Rhodopsin and G-Protein Linked Receptors, Part A,

BIOMEMBRANES A Multi- Volume Treatise Volume 2A • 1996 RHODOPSIN AND G-PROTEIN LINKED RECEPTORS This Page Intentio...

Author: A.G. Lee

14 downloads 678 Views 15MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

BIOMEMBRANES A Multi- Volume Treatise

Volume 2A •

1996

RHODOPSIN AND G-PROTEIN LINKED RECEPTORS

This Page Intentionally Left Blank

BIOMEMBRANES A Multi-Volume Treatise

RHODOPSIN AND G-PROTEIN LINKED RECEPTORS Editor: A. G. LEE Department of Biochemistry University of Southampton Southampton, England

VOLUME 2A

1996

@)JAl PRESS INC. Greenwich, Connecticut

London, England

Copyright © 1996 by JAI PRESSINC 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESSLTD. The Courtyard 28 High Street Hampton Hill, Middlesex TWl 2 1PD England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-659-2 Manufactured in the United States of America

CONTENTS (Volume 2A)

LIST OF CONTRIBUTORS

ix

PREFACE A.G. lee

...

Xlll

RHODOPSIN STRUCTURE AND FUNCTION Burton J. Litrnan and Drake C. Mitchell CHARACTERIZATION OF THE PRIMARY PHOTOCHEMICAL EVENTS IN BACTERIORHODOPSIN AND RHODOPSIN Jeffrey A. Stuart and Robert R. Birge

1

33

LIGHT-INDUCED PROTEIN-PROTEIN INTERACTIONS ON THE ROD PHOTORECEPTOR DISC MEMBRANE Klaus Peter Hofmann and Martin Heck

141

MICROBIAL SENSORY RHODOPSINS John L. Spudich and David N. Zacks

199

ALPHA-ADRENERGIC RECEPTORS David B. Bylund

227

V


CONTENTS (Volume 2B) [3-ADRENERGIC RECEPTORS

Susan M. Pellegrino, Norman H. Lee, and Claire M. Fraser

G PROTEIN-COUPLED SEROTONIN RECEPTORS

Jean C. Shih and Timothy K. Gallaher

THE MUSCARINIC ACETYLCHOLINE RECEPTORS

Petra HOgger, Wolfgang Sad6e, and Jelveh Lameh

ADENOSINE RECEPTORS

David R. Luthin, John A. Auchampach, and Joel Linden

245 281 301 321

METABOTROPIC GLUTAMATE RECEPTORS

Patrick J. O'Hara

349

GLYCOPROTEIN HORMONE RECEPTORS: NEW MOLECULAR SUPPORTS FOR OLD BIOLOGICAL FUNCTIONS

Roland Salesse and Jean Gamier

PLATELET ACTIVATING FACTOR RECEPTOR

Shivendra D. Shukla

INDEX

387 463 481

vii


LIST OF CONTRIBUTORS

John A. Auchampach

Departments of Internal Medicine and Molecular Physiology and Biological Physics University of Virginia Charlottesville, Virginia

Robert R. Birge

Department of Chemistry Syracuse University Syracuse, New York

David B. Bylund

Department of Pharmacology University of Nebraska Medical Center Omaha, Nebraska

Claire M. Fraser

Department of Molecular and Cellular Biology The Institute for Genomic Research Gaithersburg, Maryland

Timothy K. Gallaher

Department of Molecular Pharmacology and Toxicology School of Pharmacy University of Southern California Los Angeles, California

Jean Garnier

Unite d'lngenierie des Proteines IN RA-Biotech nologies Cedex, France

Martin Heck

Institut fLir Medizinische Physik und Biophysik Humboldt-Unlversit~it zu Berlin Berlin, Germany

Klaus Peter Hoffman

Institut for Medizinische Physik und Biophysik Humboldt-Universit~it zu Berlin Berlin, Germany

LIST OF CONTRIBUTORS Petra H6gger

Departments of Pharmacy and Pharmaceutical Chemistry University of California San Francisco, California

Jelveh Lameh


Norman H. Lee


Joel Linden


Burton J. Litman

Laboratory of Membrane Biochemistry and Biophysics National Institute of Health Rockville, Maryland

David R. Luthin


Drake C. Mitchell

Laboratory of Membrane Biochemistry and Biophysics National Institute of Health Rockville, Maryland

Patrick J. O'Hara

DNA Chemistry and Computer Science ZymoGenetics, Inc. Seattle, Washington

Susan M. Pellegrino


List of Contributors Wolfgang Sadie


Ronald Salesse

Unite d'lngenierie des Proteines IN RA-Biotech nologies Cedex, France

Jean C. Shih

Department of Molecular Pharmacology and Toxicology School of Pharmacy University of Southern California Los Angeles, California

Shrivendra D. Shukla

Department of Pharmacology School of Medicine University of Missouri Columbia, Missouri

John L. Spudich

Department of Microbiology and Molecular Genetics University of Texas Medical School Houston, Texas

Jeffrey A. Stuart

Department of Chemistry Syracuse University Syracuse, New York

David N. Zacks

Department of Microbiology and Molecular Genetics University of Texas Medical School Houston, Texas


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. Application of the techniques of molecular biology has provided the sequences of a very large number of membrane proteins, and has led to the discovery of superfamilies of membrane proteins of related structure. The classic example of the superfamily is the seven helix receptor superfamily, all related in structure to bacteriorhodopsin, and named after the seven trans-membrane or-helices identified in bacteriorhodopsin. This volume explores the structures and functions of this superfamily. 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

xiii


RHODOPSIN STRUCTURE AND FUNCTION

Burton J. Litman and Drake C. Mitchell

I. II.

III.

IV. V. VI.

VII.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

Rhodopsin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

3

A.

General Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

B.

Structural C o m p a r i s o n with Bacteriorhodopsin

...............

5

C. Helix-Linking Loops and Functional Domains Rhodopsin Photointermediates . . . . . . . . . . . A. Overview . . . . . . . . . . . . . . . . . . . . B. Early Photointermediates . . . . . . . . . . . . C. Late Photointermediates . . . . . . . . . . . . D. Structural Changes in MII . . . . . . . . . . .

............... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 9 9 9

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

12

O p s i n - C h r o m o p h o r e Interactions . . . . . . . . . . . . . . . . . . . . . . . . . Rhodopsin-Transducin Interactions . . . . . . . . . . . . . . . . . . . . . . . . Rhodopsin-Lipid Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 15 17

11

A.

Disk M e m b r a n e Lipid Composition

. . . . . . . . . . . . . . . . . . . . .

17

B.

Lipid Modulation o f MII Formation . . . . . . . . . . . . . . . . . . . . .

18

C.

M e m b r a n e Lipid D o m a i n Model . . . . . . . . . . . . . . . . . . . . . . .

22

Conclusions

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Biomembranes Volume 2A, pages 1-32 Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-659-2

24

BURTON J. LITMAN and DRAKE C. MITCHELL

!. I N T R O D U C T I O N The visual pigment, rhodopsin, is localized in the retinal rod outer segment (ROS) disk membrane and is a prototypical member of the seven transmembrane helical, superfamily of G protein-coupled receptors. Photoactivation of rhodopsin results in the formation of an active conformation, R*, which binds the rod cell G protein, transducin (Gt) and activates it by catalyzing the exchange of Gt-bound GDP for GTP (Figure 1, for recent reviews see Stryer, 1991; Khorana, 1992; Nathans, 1992; Hargrave et al., 1993). A single R* can activate several hundred Gt molecules during its lifetime. The ct subunit of Gt, Gta, activates the effector enzyme, a cGMP phosphodiesterase (PDE), by binding its inhibitor subunit, thus initiating cGMP hydrolysis. The reduction in cGMP concentration results in a hyperpolarization of the ROS plasma membrane, due to the reduced flux of Na ÷ ions through the cGMP-gated channels, with concomitant change in neurotransmitter release at the synaptic end of the rod cell, which generates the neuronal response to light. Rhodopsin is unique as a receptor in that it contains a covalently bound spectroscopic marker, the retinal chromophore. As will be discussed later in RhodopsinTransducin Interactions, several different investigations point to identifying R* with the metarhodopsin II (MII) photointermediate. Thus, monitoring the time-depend-

R.. G, (GTP). j f=~ GTP R.~. hV

~ .

G,,1,

GDP

R*- G, (GDP)

MII = R* +

Gt=(GTP)~%%

PDIE i

G,=(GTP)*-PDEi ] G,= (GTP)*- PDE, + PDF_=*~

//i

G, (GDP)

PDEi

Figure 1. Visual sig,nal transduction in rod cell. Light (hv) converts rhodopsin (R) to an activated form (R), which binds and activates Gt(GDP) by catalyzing the exchange of bound GDP for GTP. Gt(GTP)* then dissociates and Gt~(GTP) binds to the inactive form of the cGMP-phosphodiesterase (PDEi). This complex dissociates to yield the active subunit complex PDE~.I3and Gto~(GTP)*. PDE~, where PDE~ is the inhibitory subunit of the PDE, initiating the hydrolysis of cGMP by PDE~.I3. The lowered concentration of cGMP induces closure of cGMP-gated Na+ channels in the plasma membrane, hyperpolarizing the cell.

Rhodopsin Structure and Function

ent changes and peak intensity of the MII spectrum allows one to determine the kinetic and equilibrium properties associated with the formation of R*, which is the functional equivalent of the activated agonist-bound conformation in liganded receptor systems. A number of studies suggest that MII exists as several structural substates, which are spectrophotometrically indistinguishable, but differ both with respect to the configuration of the hydrophilic loops connecting the transmembrane helices and their interaction with the peripheral proteins of the visual transduction pathway. Various lines of research indicate, however, that the interaction of these transduction proteins with R* are reflected by the spectrophometrically monitored behavior of MII. As such, MII and R* will be used interchangeably in this review. The following topics associated with the visual transduction pathway will be discussed: rhodopsin structure, the interaction of opsin, the protein portion of rhodopsin, with the retinal chromophore, rhodopsin photochemistry, and the interaction ofphotoactivated rhodopsin with Gt, which is the first stage of amplification in the visual transduction pathway and is initiated by the formation of a R*.Gt complex. Rhodopsin is an integral membrane protein with about 50% of its mass in the phospholipid bilayer. In addition, the retinal binding site, where the initial event of photon absorption occurs in the form of the cis-trans isomerization of retinal, is at about the midpoint of the membrane bilayer. Hence, the effect of membrane lipid composition and associated physical properties on the photoactivation of rhodopsin will also be discussed.

