman et al., 1990) present on a wide range of cells including platelets, endothelial cells, neutrophils, monocytes, and some T-lymphocyte subsets (see Chapter 2). The protein contains 738 amino acids with six immunoglobulin-like C2 subgroup extracellular domains, a transmembrane domain, and a 118 amino acid cytoplasmic domain (Figure 9). There are numerous N-glycosylation sites and the protein is, in fact, quite strongly glycosylated (M^. 130 kDa). It is rapidly phosphorylated on serine or threonine residues when platelets are activated (Zehnder et al., 1992). There was no evidence for tyrosine phosphorylation. Structural similarities between PECAM and cell adhesion molecules (CAM) such as N-CAM and ICAM-1, together with its localization at intercellular junctions indicates that it may also be an adhesion molecule. Although there is now evidence for such a function (MuUer et al., 1992), the complementary molecule involved in heterophilic adhesion has not yet been identified. C. Glycoprotein VI

GPVI is a heavily 0-glycosylated protein of M^. 60-65 kDa and pi 4.5-5, that shows a characteristic sideways V-appearance on two-dimensional gels. Although

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CD31 PECAM-1

a

Ig homology/-^ units • ( a

a C

Cytoplasmic domain Pnosphorylation sites

Figure 9. Diagram of PECAM-1 (CD31) showing domain structure and homologies to related proteins.

little is known about its structure and function, it has been proposed as a collagen receptor in two studies. In one of these (Sugiyama et al., 1987), a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia had antibodies that reacted with a platelet glycoprotein of about 62 kDa. In the other case, a patient with platelets deficient in glycoprotein VI lacked both collagen-induced aggregation and adhesion (Moroi et al., 1989). Two dimensional gels were not run in either case so identification of the missing or immuno-inhibited protein as GPVI remains tentative since there are several platelet glycoproteins in this Mj. range on one dimensional gels.

D. CD9 This is the most common member on platelets of a new family of glycoproteins (Horejsi and Vlcek, 1991) including CD53 and CD63 (see following). A common feature is the presence of four membrane spanning segments and two extracellular loops, one much larger than the other and containing the oligosaccharide. ,CD9 has a mass of 24 kDa, a pi of about 6, and is found on a wide range of cells including monocytes, eosinophils, basophils, and several subclasses of B-lymphocytes (Boucheix et al, 1991; Lanza et al., 1991). No function for CD9 has yet been

254


discovered though several lines of evidence point to a signal transduction role. CD9 has been reported to associate with GPIIb-IIIa. With few exceptions, monoclonal antibodies to CD9 activate platelets.

Vll. IMMUNOLOGJCALLY RELEVANT MOLECULES A. HLA(MHCCIassl) HLA molecules are expressed on cells of the immune system and play a role in the recognition and display of foreign peptides on the cell surface. In such cells, they are recognized together with the foreign antigen, e.g. viral, and serve as a target for killer T-cells. There are two classes of MHC (major histocompatibility complex), I and II (see Chapter 4). Normally, platelets only express Class I. HLA Class I molecules are polymorphic and consist of heavy chains containing 3 immunoglobulin-like domains linked non-covalently to P2"^îc^08lobulin. Since HLA antigens are strongly immunogenic and can cause adverse reactions, efforts have been made to reduce the platelet content in erythrocyte transfusions and either to match HLA types or to devise methods for removing HLA antigens specifically from platelets used in transfusions. B. Fc Receptors, FcyRII (CD32)

Fc receptors are involved in activation of phagocytosis (see Chapter 11). Their presence on platelets suggests that phagocytosis is also a platelet function. The major Fc receptor present on platelets is the FcyRII (Kiister et al., 1990). It is a glycoprotein of about 40 kDa and is the product of at least three different genes. Activation of platelet FcyRII has been shown to induce tyrosine phosphorylation of many proteins including FcyRII itself (Huang et al., 1992). Some subtypes interact weakly with mouse IgGl and are "low responders" while others interact strongly and are "high responders" (Parren et al., 1992). There is some evidence that these subtypes may influence the platelet response in a variety of clinical conditions and that it is, therefore, useful to be able to classify them. There is also some evidence that platelets may have Fee receptors for IgE but this has not been confirmed.

Vm. COMPLEMENT PROTECTION MOLECULES Like other blood cells which may be exposed to complement in inflammatory situations, platelets have molecules on their surface that block or cause the rapid inactivation of complement components. Many, but not all of these, are linked to the membrane by glycophospholipid (GPI) anchors instead of a transmembrane protein sequence and are affected in paroxysmal nocturnal haemoglobinuria (PNH).