!!. R H O D O P S I N STRUCTURE A. General Structure

Bovine opsin consists of 348 amino acids. Hydropathy profile analysis of the amino acid sequence suggests seven transmembrane regions connected on both the cytoplasmic and intradiskal membrane surfaces by hydrophilic loops of varying lengths (Ovchinnikov et al., 1983; Hargrave et al., 1983; Nathans and Hogness, 1983). A variety of physical measurements, including infrared linear dichroism on oriented rod outer segments (Michel-Villaz et al., 1979) and circular dichroism (Shichi and Shelton, 1974; Stubbs and Litman, 1976) have demonstrated that the transmembrane segments are (z-helices, oriented essentially perpendicular to the membrane surface. Palmitoyl acyl chains, covalently bound to Cys 322 and Cys 323, are thought to anchor part of the carboxyl terminal tail to the membrane forming a putative fourth loop consisting of 11 amino acids on the cytoplasmic surface of the rod outer segment disk membrane (Ovchinnikov et al., 1988), as shown in Figure 2. An additional posttranslational covalent modification is found in the amino terminal tail at Asn 2 and Asn 15, where N-linked oligosaccharides are added (Fukuda et al., 1979; Liang et al., 1979). Glycosolation at Asn 15 appears to be required for normal binding and activation of Gt, although blocking glycosolation at Asn 2 has no effect on interaction with G t (Kaushal et al., 1994). Glycosolation does not

BURTON J. LITMAN and DRAKE C. MITCHELL H, Ac-N

-

INTRADISKAL SURFACE

~o~)I

VII

i,. | _ ~.-~ ,lu

Palmitoyl Cysteines

=q_,,U.}7=.o; ~'y2p vY~ ~. N.__)F..~"~. ! " ~ V

T,,~

CYTOPLASMIC

SURFACE

~.~..~.~, T>~ = " ~ o, ~ ~ - "ooc-~.~.~-~=~ "-'° ~:~"~ Regions of Transducin Binding

Figure 2. Model for the topography of rhodopsin in the rod outer segment disk membrane. N-linked oligosaccharides are shown attached to Asn 2 and Asn15. The heavy line connecting Cys 11° and Cys 187 denotes the single disulfide bond found in rhodopsin. Palmitic acid moieties attached to Cys322 and Cys323 anchor part of the carboxyl tail, forming the putative 4th cytoplasmic loop, i4 (from Hargrave et al., 1993). appear to be required for formation of the final, folded structure of opsin. Nonglycosolated opsin is normally palmitoylated, inserts properly into the membrane, and combines with 11-cis retinal to form the characteristic rhodopsin pigment, although it has a diminished ability to activate Gt (Kaushal et al., 1994). Opsin and 11-cis retinal combine via a protonated Schiff base linkage at Lys296 to form rhodopsin (Bownds, 1967; Wang et al., 1980; Mullen and Akhtar, 1981; Findlay et al., 1981). Linear dichroism measurements on oriented ROS show that the retinal transition dipole moment is tilted about 15° with respect to the plane of the disk membrane (Liebman, 1962; Chabre and Breton, 1979; Michel-Villaz et al., 1982). The chromophore is located about equidistant from the cytoplasmic and intradiskal surfaces of the disk membrane, as shown by fluorescence energy transfer measurements, in which a water soluble donor transferred energy to retinal with equal efficiency when placed on either side of the membrane (Thomas and Stryer, 1982). Thermal denaturation studies show that bound retinal confers structural


stability to the final, folded protein structure, as evidenced by the fact that rhodopsin is 43 kcal/mole more stable than opsin (Khan et al., 1991). The basic topography of rhodopsin in the disk membrane, first suggested by a variety of spectroscopic techniques, and later by hydropathy profile analysis of the primary structure, has been refined by a variety of experiments. In intact ROS disks, points of proteolytic cleavage are found in loops i 1, i2, and i3, and in the carboxylterminal sequence. This indicates that these regions are exposed to the cytosol of the rod cell (Trayhurn et al., 1974; Pober and Stryer, 1975; Albert and Litman, 1978; Fung and Hubbell, 1978a,b). Proteolytic cleavage sites in the amino-terminal tail and in loop e 2 are made accessible by freezing and thawing ROS disks, indicating that these regions are exposed to the intradiskal space (Martynov et al., 1983). The sites of lectin binding to the N-linked oligosaccharides in the amino-terminal tail (Rohlich, 1976), antibody binding (Molday and Molday, 1979), and chemical modification (Barclay and Findlay, 1984) are all consistent with the topography deduced from proteolysis experiments. Circular dichroism measurements indicate that rhodopsin tertiary structure is approximately 55% or-helix (Shichi and Shelton, 1974, Stubbs and Litman, 1976), consistent with the transmembrane regions being in the form of or-helices. A series of high resolution FTIR measurements on rhodopsin in ROS disk membranes confirms a helix content of about 50%, and suggests that as much as 15% of this structure is in the form of more extended 310-type helix (Garcia-Quintana et al., 1993). On the basis of FTIR measurements made in 1H20 and 2H20, these authors concluded that 61% of the protein is solvent accessible and most of the inaccessible domains are in the transmembrane helices located in the hydrophobic portion of the bilayer. They also report evidence for [3-strands and reverse turns in the hydrophilic linking regions. The consistent picture which emerges from biochemical and spectroscopic studies is a structure consisting of seven transmembrane or-helices, with the remaining 50% of the structure about evenly divided between the intradiskal and cytoplasmic membrane surfaces.

B. Structural Comparison with Bacteriorhodopsin It has generally been supposed that the three-dimensional structure ofrhodopsin, and the entire superfamily of seven transmembrane helix, G protein-coupled receptors, is similar to that ofbacteriorhodopsin, the light-driven proton pump from Halobacterium halobium, whose three-dimensional structure is known (Henderson et al., 1990). The presumed structural homology between rhodopsin and bacteriorhodopsin springs from several striking similarities between the two proteins. Both have large hydrophobic domains consisting of seven, transmembrane (x-helices, their carboxyl terminal ends are exposed to the cytoplasm and amino terminal ends are exposed extracellularly, and both have chromophores consisting of an isomer of retinal linked via a protonated Schiffbase to a lysine, approximately in the middle of the a-helix nearest the carboxyl terminus. However, there are


significant functional differences between the two proteins. In rhodopsin, retinal isomerization drives the formation of binding sites for transducin and other regulatory enzymes on the cytoplasmic surface, while in bacteriorhodopsin, retinal isomerization is coupled to vectorial proton transport. The bacteriorhodopsin ground state chromophore is in the linear all-trans configuration, while in rhodopsin, it is 11-cis retinal. In rhodopsin, photoisomerization of the chromophore from 11-cis to all-trans retinal ultimately destabilizes the opsin-retinal Schiff base linkage and the chromophore is released by opsin. In contrast, the 13-cis chromophore ofphotoexcited bacteriorhodopsin remains bound and returns to the all-trans configuration via a purely thermally driven process. Bacteriorhodopsin has no significant sequence homology with rhodopsin (Hargrave et al., 1983), or any other member of the G protein-coupled receptor superfamily (Baldwin, 1993). The two-dimensional projection structure of bovine rhodopsin at 9,h. resolution has been obtained by electron crystallography of two-dimensional rhodopsin crystals (Schertler et al., 1993). The density map, shown in Figure 3A, consists of four well defined peaks, which are interpreted as a-helices perpendicular to the membrane, and an arcshaped feature which is interpreted as three somewhat tilted helices. This structure differs from that ofbacteriorhodopsin in several respects, as shown in Figure 3B. At 9.& resolution, only two sharp density peaks are visible in the projection structure of bacteriorhodopsin, indicating that two of the rhodopsin helices are more perpendicular to the plane of the membrane than in bacteriorhodopsin. The cross section of rhodopsin (in the plane of the membrane) is less ellipsoidal and more circular than that of bacteriorhodopsin. The region between the helices, which forms the retinal chromophore binding pocket, is somewhat S-shaped in rhodopsin, while in bacteriorhodopsin it is essentially straight, a

A

B

:0

Figure 3. Projection structure of rhodopsin. A. Projection density map of a single rhodopsin molecule at 9 ,g, resolution. B. Projection density map of a single bacteriorhodopsin molecule at 9 ,g, resolution, using the same scale as in A (from Schertler et al., 1993).


Figure 4. Proposed assignment of the putative transbilayer oc-helices of rhodopsin to the projection structure (adapted from Baldwin, 1993).

probable consequence of ground state rhodopsin having a bent, 11-cis retinal chromophore, while ground state bacteriorhodopsin has a straight all-trans retinal chromophore. Baldwin (1993) has recently proposed an arrangement of the helices for rhodopsin and other G protein-coupled receptors. Following a detailed analysis of the amino acid sequences of 204 G protein-coupled receptors, the structural constraints imposed by sequence homology were used to assign particular hydrophobic stretches in the primary sequence to peaks in the projection structure of Schertler et al. (1993). Based on sequence alignment of the entire G protein-coupled receptor superfamily, three constraints on the arrangement of the helices were determined: (1) successive helices in the sequence must be adjacent in the three dimensional structure, (2) helices I, IV, and V must be most exposed to the lipid bilayer, and (3) helix III must be least exposed to the lipid bilayer. These three constraints along with the requirement for the Schiffbase counterion, Glu ~13(Zhukovsky and Oprian, 1989; Sakmar et al., 1989; Nathans, 1990a), to be near the end of the lysine side chain of Lys 296, lead to a single, best assignment of helices to the projection structure. The resulting assignment of the helices to the two dimensional structure, shown in Figure 4, makes no predictions regarding structure of the hydrophilic loops connecting the helices, but represents the most detailed three-dimensional model for rhodopsin proposed to date.

C. Helix-Linking Loops and Functional Domains Compared to the transmembrane domain, little structural information is available regarding the hydrophilic intradiskal and cytoplasmic regions of rhodopsin. Gt, rhodopsin kinase, arrestin, and phosphatases bind to regions exposed to the cyto-


plasm at various times following photolysis, thus, this surface has attracted more experimental attention than the intradiskal domain. The sites ofphosphorylation by rhodopsin kinase are located in the carboxy terminal sequence. The first and second phosphorylation sites are at Ser338 and Ser343, respectively, and higher levels of phosphorylation occur at Thr335,Thr 336,Thr349, and Thr342 (McDowell et al., 1993; Papac et al., 1993). Most of the structural information regarding the cytoplasmic surface has focused on the requirements for Gt binding and activation; this information will be covered in the section on Rhodopsin-Transducin Interactions. Rhodopsin's single disulfide bond, between Cys 110 and Cys 187, is found on the intradiskal surface (Karnik and Khorana, 1990). Although, this pair of cysteines is conserved in all visual pigments and G-protein coupled receptors, it is not strictly required for formation of functional rhodopsin. Replacement of Cys 11° and Cys 187 by alanine residues results in a properly folded, functional form ofopsin (Davidson et al., 1994), however, replacement of these cysteines with serines prevented proper folding and insertion into the bilayer (Karnik et al., 1988). A detailed mutagenesis study of the loops exposed on the intradiskal surface and the N-terminal tail indicates that this region is necessary for proper folding of opsin (Doi et al., 1990). Deletion of as few as two amino acids in any one of the three intradiskal loops or the N-terminal tail caused mutant opsins expressed in COS-1 cells to remain in the endoplasmic reticulum, indicating that the mutant proteins could not form a properly folded structure. Experiments with proteolyzed rhodopsin have shown that the retinal binding domain, formed by the transmembrane helices, and the cytoplasmic surface of rhodopsin are functionally distinct domains. Treatment of intact ROS disks with papain results in cleavage of the hydrophilic loop i3, which connects helices V and VI. This produces a large fragment, F 1, consisting of the carbohydrate-containing 5 amino terminal helices and a smaller fragment, F2, consisting of the 2 carboxyl terminal helices. These two fragments copurify on Concanavalin A-Sepharose as a tight complex, which is stabilized by helix-helix interactions alone (Albert and Litman, 1978). Proteolysis was found to have no effect on the visible CD spectrum, indicating that the retinal binding site remained intact, while changes in the far-ultraviolet CD spectrum showed that proteolysis reduced the helical content of rhodopsin by only a few percent (Albert and Litman, 1978). In addition, papain proteolysis of loop i3 had very little effect on the MI---~MII transition kinetics (Litman et al., 1982), which is somewhat surprising since retinal is attached to F2, while the Schiffbase counterion, Glu 113(see, sections opsin-chromophore interactions), is in F 1. These results suggest that a basic feature of rhodopsin's structurefunction relationship involves a one-way flow of information from the retinal binding domain out to the cytoplasmic surface. Conformation changes in the retinal binding domain, triggered by retinal isomerization, produce conformation changes at the cytoplasmic surface, which lead to formation of the Gt binding site. In contrast, proteolytic cleavage of the carboxyl terminus and the cytoplasmic loop connecting the F 1 and F2 fragments has little effect on opsin conformation changes


involving the transmembrane helices which form the retinal binding domain (Litman et al., 1982).

III.

RHODOPSIN PHOTOINTERMEDIATES A. Overview

In rhodopsin, nature has provided investigators with a G protein-coupled receptor with an intrinsic reporter of the interactions between the binding pocket of the receptor and both agonist and antagonist ligands. Absorption of a photon by rhodopsin converts 11-cis retinal, the antagonist, to all-trans retinal, the agonist, with a quantum efficiency of 0.67 (Kropf, 1967; Dartnall, 1968). Hubbard and Kropf (1958) identified retinal photoisomerization as the initial event in visual transduction, and this step is functionally equivalent to the binding ofagonist in the liganded G protein-coupled receptors. In rhodopsin, photoisomerization induces a series of charge rearrangements in the retinal binding pocket, which spawn a series of rapid absorption changes known as the photobleaching sequence. Thus, UV/VIS spectroscopy can be utilized to temporally and energetically resolve the initial series of intramolecular conformation changes, which ultimately create the conformation in the cytoplasmic surface which binds G c The sequence ofrhodopsin photointermediates is shown in Figure 5. Blue shifted intermediate (BSI) and metarhodopsin II' (MII') are not observed in low temperature equilibrium studies, but are observed in time-resolved experiments at room temperature. The wavelength maxima and transition times given in Figure 5 are those observed in time resolved measurements for rhodopsin at ambiem temperature (~-20 °C) and neutral pH. Photoproducts such as hypsorhodopsin, which form only under high light intensities or low temperatures, have been excluded.

B. Early Photointermediates Uncertainty persists regarding the exact nature of photorhodopsin, as to whether it is a single species, and whether it can fall back to rhodopsin or must complete the photointermediate cascade (Lewis and Kliger, 1992). One or more red-shifted precursors of bathorhodopsin were observed at low temperatures (Peters et al., 1977) and in a number of ambient temperature kinetic measurements (Monger et al., 1979; Kandori et al., 1989; Yan et al., 1991). Following Lewis and Kliger (1992), the term photorhodopsin is used here to refer to one or more red-absorbing photointermediates, which are detected on the picosecond timescale. Bathorhodopsin (batho) is the first photointermediate which was demonstrated to have an all-trans chromophore (Mathies et al., 1976; Hug et al., 1988). Batho stores about 36 kcal/mol (Cooper, 1979; Boucher and Leblanc, 1985; Shick et al., 1987), or about 60% of the 57 kcal/mol of incident photon energy for photons of

10

BURTON J. LITMAN and DRAKE C. MITCHELL R h o d o p s i n (498)

P h o t o r h o d o p s i n (.--.560) 4 ps B a t h o r h o d o p s i n (540) 35 ns Blue Shifted I n t e r m e d i a t e (477)

+

200 ns

Lumirhodol~sirt (497)

~ M e t a II' (380)

ff

90 ~ts

Meta I (478)

(?) I ms M e t a r h o d o p s i n II (380) -- 5 rain M e t a r h o d o p s i n III (465) ,~

--- 10 rnin

all trans retinal + opsin

Figure 5. The rhodopsin photoreaction cascade. Numbers in parenthesis next to individual species indicate their approximate wavelength of maximum absorbance in nanometers. -limes for transitions between species correspond to conditions of 20-25 °C, pH 7.0 in ROS disk membranes. Transitions involving Lumirhodopsin, Metarhodopsin I, Metarhodopsin II, and Metarhodopsin I1' that have been proposed, but not fully characterized are marked (?).

500 nm wavelength. This energy drives all subsequent transitions, and is stored via distortion of the all-trans chromophore (Birge et al., 1988; Smith et al., 1991). It has been demonstrated in native rhodopsin at room temperature that batho rapidly establishes an equilibrium with BSI (Hug et al., 1990; Lewis et al., 1990; Randall et al., 1991). BSI is higher in both enthalpy and entropy than batho (Hug et al., 1990). Thus, at low temperature, the rate of back reaction to batho is sufficiently large that BSI never accumulates and at room temperature, where the batho ~-~ BSI equilibrium constant is 1.4, decay to lumirhodopsin is fast enough that a spectral peak due to BSI is never observed. A study of BSI in rhodopsin, where retinal was replaced with a variety of retinal analogs, showed that the rate of BSI formation is strongly chromophore dependent, but the rate of BSI decay is largely chromophore independent (Randall et al., 1991). This suggests that the rate


11

limiting step in BSI decay involves a protein relaxation, indicating that the BSI to lumirhodopsin (lumi) transition is the earliest step in the photoreaction cascade that is associated with a protein conformation change. C. Late Photointermediates

In contrast with the events leading up to lumirhodopsin (lumi), the sequence of events following formation of lumi are not as well characterized, as shown by the transitions marked (?) in Figure 5. The 'classical' scheme, based on low-temperature measurements, of lumi decaying to MI, which then forms a quasi-stable equilibrium with MII (Mathews et al., 1963) is unable to account for ambient temperature kinetic measurements conducted with a high level of signal to noise on rhodopsin in disk membrane suspensions (Stewart et al., 1977; Lewis et al., 1981; Straume et al., 1990; Thorgeirsson et al., 1992, 1993). To adequately describe absorbance changes on the time scale of lumi, MI, and MII, ambient temperature kinetic measurements require three or more discrete exponential decays, rather than the two exponential decays predicted by the 'classical' scheme. The kinetic behavior of photointermediates, which occur on the submicrosecond timescale, is identical for rhodopsin in ROS disk membranes or solubilized in detergent, however all transitions beyond lumi are dramatically accelerated in detergents (Applebury et al., 1974, Litman et al., 1981). Artifacts due to light scattering, that are inherent in UV/VIS measurements on membrane suspensions, have made it difficult to acquire data of sufficient signal-to-noise to test detailed photoreaction models. A series of recent investigations utilizing multichannel detection with microsecond time resolution makes it clear that the kinetic complexity observed on the lumi to MII timescale is due to a decay product of lumi, which is isochromic with MII (Thorgeirsson et al., 1992, 1993). The existence of two, isochromic species of MII was previously suggested in studies ofMI ~-~ MII kinetics (Hoffmann et al., 1978; Straume et al., 1990), pH dependence (Parkes and Liebman, 1984), and the kinetics of proton uptake by photoactivated rhodopsin (Arnis and Hofmann, 1993). The work by Kliger and coworkers indicates that the fastest absorbance increase at 380 nm is due to a photointermediate with an absorbance maximum at 380 nm, which forms directly from lumi (Thorgeirsson et al., 1992, 1993). They have proposed several kinetic models to link the new intermediate, designated MII' in Figure 5, with lumi, MI, and MII. The decay scheme beyond lumi, shown in Figure 5, summarizes the essential features of the models which have been tested to date, and points out the transitions which are still unresolved. A definitive model, which accounts for both the kinetics and the observed equilibrium between 480 nm and 380 nm absorbing species, over a range of temperatures and pH values, has not been unambiguously determined. The sequence of events beyond MII is well understood in terms of the scheme shown in Figure 5. Below pH 7.7, MII can decay either to MIII or directly to opsin + retinal387 (Blazynski and Ostroy, 1984). Stabilization of MII by G t decreases

12


formation of MIII from MII, and favors decay of MII to opsin + retinal via a pathway which bypasses Mill (Pfister et al., 1983; Hofmann et al., 1983). An equilibrium between MII and Mill was first suggested to account for linear dichroism measurements of the slow intermediates (Chabre and Breton, 1979) and is supported by kinetic measurements of MII decay in the presence of G t (Pfister et al., 1983; Hofman et al., 1983). The equilibrium between MII and MIII was directly demonstrated by showing that photolyzed rhodopsin, which appeared to have decayed to MIII, could be converted into a mixture of MII and MIII by the addition of G t (Kibelbek et al., 1991). Complex formation between MII and G t is well documented (see Rhodopsin-Transducin Interactions) and the formation of the MII.G t complex shifted the equilibrium, which strongly favors Mill, towards MII. At neutral pH, MIII decays to opsin + retinal (Blazynski and Ostroy, 1984).