Platelet Membrane Glycoproteins A.

255

Decay Accelerating Factor (DAF, CD55)

DAF has a mass of about 70 kDa and is found in a wide range of blood and endothelial cells that come in contact with complement. It is involved in regulation of the formation of C3 and C5 convertases, which are amplification enzymes of the complement cascade. DAF has been shown to have a GPI anchor which on platelets is readily cleaved by phosphatidylinositol specific phospholipase C. B. CD59

CD59 has a M^. of about 18 kDa and inhibits complement lysis of platelets and other blood cells by preventing C8 activation of C9 and C9 polymerization to form a pore. It is more critical than DAF for defense against complement, but like DAF it is also GPI-anchored and missing in PNH (Taguchi et al., 1990). The platelet molecule has been isolated and characterized (Morgan, 1992). It is cleavable by PI-PLC. C. C8 Binding Protein (C8bp)

CSbp has a Mj. of about 50 kDa in platelets (about 66 kDa in erythrocytes, possibly due to glycosylation differences) and inhibits complement lysis of platelets and other blood cells by binding to C8. It is also GPI-anchored and missing in PNH (Blaas et al, 1988). D. Membrane Cofactor Protein (MCP, CD46)

CD46 consists of two nondisulphide-linked chains (56/66 kDa) with an acid pi (4.8-5.5) due to extensive 0-glycosylation (Vanderpuye et al., 1992). Two bands are immunologically and structurally related but the relative contribution of each band is controlled by a two-allele system which is inherited in an autosomal codominant way. The structure is very similar to DAF and MCP has an activity that largely complements that of DAF in that it binds C3b to C4b and acts as cofactor for their first cleavage. Unlike the other complement defence proteins described above, MCP is linked to the membrane by a transmembrane protein segment that may be involved in signal transduction. The structure of MCP has been extensively studied (Liszewski et al, 1991).

X, GRANULE MEMBRANE GLYCOPROTEINS These glycoproteins are present on the inner side of granules (a-, 8-, and lysozomes) and are expressed on the platelet surface after fusion during activation. Therefore, they make good markers for platelet activation. Their expression is dependent on fairly strong activation leading to release and they are more permanent than some other markers such as conformational changes in GPIIb-IIIa which can be revers-

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ible. It is always useful to be able to compare several different activation markers because we still know very little about the biochemistry of these glycoproteins and their dynamics may vary considerably. It is also possible, because of this, that their expression may vary among different diseases or clinical conditions. It is, therefore, very important that a wide range of markers be examined to accumulate as many data as possible in various clinical situations where platelet activation is suspected. Only by applying a variety of techniques to this problem will a consensus eventually be reached as to the best approach. A. P-seiectin(GMP-140, PADGEM)

P-selectin is the platelet member of a family that includes the endothelial cell (E-) and leukocyte (L-) representatives (Johnston et al, 1989). In the platelet and endothelial cell, these are granule membrane proteins which are only expressed on the cell surface after activation and are, therefore, valuable markers. Their biological function is adhesion between cells via heterotypic attachment to a molecule on the partner cell. The sequence of P-selectin shows domains related to other well-known proteins and, in particular, the N-terminal outer domain is related to structures with lectin function in other proteins (see Chapter 7). Based on this, an oligosaccharide was sought as partner structure and was identified as sialyl Le^ structures (Larsen et al., 1990). P-selectin is the prototype marker on platelets for release from a-granules and more studies have been done on its surface expression in various situations than with other, more recent markers. As a result, a considerable literature has accumulated concerning a variety of clinical disorders. B. GMP-33

GMP-33 was identified as a platelet a-granule membrane protein by Metzelaar et al. (1992). Activated platelets display about 19,000 molecules. Expression appears to be restricted to platelets. C.