D. Structural Changes in MI! MII is considered the most important photointermediate species because of its identification with R* (Emeis et al., 1982; Bennett et al., 1982; Kibelbek et al., 1991). A wide variety of physical measurements show that MII is the most structurally, as well as functionally, unique rhodopsin photointermediate. In MII, the Schiff base linking the retinal chromophore to tys 296 becomes deprotonated (Doukas et al., 1978), and in the native protein this deprotonation is required for formation of MII (Longstaff et al., 1986). The first evidence for conformational changes in the MI-MII transition was the susceptibility of the retinal chromophore in MII to attack by hydroxylamine (Falk and Fatt, 1966) and the reduction of the Schiffbase by sodium borohydride (Akhtar et al., 1965). The MI-MII equilibrium is pressure dependent, with high pressure favoring MI (Lamola et al., 1974; Attwood & Gutfreund, 1980). In purified ROS disks, the pressure dependence was used to calculate a reaction volume of 108 ml/mol for the MI to MII transition (Attwood and Guffreund, 1980). Several unique structural features of MII have been elucidated by a number of investigations utilizing FTIR difference spectroscopy. MII appears to have a more open, less rigid structure than MI, and appears to return to a more rhodopsin-like structure during the transition to Mill (DeGrip et al., 1985, Rothschild et al., 1987). The result is that MII has a unique peptide backbone conformation, resulting from a few peptide bonds undergoing a secondary structural rearrangement (Rothschild et al., 1987; Klinger and Braiman, 1992). This structural rearrangement and the expansion of MII relative to MI is consistent with a small movement of helices IV and VI towards the cytoplasmic surface during the MI to MII transition (Pellicone et al., 1985). Acid pH favors MII over MI (Mathews et al., 1963) and photolyzed rhodopsin was reported to take up protons from the aqueous medium with kinetics similar to the formation ofMII (Wong and Ostroy, 1973). Amore recent study of the kinetics of proton uptake and MII formation for rhodopsin in octyl glucoside and dodecyl


13

maltoside demonstrates that proton uptake from the aqueous medium occurs after deprotonation of the retinal Schiffbase and formation ofMII (Arnis and Hofmann, 1993). The authors postulate that this proton-dependent transition from MII a to MII b corresponds to formation of the loop conformation on the cytoplasmic surface which binds G t. Conformation changes in the cytoplasmic loops in MII have been observed by a variety of spectroscopic techniques. A comparison of the FTIR spectra of proteolyzed and unmodified rhodopsin showed that loops i2 and i 3 adopt a well-defined conformation in rhodopsin, but become more random and flexible in MII (Ganter et al., 1992). Conformation changes in loops i2 and i4 have been directly observed using nitroxide spin labels attached to cysteine residues at positions 140 in loop i2 and 316 in loop i4. Formation of MI produced no changes in the EPR spectra of these two attached nitroxides, while formation of MII produced significant changes in their EPR spectra (Resek et al., 1993). Conformational changes in loop i2 in MII were also indicated by a time-resolved EPR study which detected conformational changes at Cys 14° with a time constant and activation energy consistent with MII formation (Farahbakhsh et al., 1993).

IV. OPSIN-CHROMOPHORE INTERACTIONS Interactions between opsin and the retinal chromophore may be divided into factors which modulate the static, or ground state, spectrum of rhodopsin, and those that modulate the dynamic, or post-photolysis, spectral characteristics. The static absorption spectrum ofrhodopsin is determined by the unique electronic environment that opsin provides for the retinal chromophore. In ground state bovine rhodopsin, the opsin-chromophore interactions lead to an opsin-induced spectral shift of 58 nm to the red, from the benchmark of 440 nm for a protonated retinylidene Schiff base in methanol (Blatz et al., 1972), to a maximum absorbance at 498 nm. The specific amino acids responsible for this opsin shift have not been identified. In recent years site-directed mutagenesis has been used to gain much information about interactions between specific amino acids and the retinal chromophore. Several charged amino acids have been eliminated from consideration by an extensive site-directed mutagenesis study. Six amino acids from helices II, III, or IV of bovine rhodopsin, Asp 83, Met 86, G1u 122, GIH134, Arg 135, and His 211, were altered by a total of 14 different mutations (Nathans, 1990b). All of the mutant proteins had an absorbance maximum near 498 nm, indicating that none of these six charged residues contribute to the opsin shift. The central question of rhodopsin's structure-function relationship is the mechanism by which retinal isomerization in the interior of the protein triggers conformational changes on the cytoplasmic surface. This process necessarily begins with dynamic, or transitory, interactions between retinal and the amino acid side chains which make up the retinal binding pocket. Thus, knowledge of the identity and disposition of the amino acids, which interact directly with retinal, is necessary to

14


understanding the molecular mechanism which couples retinal isomerization to conformation changes of the entire protein. A major advance in this area is the identification of G l u 133 as the retinylidene Schiff base counterion (Sakmar et al., 1989; Zhukovsky and Oprian, 1989; Nathans, 1990b). This determination principally rests on the fact that replacement of Glu 133with Gln causes a blue-shift of the chromophore spectrum by 120 nm to a maximum at 380 nm and, lowers the pK of the Schiff base to such an extent that at neutral pH, the Schiff base nitrogen is no longer protonated (Sakmar et al., 1989; Zhukovsky and Oprian, 1989). Robinson et al. (1992) proposed that rhodopsin with an 11-cis chromophore is constrained in an inactive conformation, unable to bind and activate transducin, by a salt bridge between the retinylidene Schiffbase nitrogen and Glu 113.They reasoned that retinal isomerization moves the Schiff base nitrogen away from Glu 113, breaking a salt bridge, and thus allowing deprotonation of the retinylidene Schiff base nitrogen and adoption of the active MII conformation. The issue of whether or not disruption of the Schiffbase proton, Glu 113charge pair is required for formation of R* will be discussed further in the Rhodopsin-Transducin Interactions section. In one, detailed study, a series of rhodopsin mutants, each with a single amino acid replacement, was tested for their ability to bind 11-cis retinal, generate chromophore with a native-like absorption spectrum, and activate G t (Nakayama and Khorana, 1991). The results of this study, along with those of an earlier study using a photoactivatible analog of retinal (Nakayama and Khorana, 1990), indicated that Glu 122,Trp 126,Trp265and Tyr268interact strongly with the retinal chromophore. In the structure proposed by Baldwin (1993) Glu 122and Tyr268 face the intrahelical cleft, which forms the retinal binding pocket, and Trp 265 is positioned in a way that would allow it to form part of the pocket for the [3-ionone ring, as suggested by Nakayama and Khorana (1991). Two mutations, D83N and W 161L, had no effect on the absorption spectnnn of unbleached rhodopsin, suggesting that Asp 83 and Trp 161 have no interaction with the retinal chromophore, and in the proposed structure these two residues do not face the intrahelical cleft; Asp 83 faces helix I and Trp161 faces helix III. In a second study, each of rhodopsin's six histidine residues was replaced one at a time by phenylalanine and/or cysteine (Weitz and Nathans, 1992). All of the resulting mutant proteins formed pigments with normal ground state spectral properties, but following photolysis the mutants H211F and H211C were unable to form the photointermediate MII. Photobleaching of these two mutants produced only MI at pH 5.8 and pH 7.8. The authors proposed that protonation of His 211 destabilizes the ionic interaction between the protonated Schiffbase of retinal and its counterion, Glu 113,thereby favoring formation of MII. However, in Baldwin's (1993) proposed rhodopsin structure His 211in helix IV is more than a helix diameter away from Glu 113 in helix III. Experiments in which the retinylidene Schiffbase was methylated, and thereby made unable to undergo deprotonation, demonstrated that deprotonation of the retinylidene Schiffbase is required for formation of MII and binding of Gt in native


15

rhodopsin (Longstaff et al., 1986). Thus, Schiff base deprotonation is directly involved in coupling retinal isomerization to overall protein conformational changes, and underlines the importance of identifying the amino acid which acts as an acceptor for the Schiff base proton. The identity of this residue has not been unambiguously determined. However, a comparison of the FTIR spectra of native rhodopsin and the site-directed mutants D83N, E 122Q, and D83N/E 122Q lead to the suggestion that Glu 113, which serves as the Schiffbase counterion, is also the acceptor for the net proton transfer from the Schiff base to opsin in MII, possibly with a water molecule acting as an intermediary (Fahmy et al., 1993). Recent site-directed mutagenesis studies show that Schiff base deprotonation is structurally distinct from formation of R* (Zvyaga et al., 1993, 1994). The Schiff base counterion was moved one helix turn by construction of the triple replacement mutant G 113A/A 117G/G 122N. The resulting mutant rhodopsin formed native-like pigment, and in response to light activated G t and formed MI and MII in a quasistable equilibrium. However, the pH dependence of the MR--~MII equilibrium was reversed, with alkaline pH favoring MII (Zvyaga et al., 1993). The altered pH dependence was interpreted as demonstrating that movement of the counterion had uncoupled retinylidene Schiffbase deprotonation from the influence of one or more histidines. These results suggest that while a specific acceptor for the retinylidene Schiffbase proton probably controls the transition from MI to MII in the presence of the Glu 113 counterion, it is not strictly required for MII formation when the counterion is absent.

V. RHODOPSIN-TRANSDUCIN INTERACTIONS Several independent lines of evidence associate the Gt-activating conformation of photoactivated rhodopsin, R*, with the photointermediate MII. These include the following: (1) when rhodopsin is bleached in the presence of G t and the absence of GTP, formation of an excess of MII is observed relative to that amount of MII that would be present in the absence of G t. The excess MII is proportional to the amount of added G t and the effect saturates at a 1:1 ratio of bleached rhodopsin t o G t (Emeis et al., 1982). (2) IfG t is added to a bleached rhodopsin sample, which has progressed to the MIII photointermediate, then the presence of a MII spectral component is observed (Kibelbek et al., 1991). The preceding observations can be attributed to the formation of a stable MII.G t complex in the absence of GTP, which has an absorption spectra identical to that of uncomplexed MII, and is thus observed spectrally as an additional MII contribution. Monitoring the amount of excess MII as a function of Gt, concentration allows one to determine the binding constant of the MII.G t complex (Kahlert and Hofmann, 1991). (3) The spectrophotometric decay of MII was found to occur with essentially the same decay constant as the loss of Gt-activating activity associated with R*, strongly identifying R* with MII (Kibelbek et al., 1991). Given the preceding evidence, MII is generally assumed to be synonymous with R*.

16


In the previous section on opsin-chromophore imeractions, those structural features important for MII formation were discussed. The initial stage of signal amplification in the visual transduction pathway is the interaction of MII with G t to form a MII.G t complex (Figure 1). Interactions of G t with MII occur on the cytosolic surface of the disk membrane, where G t is bound as a peripheral protein. The cytosolic surface ofrhodopsin is formed from the four hydrophilic loops il, i2, and i3 connecting helices I and II, IV and V, and VI and VII, respectively, and 14, the putative fourth loop in the carboxyl terminus formed by the palmitoyl chains at Cys 322 and Cys 323 partitioning into the lipid of the disk membrane, as depicted in Figure 2. Various segments of the cytosolic surface of rhodopsin have been identified as being involved in G t interactions. Limited proteolysis experiments, which removed segments of the carboxyl terminus beyond the putative i4 loop, had little effect on G t binding to the disk membrane (KOhn and Hargrave, 1981). Similar limited proteolysis was reported to produce a 40% increase of PDE activity, presumably as the result of more efficient G t activation (Aton and Litman, 1984). Phosphorylation of a number of ser and thr residues in this region is thought to be responsible for the down regulation ofrhodopsin's receptor function (Miller et al., 1986; Wilden et al., 1986). G t binding studies demonstrate that the affinity of MII for G t decreases with increasing phosphorylation level, while the amount of MII changes very little (Mitchell et al., 1992a). Hence, the region of the carboxyl terminus beyond the putative i4 loop appears to sterically hinder G t binding and when phosphorylated, plays a major role in down regulating G t binding. Small peptides simulating the sequence of regions of the i2, i3, and i4 loops were found to reduce G t binding to MII, suggesting that these loops are involved in the formation of the G t binding site (Konig et al., 1989). Studies of a variety of mutant rhodopsins, having both single amino acid mutations and sequence deletions or replacements, have further identified several amino acid residues and hydrophilic loops as sites of G t interaction. Franke et al. (1990) examined three mutants, all of which form pigments with native-like absorption spectra, but differed greatly in their interaction with G t. When residues 140-152 in loop i 2 were replaced with a sequence of residues from the amino terminus, G t binding was detected, but no light stimulated GTPase activity, which is an indirect measure of G t activation, was observed. A deletion of residues 237-249 in i 3 also showed G t binding without activation. Residues Glu TM and Arg 135, Which form a charge pair at the hydrophilic interface of helix III, appear to be critical for G t binding. When the order of these residues is changed to Arg TM and Glu 135, no G t binding was observed. Replacement of these two amino acids by Val also resulted in a lack of G t activation (Franke et al., 1992). These studies show that G t binding and activation represent separate steps involving different segments in the rhodopsin structure and suggest that the charged pair, Glu TM and Arg 135, is necessary for G t binding, while loops i2 and i3 are required for G t activation.


17

One of the most interesting results obtained from mutagenesis experiments was the observation of a constitutively active rhodopsin molecule in the absence of any bound retinal (Robinson et al., 1992). In these experiments, mutants, which prevented the formation of the charge pair between the protonated Schiff base at the Lys 296 retinal attachment site and Glu 113, were expressed. An El l3Q mutant was produced, which recombined with a propylamine derivative of vitamin A aldehyde to form a bleachable pigment capable of activating G r The striking finding was that in the absence of any bound retinal the protein was still capable of activating G t. A K296G mutant showed the same properties as the E 113Q mutant. These observations led to a proposal that the charge pair formed by Glu 113 and the protonated Schiff base restricted the formation of the active conformation of rhodopsin. After the release of retinal, a charge pair between Glu 113 and Lys 296 fulfilled this role. Removal of the constraint imposed by the charge pair in the mutants allowed the formation of a constitutively active opsin. Although removal of the Schiff base proton during the activation step appears to be required in rhodospins which can form a charge pair, it may not be necessary when the charge pair is not formed. A mutagenesis experiment, in which the double mutant E 113A/A117E was formed, shows that a MI-like photointermediate, whose spectrum indicates the presence of a protonated Schiffbase, is capable of activating G t (Zvyaga et al., 1994). It would appear that the lack of charge pair formation, negates the need to deprotonate the Schiff base in order to form a Gt-activating conformation.

VI.

R H O D O P S I N - L I P I D INTERACTIONS A. Disk Membrane Lipid Composition

The native disk membrane is relatively simple from a compositional perspective. Approximately 90-95% of the integral membrane protein of the disk is rhodopsin (Papermaster and Dreyer, 1974; Smi.th et al., 1975). The lipid composition consists primarily of about 42% phosphatidylethanolamine (PE), 40% phosphatidylcholine (PC), 16% phosphatidylserine (PS), some minor amounts of phosphatidylinositol, and 12-14 mol% cholesterol (Anderson et al., 1976; Stone et al., 1979). The amino-containing lipids are thought to be preferentially distributed towards the cytoplasmic surface of the disk (Litman, 1982). Cell membranes in the retina, as well as those of other neuronal tissue, are unique in their high content of polyunsaturated phospholipid acyl chains (Salem, 1989). In most of these cells, the dominant polyunsaturate is 20:4n6, which is confined primarily to PE and PS. The disk membrane has about a 75/1 phospholipid to rhodopsin ratio (Stubbs and Litman, 1978) and is exceptional in that approximately 47 mol% of its phospholipid acyl chains are derived from docosahexaenoic (22:6n3) acid, with about 16%, 20%, 5% and 3.6 mol% of 16:0, 18:0, 18" ln9, and 20:4n6 respectively (Stone et al., 1979). In addition, the 22:6n3 is prevalent in all three major phospholipid classes.

18


Consistent with the high level of acyl chain unsaturation, Cone and coworkers determined rhodopsin to have a high degree of both rotational (Cone, 1972) and translational (Poo and Cone, 1974) freedom, indicating that the disk membrane is a prime example of the Singer-Nicholson fluid mosaic membrane (Singer and Nicholson, 1972).

B. Lipid Modulation of Mll Formation Early studies ofrhodopsin photochemistry demonstrated that MII formation was dependent on both the presence and type of lipid associated with rhodopsin. In delipidated ROS membranes, photolysis of rhodopsin did not progress beyond the MI photointermediate (Applebury et al., 1974). MII formation was also found to be faster in more unsaturated PCs than in disaturated PCs (O'Brien et al., 1977; Mitchell et al., 1992b). The rate of meta II formation increased dramatically when disk membranes were detergent solubilized and was shown to be dependent on the amount ofphospholipid in the rhodopsin-containing micelles (Litman et al., 1981). More recent studies, in which the MI-MII equilibrium constant, Keq, was measured, show that the amount of MII formed is increased by higher levels of acyl chain unsaturation (Wiedmann et al., 1988; Mitchell et al., 1992b; Gibson and Brown, 1993) and decreased by increased bilayer cholesterol contem (Mitchell et al., 1990). Early reports of the lack of MII formation in disaturated PCs led to the speculation that acyl chain unsaturation was a requirement for meta II formation (Baldwin and Hubbell, 1985a,b). Subsequently, it was shown that MII not only formed in di- 14:0 PC (DMPC), but was capable of activating Gt (Mitchell et al., 1991). Thus, it would appear that no requirement of unsaturation exists for either MII formation or subsequent Gt activation. The question then remains as to the role of the preponderance of 22:6 acyl chains in the disk membrane phospholipids and what special properties are imparted to membranes by these polyunsaturated phospholipids. Several properties of phospholipids, related to both head group and acyl chain content, have been examined relative to their ability to influence the function of integral membrane proteins. Head group effects are in general associated with either a surface potential imparted by charged lipids or the force exerted on a membrane protein, when phospholipids such as PE, which have a propensity to form an inverted tubular hexagonal type II (Hn) phase, are confined to a planar bilayer (Gruner, 1985), whereas acyl chain effects are expressed as variation in membrane thickness (Moufitsen and Bloom, 1984) and acyl chain packing free volume (Mitchell et al., 1990, 1992b).

Phospholipid Head Groups In mixtures of 16:0, 18:1 PC (POPC) and POPE, the Keq for MII formation increases as a function of the mole fraction of POPE (Gibson and Brown, 1993; Mone and Litman, 1991). At 37 ° C, Keq increases by about 60% in a 1:1 POPCPOPE relative to pure POPC in reconstituted vesicles (Mone and Litman, 1991).


19

Whether this effect is related to the tendency of PE to form HII phase remains to be determined. The presence of PS also enhances the formation of MII (Gibson and Brown, 1991; Mone and Litman, 1991). In a 1:1 POPS-POPC mixture at 37 ° C, Keq increased by about 4 fold, relative to a pure POPC vesicle system (Mone and Litman, 1991). The influence on Keq of 16:0, 18:1 phosphatidic acid, another negatively charged phospholipid, was similar to that of POPS. The effect of PS on Keq is explained by the predicted effect of varying the vesicle surface potential on the pK a of an amino acid side chain of rhodopsin, which is critical with respect to the MI to MII conversion (Mone and Litman, unpublished). Thus studies incorporating PE into PC bilayers suggest that the presence ofphospholipids with a strong tendency to form an H H phase in the bilayer may influence protein conformation, while studies incorporating PS into PC bilayers clearly show that vesicle surface potential can have a marked effect on protein conformation. By affecting the level of activated receptor formed, both these membrane properties can strongly influence the level of function in a signalling pathway.

Phospholipid Acyl Chains The best correlation between PC acyl chain composition and MII formation obtained to date is with a parameter referred to as the fractional volume, fv; this is derived from the dynamic fluorescence anisotropy properties of the hydrophobic probe, DPH (1,6-diphenyl-l,3,5-hexatriene) (Straume and Litman, 1987a,b). F v was used to characterize the relative acyl chain packing free volume of a variety of lipid bilayers. Acyl chain packing free volume is related to bilayer compressibility and appears to be a primary factor in modulating conformational changes of integral membrane proteins. F v relates to phospholipid acyl chain packing properties in the following way. In an ordered ensemble ofphospholipid molecules, the tight packing of the acyl chains limits the solid angle over which a free tumbling probe, such as DPH, can distribute; this is typical of the gel state. However, when the chains are more disordered, such as in the liquid crystalline phase, DPH molecules can distribute over a much larger solid angle. Fractional volume is defined as the volume over which DPH can distribute in the anisotropic structure of the bilayer relative to the volume it distributes over in an isotropic medium such as a liquid hydrocarbon (Straume and Litman, 1987a,b). In phospholipid bilayers in the liquid crystalline phase, fv was found to increase with increasing temperature (Figure 6A), and decrease with increasing cholesterol content (Figure 6B), reflecting the temperature-induced disordering and cholesterol-induced ordering of the acyl chains, observed by other physical techniques. F v also increases going from di(14:0)PC to di(20:4)PC (Figure 6C). This indicates that higher levels of polyunsaturation result in decreased restriction of the angular distribution of DPH, which is associated with increased acyl chain packing free volume. This is in agreement with the weaker interactions between acyl chains of polyunsaturated phospholipids observed in differential scanning calorimetry

20


B

0.16 -

0.14 -

rv 0.12 -

0.10 -

0.08

I

10

20

30

40

Temperature

(°C)

0

10

20

Mol% cholesterol

30

I

di(14:0)

I

I

16:0,22:6

16:0,18:1

di(20:4)

Figure 6. Illustration of changes in fractional volume, fv, in response to factors known to modify phospholipid acyl chain packing properties. A. Temperature vs. fv for rhodopsin-containing (16:0,22:6)PC (PDPC) vesicles. B. Bilayer mol% cholesterol vs. fv at 37 °C for rhodopsin-containing egg PC vesicles. C. Acyl chain composition vs. fv at 37 °C. Data in A, B, and C. (DMPC only) are taken from Mitchell et al., 1992b. The remaining data in C is unpublished data of Mitchell and Litman.