Lysosome-associated Membrane Protein-1 (LAMP-1) and -2 (LAMP-2)

These two closely related glycoproteins are found in platelet lysosomal membranes and become expressed on the platelet surface on activation (Febbraio and Silverstein, 1990). They both have a M^. between 95 and 120 kDa and are heavily glycosylated (Carlsson et al, 1988). Monoclonal antibodies have been made against both and they have been used in flow cytometry studies on platelet activation in various disease situations. D. CD63(LIMP) This lysosomal integral membrane glycoprotein with Mj. 30-60 kDa was first described as a platelet activation marker and was afterwards found to be a lysosomal


257

protein with a structure related to CD9 (Horejsi and Vlcek, 1991). CD63 contains four transmembrane regions and is rich in cysteines (Metzelaar et al., 1991). An identical molecule, ME491, has been found on the surface of several types of highly metastatic tumors (Azorsa et al., 1991). Flow cytometry studies with monoclonal antibodies to CD63 have been used to study platelet activation in clinical disorders and increased levels of expression were found in deep venous thrombosis and diabetes mellitus. E. Granulophysin

A glycoprotein specific to platelet dense granule membranes and expressed on the surface after platelet activation and release was recently described and called granulophysin. It has a mass of40 kDa and was shown to be deficient in the Hermansky-Pudlak syndrome and present in lower amounts in the Chediak-Higashi syndrome, both of which are associated with albinism. Other forms of dense granule disorders had normal amounts (Shalev et al, 1992). Granulophysin has some immunological similarity to synaptophysin, a 38 kDa synaptic vesicle protein involved in exocytosis.

IX. CONCLUSIONS Platelets contain a large number of membrane glycoproteins involved in various adhesive and other recognition interactions. Many of these represent interesting targets for pharmacological manipulation. Already, a number of inhibitors of GPIIb/IIIa, the aIIb-P3 integrin, are in clinical testing and show promise for treatment of acute and chronic diseases involving platelet activation. However, it is clear that other molecules acting in different stages of haemostasis are also worth investigation, as well as those interacting with other cells and important in processes such as inflammation. The next decade will see a rapid expansion of our knowledge of structure/fixnction relationships throughout this area which should allow the development of inhibitors with potentially new applications in different situations.

ACKNOWLEDGMENTS Support for the work described here that was carried out at the Theodor Kocher Institute came from the Swiss National Science Foundation Grant 31-32416.91, by a grant from HofFmann-La Roche Ltd., and by the supply of buffy coats from the Central Laboratory of the Swiss Red Cross Blood Transfusion Service. This support is gratefully acknowledged.

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IMMUNOGLOBULIN Fc RECEPTORS: DIVERSITY, STRUCTURE, AND FUNCTION

P. Mark Hogarth and Mark D. Hulett

I. Introduction II. Receptors for IgGiFcyR . A. General Comments B. Affinity and Specificity for IgG C. FcyR Protein Structures and Genes D. Tissue Distribution of FcyR E. Signal Transduction by FcyR F. The Interaction Between Ig and FcyR G. The FcyR Binding Regions of IgG III. Receptors for IgE:Fc8R A. General Comments B. Affinity and Specificity for IgE C. Protein Structure and Genes of FcsR D. Tissue Distribution of FcsR E. Interaction Between IgE and FceR F. Signal Transduction of FceR IV. Other FcR A. FcaRI B. Neonatal-intestinal Ig Receptor FcRn V. Concluding Remarks References Biomembranes Volume 3, pages 269-314. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-660-6. 269

270 272 272 275 276 279 281 284 287 289 289 289 290 293 294 295 298 298 299 300 300