(Keough and Kariel, 1987) and bilayer compressibility (Needham and Nunn, 1990) measurements. The temperature dependence of Keq for the MI-MII equilibrium and fv were determined in parallel spectrophotometric and fluorescence measurements made on reconstituted vesicles containing approximately a 100/1 ratio of phospholipid to rhodopsin. A linear relationship was found between Keq and fv for each lipid bilayer examined. The slope of these correlation lines were found to increase with increasing degree of sn-2 unsaturation (Figure 7A). The slopes of the correlation lines for 16:0, 20:4 PC (PAPC) and 16:0, 22:6n3 PC (PDPC) are 18 and 41 respectively, demonstrating that the more polyunsaturated PC has a 2.3 fold greater propensity to promote the formation of MII. These results demonstrate that phospholipids containing polyunsaturated acyl chains have an enhanced ability to Utilize acyl chain packing free volume to promote the formation ofMII. PAPC and PDPC are reported to have very similar hydrophobic thickness (Mclntosh and Brown, unpublished). Therefore, this observation indicates that membrane protein conformational equilibria can be modulated by variation of acyl chain unsaturation under conditions of constant hydrophobic thickness. The experiments summarized in


21

Figure 7 demonstrate that the extent of MII formation shows very good correlation with the increase in acyl chain packing free volume induced by increasing levels of unsaturation in the sn-2 chain.

A Ke q

4 3

1.0

B

Koq 0.5

0.0

0.00

0.05

0.10

0.15

fv-fv ° Figure 7. The MI MII equilibrium constant, Keq, as a function of the normalized fractional volume, fv - f v°. The parameter f v° is fv intercept value of the Keq vs fv correlation line. Subtraction of fv ° shifts all lines to a common origin, making a comparison of the variation in slopes more direct. A. Effect of phospholipid acyl chain composition for rhodopsin in di(14:0)PC (DMPC)(o), egg PC (rq), (16:0, 20:4)PC (PAPC) (A), (16:0, 22:6)PC (PDPC) (,), di(20:4)PC (DAPC) (o), and di(22:6)PC (DDPC) (m) vesicles at a rhodopsin to lipid ratio of 100/1. All points resulted from measurements 10, 20, 30, and 37 °C, except for DMPC which was studied at 26, 30, 37, and 45 °C. B. Effect of bilayer cholesterol for 30 mol% cholesterol in (16:0, 20:4)PC (o), 15 mol% cholesterol in egg PC (m), and 30 mol% cholesterol in egg PC (A). Solid lines are the correlation lines due to variation of temperature for rhodopsin in the host phospholipid, as shown in A. Panel A is reproduced from Litman and Mitchell (1996), data in panel B is from Mitchell et al., 1992b.

22


Cholesterol Cholesterol is one of the most studied molecules in lipid bilayers. The dependence of Keq and fv on bilayer cholesterol content in egg PC and PAPC is shown in Figure 7B. The Keq, fv data points are for the cholesterol-containing systems and lie on the correlation lines obtained for cholesterol-free egg PC-and PAPC by temperature variation. Thus, the cholesterol-induced decrease in fv induces a proportional decrease in Keq, and the extent of change in Keq for an incremental decrease in fv is determined by the properties of the host phospholipid. These data demonstrate that cholesterol modulates membrane properties by decreasing acyl chain packing free volume within the constraints imposed by the acyl chain composition. In PC systems, the acyl chain composition appears to be the primary factor in determining the bilayer physical properties, which regulate MII formation (Mitchell et al., 1992b). The lipid bilayer serves as the solvating medium for integral membrane proteins and will therefore determine the conformational degrees of freedom available to these proteins. The conversion of MI to MII has associated with it a positive volume change of 100cc/mole (Attwood and Gutfreund, 1980). Cholesterol, which has been shown to decrease acyl chain packing free volume, decreases the amount of MII formed relative to a cholesterol free membrane. Hence, access to conformations with larger volumes is not favored by factors which reduce acyl chain packing free volume. Given that denatured structures are also associated with larger volume conformations, one would predict that cholesterol would increase the thermal stability of an integral membrane protein by stabilizing the native conformation. In experiments where 30% cholesterol was incorporated into egg PC vesicles containing rhodopsin, cholesterol induced a shift in the T m for rhodopsin from 68 ° C to 71.6 ° C (Mone and Litman, 1990). Thus, factors which are favorable to the stabilization of a protein, do not necessarily promote the function of the protein, which may require a certain degree of conformational flexibility to form its active state. The properties of the lipid bilayer must be optimized to promote both protein stability and function.

C. Membrane Lipid Domain Model The Keq, fv correlation data can be explained by the model shown in Figure 8. Raman vibrational spectroscopy data indicate a propensity for phospholipids, which contain a saturated sn-1 chain and a polyunsaturated sn-2 chain, to form microdomains as the level of unsaturation in the sn-2 position increases (Litman et al., 1991). This tendency is driven by the stronger sn-1, sn-1 saturated chain interactions relative to the weaker sn-2, sn-2 polyunsaturated chain interactions. As a result of these interactions sn-1 chains are oriented towards the interior of the domains and preferentially interact with one another rather than with the sn-2 chains. Rhodopsin and its surrounding layer of phospholipids can be viewed as an independent domain within the membrane.

/ sn'l

t

b D

(

CDq3~

D

Figure 8. Top view of the microdomain model for acyl chain packing in a polyunsaturated phospholipid bilayer containing rhodopsin. The small ellipses represent PC molecules in which the sn-1 chain and sn-2 chain are designated by the filled and unfilled sectors, respectively. In the liquid crystalline phase, the domains have a characteristic lifetime and size, which can be influenced by the partitioning of lipid soluble molecules, such as cholesterol, into the bilayer. Rhodopsin is shown surrounded by a rapidly exchanging boundary layer of lipid, creating a protein-containing domain. The sn-1 chains are oriented towards the tightly packed interior of the domain, while the sn-2 chains are at the domain boundary and determine the lateral compressibility properties of the bilayer. 23

24


Domain-domain interactions depend on the nature of the sn-2 chain and will strongly influence the lateral compressibility of the membrane. If some portion of the volume expansion associated with the MI to MII conversion is associated with the conformation change in rhodopsin, then the magnitude of Keq will depend on the membrane compressibility. As such, the slope of t h e Keq - fv correlation lines will depend on the acyl chain at the sn-2 position, which is in good agreement with experimental observations. Cholesterol shows a preferential interaction with the saturated sn- 1 chain relative to the polyunsaturated sn-2 chain. This is demonstrated by observations that cholesterol has little effect on either the enthalpy of the gel to liquid cr3/stalline phase transition of dipolyunsaturated lipids (Kariel et al., 1991) or the compressibility modulus ofdi 20:4 PC (Needham and Nunn, 1990). In our model, cholesterol is expected to partition into the interior of the microdomain so as to interact with the saturated sn-1 chain, where it can effect the stability, size, and volume of the domains, thus altering the physical properties of the bilayer. However, if the basic domain structure persists, the lateral packing properties of the system will continue to depend on the sn-2 chain interactions. Cholesterol is observed to shift the system along the correlation line determined by the sn-2 acyl chain, in good agreement with the model.

VII. CONCLUSIONS The unique ability to monitor the conformation changes associated with the functional activation of rhodopsin provide an insight into the forces modulating this process. The extent of formation of MII is influenced by both intramolecular and intermolecular interactions. G l u 113 appears to form a salt bridge with the protonated Schiff base in unbleached rhodopsin and with Lys296 in retinal-free opsin. This salt bridge prevents formation of the active conformation of opsin. In mutagenesis experiments, changing either G l u 113 o r Lys296 so as to prevent formation of the salt bridge was found to produce a constituitively active form of opsin, which can activate Gt in the absence of retinal. The role of photoisomerization of retinal is to induce a conformation change in the retinal binding domain of opsin, which leads to the loss of the Schiffbase proton and disruption of the salt bridge, resulting in formation of MII. These findings demonstrate the importance of intramolecular charge interactions in regulating function in this receptor superfamily. Among intermolecular forces, compositionally determined acyl chain packing properties were found to play a major role in modulating MII formation. The ROS disk membrane phospholipid acyl chain composition is approximately 50% 22:6n3. MII formation was found to increase with increasing phospholipid polyunsaturation. An explanation of the cholesterol and phospholipid acyl chain dependence of MII formation is found in a model of membrane lipid packing. This model suggests that lipids in biological membranes pack in lateral microdomains, whose formation


25

is driven by the interaction of saturated sn-1 chains, which is stronger than the interaction of polyunsaturated sn-2 chains. It is significant that the vast majority of the molecular diversity of naturally occurring phospholipids is a result of substitution at the sn-2 position, whereas the sn-1 position shows little heterogeneity and is generally either 16:0 or 18:0. The critical nature of the high content of 22:6n3 acyl chains in ROS disk phospholipids is demonstrated in experiments to determine the effects of a deficiency in this acyl chain. In newborn rhesus monkeys, whose mothers were maintained on an 18:3n3 deficient diet, plasma 18:3n3 was absent and relative to controls, plasma 22:6n3 fell to about 9% in 8 weeks and 6% after 12 weeks. The visual acuity, compared to control infants, fell to about one-half in the 8 to 12 week time period (Neuringer et al., 1984). The role of the 22:6n3 acyl chains appears to be structural, in that higher levels of polyunsaturation promote a higher yield of MII, which would produce a higher level of activity in the visual transduction pathway.

REFERENCES Akhtar, M., Blosse, R T., & Dewhurst, P. B. (1965). The reduction of a rhodopsin derivative. Life Sci. 4, 1221-1226. Applebury, M. L., Zuckerman, D. M., Lamola, A. A., & Jovin, T. M. (1974). Rhodopsin. Purification and recombination with phospholipids assayed by the metarhodopsin I leads to metarhodopsin II transition. Biochemistry 13, 3448-3458. Albert, A. D., & Litman, B. J. (1978). Independent structural domains in the membrane protein bovine rhodopsin. Biochemistry 17, 3893-3900. Anderson, R. E., Landis, D. J., & Dudley, P. A. (1976). Essential fatty acid deficiency and renewal of rod outer segments in the albino rat. Inv. Ophthal. 15, 232-236. Amis, S., & Hofmann, K. P. (1993). Two different forms ofmetarhodopsin II: Schiffbase deprotonation precedes proton uptake and signaling state. Proc. Natl. Acad. Sci. USA 90, 7849-7853. Aton, B., & Litman, B. J. (1984). Activation of rod outer segment phosphodiesterase by enzymatically altered rhodopsin: a regulatory role for the carboxyl terminus of rhodopsin. Exp. Eye Res. 38, 547-559. Attwood, P. V., & Guffreund, H. (1980). The application of pressure relaxation to the study of the equilibrium metarhodopsin I and II from bovine retinas. FEBS Lett. 119, 323-326. Baldwin, J. (1993). The probable arrangement of the helices in G protein-coupled receptors. EMBO J.I 4, 1693-1703. Baldwin, P. A., & Hubbell, W. L. (1985a). Effects of lipid environment on the light-induced conformational changes of rhodopsin 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholine recombinant membranes. Biochemistry 24, 2624-2632. Baldwin, P. A., & Hubbell, W. L. (1985b). Effects of lipid environment on the light-induced conformational changes of rhodopsin. 2. Roles of lipid chain length, unsamration, and phase state. Biochemistry 24, 2633-2639. Barclay, P. L., & Findlay, J. B. (1984). Labelling of the cytoplasmic domains of bovine rhodopsin with hydrophilic chemical probes. Biochem. J. 220, 75-84. Bennett, N., Michel-Villaz, M., & Kfihn, H. (1982). Light-induced interaction between rhodopsin and the GTP-binding protein. Metarhodopsin II is the major photoproduct involved. E. J. Biochem. 127, 97-103.

26


Birge, R. R., Einterz, C. M., Knapp, H. M., & Murray, L. E (1988). The nature of the primary photochemical events in rhodopsin and isorhodopsin. Biophys. J. 53, 367-385. Blatz, P. E., Mohler, J. H., & Navangul, H. V. (1972). Anion-induced wavelength regulation of absorption maxima of Schiffbases of retinal. Biochemistry 11,848-855. Blazynski, C., & Ostroy, S. E. (1984). Pathways in the hydrolysis of vertebrate rhodopsin. Vis. Res. 24, 459-470. Boucher, E, & Leblanc, R. M. (1985). Energy storage in the primary photoreaction of bovine rhodopsin. A photoacoustic study. Photochem. Photobiol. 41,459-465. Bownds, D. (1967). Site of attachment of retinal in rhodopsin. Nature 216, 1178-1181. Chabre, M., & Breton, J. (1979). The orientation of the chromophore of vertebrate rhodopsin in the "meta" intermediate states and the reversibility of the meta II-meta III transition. Vis. Res. 19, 1005-1018. Cone, R. A. (1972). Rotational diffusion of rhodopsin in the visual receptor membrane. Nature New Biol. 236, 39-43. Cooper, A. (1979). Energy uptake in the first step of visual excitation. Nature 282, 531-533. Dartnall, H. J. A. (1968). The photosensitivities of visual pigments in the presence of hydroxylamine. Vis. Res. 8, 339-358. Davidson, F. F., Loewen, P. C., & Khorana, H. G. (1994). Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. Proc. Natl. Acad. Sci. USA 9 l, 4029-4033. DeGrip, W. J., Gillespie, J., & Rothschild, K. J. (1985). Carboxyl group involvement in the meta I and meta II stages in rhodopsin bleaching. A Fourier transform infrared spectroscopic study. Biochim. Biophys. Acta 809, 97-106. Doi, T., Molday, R. S., & Khorana, H. G. (1990). Role of the intradiscal domain in rhodopsin assembly and function. Proc. Natl. Acad. Sci. USA 87, 4991-4995. Doukas, A. G., Aton, B., Callender, R. H., & Ebrey, T. G. (1978). Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II. Biochemistry 17, 2430-2435. Emeis, D., Kuhn, H., Reichert, J., & Hofmann, K. P. (1982). Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium. FEBS Lett. 143, 29-34. Fahmy, K., Jager, F., Beck, M., Zvyaga, T. A., Sakmar, T. P., & Siebert, F. (1993). Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants. Proc. Natl. Acad. Sci. USA 90, 10206-10210. Falk, G., & Fatt, P. (1966). Rapid hydrogen ion uptake of rod outer segments and rhodopsin solutions on illumination. J. Physiol.(Lond) 183, 211-224. Farahbakhsh, Z. T., Hideg, K., & Hubbell, W. L. (1993). Photoactivated conformational changes in rhodopsin: a time-resolved spin label study. Science 262, 1416-1419. Findlay, J. B., Brett, M., & Pappin, D. J. (1981). Primary structure of C-terminal functional sites in bovine rhodopsin. Nature 293, 314-317. Franke, R. R., Konig, B., Sakmar, T. P., Khorana, H. G., & Hofmann, K. P. (1990). Rhodopsin mutants that bind but fail to activate transducin. Science 250, 123-125. Franke, R. R., Sakmar, T. P., Graham, R. M., & Khorana, H. G. (1992). Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin. J. Biol. Chem. 267, 14767-14774. Fukuda, M. N., Papermaster, D. S., & Hargrave, P. A. (1979). Rhodopsin carbohydrate. Structure of small oligosaccharides attached at two sites near the NH2 terminus. J. Biol. Chem. 254, 82018207.


27

Fung, B. K., & Hubbell, W. L. (1978a). Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments. Biochemistry 17, 4396-4402. Fung, B. K., & Hubbell, W. L. (1978b). Organization of rhodopsin in photoreceptor membranes. 2. Transmembrane organization of bovine rhodopsin: evidence from proteolysis and lactoperoxidase-catalyzed iodination of native and reconstituted membranes. Biochemistry 17, 4403-4410. Ganter, U. M., Charitopoulos, T., Virmaux, N., & Siebert, F. (1992). Conformational changes ofcytosolic loops of bovine rhodopsin during the transition to metarhodopsin-II: an investigation by Fourier transform infrared difference spectroscopy. Photochem. Photobiol. 56, 57-62. Garcia-Quintana, D., Garriga, P., & Manyosa, J. (1993). Quantitative characterization of the structure of rhodopsin in disc membrane by means of Fourier transform infrared spectroscopy. J. Biol. Chem. 268, 2403-2409. Gibson, N. J., & Brown, M. F. (1991). Role of phosphatidylserine in the MI-MII equilibrium of rhodopsin. Biochem. Biophys. Res. Comm. 176, 915-921. Gibson, N. J., & Brown, M. F. (1993). Lipid headgroup and acyl chain composition modulate the MI-MII equilibrium of rhodopsin in recombinant membranes. Biochemistry 32, 2438-2454. Gruner, S. M. (1985). Intrinsic curvature hypothesis for biomembrane lipid composition: A role for nonbilayer lipids. Proc. Natl. Acad. Sci. USA 82, 3665-3669. Hargrave, P. A., McDowell, J. H., Curtis, D. R., Wang, J. K., Juszczak, E., Fong, S. L., Rao, J. K., & Argos, P. (1983). The structure of bovine rhodopsin. Biophys. Struc., & Mech. 9, 235-244. Hargrave, P. A., Hamm, H., & Hofmann, K. P. (1993). Interaction of rhodopsin with the G-protein, transducin. Bioessays 15, 43-50. Henderson, R., Baldwin, J. M., & Ceska, T. A. (1990). Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929. Hoffmann, W., Siebert, F., Hofmann, K. P., & Kreutz, W. (1978). Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments. Biochim. Biophys. Acta 503,450-461. Hofmann, K. P., Emeis, D., & Schnetkamp, P. P. (1983). Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes. Biochim. Biophys. Acta 725, 60-70. Hubbard, R., & Kropf, R. (1958). The action of light on rhodopsin. Proc. Natl. Acad. Sci. USA 44, 130-139. Hug, S. J., Lewis, J. W., & Kliger, D. S. (1988). Evidence for a common batho intermediate ofrhodopsin and isorhodopsin. J. Am. Chem. Soc. 110, 1998-1999. Hug, S. J., Lewis, J. W., Einterz, C. M., Thorgeirsson, T. E., & Kliger, D. S. (1990). Nanosecond photolysis of rhodopsin: evidence for a new, blue-shifted intermediate. Biochemistry 29, 14751485. Kahlert, M., & Hofmann, K. P. (1991). Reaction rate and collisional efficiency of the rhodopsintransducin system in intact retinal rods. Biophys. J. 59, 375-386. Kandori, H., Shichida, Y., & Yoshizawa, T. (1989). Absolute absorption spectra of batho- and photorhodopsins at room temperature. Picosecond laser photolysis of rhodopsin in polyacrylamide. Biophys. J. 56, 453-457. Kariel, N., Davidson, E., & Keough, K. (1991). Cholesterol does not remove the gel liquid crystalline phase transition ofphosphatidylcholines containing two polyenoic acyl chains. Biochim. Biophys. Acta 1062, 70-76. Karnik, S. S., Sakmar, T. P., Chen, H. B., & Khorana, H. G. (1988). Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc. Natl. Acad. Sci. USA 85, 8459-8463. Karnik, S. S., & Khorana, H. G. (1990). Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187. J. Biol. Chem. 265, 17520-17524. Kaushal, S., Ridge, K. D., & Khorana, H. G. (1994). Structure and function in rhodopsin: the role of asparagine-linked glycosylation. Proc. Natl. Acad. Sci. USA 91, 4024-4028.