270

P. MARK HOGARTH and MARK D. HULETT

I. INTRODUCTION Receptors for immunoglobulins, Fc receptors (FcR), play a fundamentally important role in immunity and resistance to infection by providing the means of linking humoral immunity to cellular effector mechanisms. Indeed, as will be seen below, these widespread and abundant receptors participate in the removal and destruction of antibody-coated particles (pathogens, cellular components, etc.); activate and induce cells to kill antibody-coated targets; are induced by, and, after cross linking, induce the production and release of cytokines and inflammatory mediators; transport immunoglobulins; and are also involved in the regulation of immunity. Fc receptors are cell membrane molecules that specifically bind the Fc portion of an immunoglobulin heavy chain. These receptors have been defined on leukocytes for all Ig classes—ÎgM, IgD, IgG, IgE, and IgA for which they are named, that is, the IgG receptors are FcyR, IgE receptors are FcsR, and so forth. Where more than one class of receptor is defined for an Ig isotype, the receptors are designated thus: FcyRI, FcyRII, or FcsRI, FcsRII, and so forth. The exception to these rules are the receptors expressed on non-leukocytes—^that is, the receptor for polymeric Ig called the poly Ig receptor, or a unique receptor in the gut of neonatal rats, the FcRn. In addition to their specificity for Ig classes, Fc receptors are also classified on the basis of reactivity with monoclonal antibodies and their affinity for Ig, for example, the high affinity FcyRI binds monomeric IgG with an affinity of ~10^M~^ whereas the FcyRII has no detectable binding of Ig monomer, but avidly binds IgG coated particles-immune complexes. The Fc receptor classes defined in Table 1 have been clearly identified and their properties as a class, for example, FcyRI or FcyRII are well defined. The last decade has seen spectacular advances in our understanding of the nature and diversity of Fc receptors—especially since the use of monoclonal antibodies, microchemical, and molecular genetic-techniques. These studies have revealed surprising numbers and diversity of receptors, several of which are only partly characterized (especially among FcyRII forms). Indeed, some of these have been defined only on the basis of molecular cloning and genuine protein products are yet to be identified. However, as the work progresses, the physiological roles of individual receptor isoforms is gradually being determined. This review will discuss the FcR classes and the structure, fimction, and properties of the members of each class as far as they are known. The emphasis of the review will be the human leukocyte FcR and where appropriate, reference to the mouse FcR will be made. The non-leukocyte FcR will also be covered. Readers are also referred to many excellent reviews that have addressed FcR structure and function (Metzger et al., 1986, 1992; Mellman et al., 1988; Unkeless et al., 1988; Kinet, 1989,1990; Kinet and Metzger, 1990; Ravetch and Anderson, 1991; Ravetch and Kinet, 1991; Van de Winkel and Anderson, 1991; Fridman et al., 1992; Van de Winklel and Capel, 1993; Hulett and Hogarth, 1994).

Table 1. Summary of Properties of FcR Classes # o f isoforms

FcR Types Leukocyte

lg Class bound IgG

Protein Family FcR Classes

Mouse

Human

Affinity for IRIM)'

Ig superfamily FcyRl Ig superfamily FcyRll

Ig superfamily FcyRlll

IgE

Ig superfamily FcERI

Type I1 lectins FcsRll IgA

I@

Ig superfamily Fca ?

FcGR low

Non-leukocyte

IgGIlgA polymeric IgG

Ig superfamily polylgR

Ig superfamily FcRn (MHC like)

avid

Comments Only FcyR that binds monomeric Ig. Avidly binds Ig-coated particles; no detectable binding of monomeric Ig. Avidly binds Ig-coated particles. Some monomeric Ig binding, but isoforrn dependant. Very high affinity for IgE. Differences in binding of hlgE and rlgE to h. FcsRI, not so to rat FcERI. Related to FqR. Related to type II lectins, but not other FcR. Lower affinity for IgE. Related to FcyR and FcsRI. Not cloned, but has lectin-like activity. Avidly binds IgM complexes, not cloned, sensitive to proteolysis. Cleavage product of poly-lg receptor is secretory component associated with secretory IgNlgM. Related to MHC class I molecules; associated with P-2 microglobulin.

272

P. MARK HOGARTH and MARK D. HULETT

II. RECEPTORS FOR IgGrPcYR A. General Gomments

In the 20 or so years since it was observed that Ig-coated particles bound to leukocytes in a specific (receptor-mediated) fashion, the IgG receptors have emerged as the largest subfamily of FcR, consisting of three classes, FcyRI, FcyRII, and FcyRIII (Tables 2-5). The three FcyR classes, FcyRI, FcyRII, and FcyRIII, are distinguished on the basis of: (a) affinity for monomeric IgG, (b) monoclonal antireceptor antibodies, and more recently by (c) primary amino acid sequences. Tissue distribution and specificity for Ig also assist in defining and classifying the FcyR (Table 2; for review see Mellman et al., 1988; Unkeless et al, 1988; Ravetch and Anderson, 1991; Ravetch and Kinet, 1991; Van de Winkel and Anderson, 1991; Van de Winklel and Capel, 1993). It is surprising that in 1994, IgG FcR (now recognized to be three classes with a total of at least ten isoforms) are often referred to as "the FcR," singular! As will be seen below, the FcyR are a series of highly evolved specialized receptors with related extracellular (Ig binding) regions, but with cytoplasmic tails or accessory subunits that presumably mediate unique signal transduction events that result in a spectrum of biological responses. While the molecular analyses have been spectacular in revealing the numbers and biochemistry of human FcyR isoforms (Table 3), they have also posed additional questions such as the reason for the numbers and diversity of these receptors, and the effects (if any) on FcyR function of small sequence variation in the extracellular domains of different FcyRII isoforms. A further challenge for those studying FcR function is to dissect the wealth of molecular data in a physiological context. Much of the data relating to the structure of FcyR have come from molecular cloning of individual receptors and much of the analysis of the cellular function has come from experiments using cells which often express two or more FcyR. The co-expression of FcyR has complicated many analyses, as historically it has been difficult to assign particular fiinctions to individual receptor isoforms—especially as different receptor isoforms or families are likely to cooperate in mediating a biological event. This problem has, to some extent, been resolved using several approaches. Monoclonal antibodies have been used to either block the fimction of one receptor class while leaving a second receptor class unaffected or to target quasi-immune complexes (e.g., monoclonal Ab conjugated to erythrocytes) to individual receptor classes, that is, FcyRII. In addition, transfection of isolated FcyR cDNA into cells has been usefiil. Certainly transfection into FcyR" cells, such as fibroblasts, enables a clear analysis of certain functions, for example, ligand binding. Such non-hemopoietic cells, however, may lack factors essential to, or that modify the molecular or cellular function of the FcR. However, a combination of molecular strategies including the use of interspecies cell lines and chimeric receptors, MAb, FcyR" hemopoietic cell lines, as well

Table 2. Functional Characteristics o f H u m a n Leukocyte Fc Receptors Fcy RI Characteristic Affinity for monomeric Ig (M-') Specificity for Ig

a

bl,b2

108-lo9

c

a

b l , b2, b3

c

?

>2

?

?

Mouse 2a, 3>>1,2b

?

?

?

?

Tissue monocytesb distribution rnacrophagesb neutrophilse eosinophilse

Fcy Rlll

FcyRll

a 2

lo7

FcERI

2a>2b>>l

neutrophils mast cells neutro- T, B cells eosinophilsC basophils phils macrophage eosinophils Langerhan's cells platelets Eosinophils NK cells Monocytesg Langerhan's cells

Table 3. Characterization o f H u m a n Leukocyte Fc Receptors Characteristic Genes (~lleles)' Chromosomal location Transcripts Receptor formj Ig-like domains

Associatedsubunits

Fcy Rll

FcyRI A lq21 .I

Ba lq21 .I

a M 3

bl b2 M S 2 2 b

y

y

-

FcyRIII

Ca lq21 .I

A(HWLR) 1q23-24

c S 2 b

a1 M 2

-

?

B lq23-24

a2a b l S M 2 2

b2 M 2

?

?

none

C Iq 23-24 c b3a M M 2 2

?

?

A lq23

B(NAIlNA2) lq23

a M 2

b M~ 2

yg,

cg,

FcERI

FcaRl

A A Iq 19q13.4 23-24 a a M M 2 2

none

Y, P

Y

5MOkDf

45-65

55-75

4or6'6

6

1

FCERll A 19 a M~ C-type lectinh no

b M~ lectinh

no

P" Mr

7

Number of glycans

6

2

-

-

-

-

-

-

40

30'

40

40

40

40

2

2

3

3

3

3

5080kD 5

N

u

P;

Notes: aProteinproduct not known, but properties proposed on the basis of predicted amino acid and nucleotide sequences. %anslation termination in third domain at different positions. 5 i z e predicted on that known for engineered recombinant soluble FcyRII. d ~anchor. ~ l eCan associate with FcaRlD in transfectants. 'Mrfvaries with NAlfNA2 allotype due to number N glycan sites-see text. 9=FcaRly subunit, 6TCR-I; subunit, $=FcsRI-P subunit. hype I1 membrane protein of the C-type lectin family. 'Alleles only shown for polyrnorphisms where inheritance confirmed. iM, Membrane anchored; S, soluble.

45-50 1

45-50

Immunoglobulin Fc Receptors

275

as the future use of FcR" mice developed by gene knock-out using homologous recombination, will be very useful in further defining the structure and function of FcyR. B. Affinity and Specificity for IgG

FcyRI has a high affinity (~10~^M) for monomeric IgG and also avidly binds immune complexes (Anderson and Abraham, 1980; Anderson, 1982; Kurlander and Batker, 1982; Allen and Seed, 1989). However, FcyRII is a lower affinity receptor (1>4>>2 3>l >2>>4 mouse 2a>>l, 1=2a=2b>>3 2b>3

IgC: 3=1>2>>4 1=2a=2b>>3

Tissue distribution

monocytes macrophages neutrophils

macrophages N K cells y6 T cells

N CO

0

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