28


Keough, K., & Kariel, N. (1987). Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chairs. Biochim. Biophys. Acta 902, 11-18. Khan, S. M., Bolen, W., Hargrave, P. A., Santoro, M. M., & McDowell, J. H. (1991). Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes. E. J. Biochem. 200, 53-59. Khorana, H. G. (1992). Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J. Biol. Chem. 267, 1-4. Kibelbek, J., Mitchell, D. C., Beach, J. M., & Litman, B. J. (1991). Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin. Biochemistry 30, 6761-6768. Klinger, A. L., & Braiman, M. S. (1992). Structural comparison of metarhodopsin II, metarhodopsin III, and opsin based on kinetic analysis of Fourier translbrm infrared difference spectra. Biophys. J. 63, 1244-1255. Konig, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A., & Hofmann, K. P. (1989). Three cytoplasmic loops of rhodopsin interact with transducin. Proc. Natl. Acad. Sci. USA,86, 68786882. Kropf, A. (1967). Intramolecular energy transfer in rhodopsin. Vis. Res. 7, 811-818. Ktihn, H., & Hargrave, P. A. (1981). Light-induced binding of Guanosinetriphosphate to bovine photoreceptor membranes: Effect of limited proteolysis of the membranes. Biochemistry 20, 2410-2417. Lamola, A. A., Yamane, T., & Zipp, A. (1974). Effects of detergents and high pressures upon the metarhodopsin I ~ metarhodopsin II equilibrium. Biochemistry 13, 738-745. Lewis, J. W., & Kliger, D. S. (1992). Photointermediates of visual pigments. J. Bioenerg. Biomem. 24, 201-210. Lewis, J. W., Winterle, J. S., Powers, M. A., Kliger, D. S., & Dratz, E. A. (1981). Kinetics of rhodopsin photolysis intermediates in retinal rod disk membranes. I. Temperature dependence of lumirhodopsin and metarhodopsin I kinetics. Photochem. Photobiol. 34, 375--384. Lewis, J. W., Hug, S. J., Wallace-Williams, S. E., & Kliger, D. S. (1990). Direct evidence for an equilibrium between early photolysis intermediates of rhodopsin. J. Am. Chem. Soc. 112, 6711-6712. Liang, C. J., Yamashita, K., Muellenberg, C. G., Shichi, H., & Kobata, A. (1979). Structure of the carbohydrate moieties of bovine rhodopsin. J. Biol. Chem. 254, 6414--6418. Liebman, P. A. (1962). In situ microspectrophotometric studies on the pigments of single retinal rods. Biophys. J. 2, 161-178. Litman, B. J., Kalisky, O., & Ottolenghi, M. (1981). Rhodopsin -phospholipid interactions: dependence of rate of the meta I to meta II transition on the level of associated disk phospholipid. Biochemistry 20, 631-634. Litman, B. J., Aton, B., & Hartley, J. B. (1982). Functional domains of rhodopsin. Vis. Res. 22, 1439-1442. Litman, B. J. (1982). Methods for determining rod outer segment disk phospholipid transmembrane topology. Meth. In Enzymol. 81,309-315. Litman, B. J., Lewis, E. N., & Levin, I. W. (1991). Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions. Biochemistry 30, 31 3-319. Litman, B. J., & Mitchell, D. C. (1996). A role for phospholipid polyunsaturation in modulating membrane protein function. Lipids. In press. Longstaff, C., Calhoon, R., & Rando, R. (1986). Deprotonation of the Schiffbase ofrhodopsin is obligate in the activation of the G protein. Proc. Natl. Acad. Sci. 83, 4209-4213. Martynov, V. I., Kostina, M. B., Feigina, M. I., & Miroshnikov, A. I. (1983). Study of the molecular organization of visual rhodopsin in photoreceptor membranes by limited proteolysis. Bioorganich. Khim. 9, 734-745.


29

Mathews, R., Hubbard, R., Brown, P., & Wald, G. (1963). Tautomeric forms of metarhodopsin. J. Gen. Phys. 47, 215-240. Mathies, R., Oseroff, A. R., & Stryer, L. (1976). Rapid-flow resonance Raman spectroscopy of photolabile molecules: rhodopsin and isorhodopsin. Proc. Natl. Acad. of Sci. USA 73, 1-5. McDowell, J. H., Nawrocki, J. P., & Hargrave, P. A. (1993). Phosphorylation sites in bovine rhodopsin. Biochemistry 32, 4968--4974. Michel-Villaz, M., Saibil, H. R., & Chabre, M. (1979). Orientation ofrhodopsin alpha-helices in retinal rod outer segment membranes studied by infrared linear dichroism. Proc. Natl. Acad. Sci. USA 76, 4405-4408. Michel-Villaz, M., Roche, C., & Chabre, M. (1982). Orientational changes of the absorbing dipole or retinal upon the conversion of rhodopsin to bathorhodopsin, lumirhodopsin, and isorhodopsm. Biophys. J. 37, 603-616. Miller, J. L., Fox, D. A., & Litman, B. J. (1986). Amplification ofphosphodiesterase activation is greatly reduced by rhodopsin phosphorylation. Biochemistry 25, 4983--4988. Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990). Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids. Biochemistry 29, 9143-9149. Mitchell, D. C., Kibelbek, J., & Litman, B. J. (1991). Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gr Biochemistry 30, 37-42. Mitchell, D. C., Kibelbek, J., & Litman, B. J. (1992a). Effect ofphosphorylation on receptor conformation: the metarhodopsin I in equilibrium with metarhodopsin II equilibrium in multiply phosphorylated rhodopsin. Biochemistry 31, 8107-8111. Mitchell, D. C., Straume, M., & Litman, B. J. (1992b). Role of sn-l-saturated, sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium. Biochemistry 31,662-670. Molday, R. S., & Molday, L. L. (1979). Identification and characterization ofmultiple forms ofrhodopsin and minor proteins in frog and bovine rod outer segment disc membranes. Electrophoresis, lectin labeling, and proteolysis studies. J. Biol. Chem. 254, 4653-4660. Mone, A. P., & Litman, B. J. (1990). Cholesterol increases the stability of rhodopsin in egg PC vesicles to thermally induced unfolding. Biophys. J. 57, 74a. Mone, A. P., & Litman, B. J. (1991). Variation of phospholipid headgroup composition modulates the formation of metarhodopsin II. Biophys. J. 59, 621 a. Monger, T. G., Alfano, R. R., & Callender, R. H. (1979). Photochemistry ofrhodopsin and isorhodopsin investigated on a picosecond time scale. Biophys. J. 27, 105-115. Mouritsen, O. G., & Bloom, M. (1984). Mattress model of lipid-protein interactions in membranes. Biophys. J. 46, 141-153. Mullen, E., & Akhtar, M. (1981). Topographic and active-site studies on bovine rhodopsin. FEBS Lett. 132, 261-264. Mullen, E., & Akhtar, M. (1983). Structural studies on membrane-bound bovine rhodopsin. Biochem. J. 211, 45-54. Nakayama, T. A., & Khorana, H. G. (1990). Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal. J. Biol. Chem. 265, 15762-15769. Nakayama, T. A., & Khorana, H. G. (1991). Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin. J. Biol. Chem. 266, 4269-4275. Nathans, J., & Hogness, D. S. (1983). Isolation, sequence analysis, and intro-econ arrangement of the gene encoding bovine rhodopsin. Cell 34, 807-8 14. Nathans, J. (1990a). Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin. Biochemistry 29, 9746-9752. Nathans, J. (1990b). Determinants of visual pigment absorbance: role of charged amino acids in the putative transmembrane segments. Biochemistry 29, 937-942. Nathans, J. (1992). Rhodopsin: Structure function, and genetics. Biochemistry 31, 4923-4931.

30


Needham, D., & Nunn, R. S. (1990). Elastic deformation and failure of lipid bilayer membranes containing cholesterol. Biophys. J. 58, 997-1009. Neuringer, M., Condor, W. E., Van Patten, C., & Barstad, L. (1984). Dietary omega-3 fatty acid deficiency and visual loss in infant Rhesus monkeys. J. Clin. Invest. 73,272-276. O'Brien, D. F., Costa, L. F., & Ott, R. A. (1977). Photochemical functionality ofrhodopsin-phospholipid recombinant membranes. Biochemistry 16, 1295-1303. Ovchinnikov, Y. A., Abdulaev, N. G., Feigina, M. I., Artamonov, I. D., & Bogachuk, A. S. (1983). Visual rhodopsin. III. Complete amino acid sequence and topography in a membrane. Bioorgan. Khim. 9, 1331-1340. Ovchinnikov Y. A, Abdulaev, N. G., & Bogachuk, A. S. (1988). Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated. FEBS Lett. 230, 1-5. Papac, D. I., Oatis, J. E., Jr., Crouch, R. K., & Knapp, D. R. (1993). Mass spectrometric identification of phosphorylation sites in bleached bovine rhodopsin. Biochemistry 32, 5930-5934. Papermaster, D. S., & Dreyer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry 13, 2438-2444. Parkes, J. H., & Liebman, P. A. (1984). Temperature and pH dependence of metarhodopsin I--metarhodopsin II kinetics and equilibrium in bovine rod disk membrane suspensions. Biochemistry 23, 5054-5061. Pellicone, C., Nullans, G., & Virmaux, N. (1985). Localization of light-induced conformational changes in bovine rhodopsin. FEBS Lett. 181,179-183. Peters, K., Applebury, M. L., & Rentzepis, P. M. (1977). Primary photochemical event in vision: proton translocation. Proc. Natl. Acad. Sci. USA 74, 3119-3123. Pfister, C., Kuhn, H., & Chabre, M. (1983). Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin. E. J. Biochem. 136, 489-499. Pober, J. S., & Stryer, L. (1975). Light dissociates enzymatically-cleaved rhodopsin into two different fragments. J. Mol. Biol. 95, 477-481. Poo, M., & Cone, R. A. (1974). Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature 247, 438--441. Randall, C. E., Lewis, J. W., Hug, S. J., Bjorling, S. C., Eisner-Shanas, I., Friedman, N., Ottolengi, M., Sheves, M., & Kliger, D. S. (1991). A New photolysis intermediate in artificial and native visual pigments. J. Am. Chem. Soc. 113, 3473-3485. Resek, J. F., Farahbakhsh, Z. T., Hubbell, W. L., & Khorana, H. G. (1993). Formation of the meta II photointermediate is accompanied by changes in the cytoplasmic surface ofrhodopsin. Biochemistry 32, 12025-12032. Robinson, P. R., Cohen, G. B., Zhukovsky, E. A., & Oprian, D. D. (1992). Constitutively active mutants of rhodopsin. Neuron 9, 71 9-725. Rohlich, P. (1976). Photoreceptor membrane carbohydrate on the intradiscal surface of retinal rod disks. Nature 263, 789-791. Rothschild, K. J., Gillespie, J., & DeGrip, W. J. (1987). Evidence for rhodopsin refolding during the decay of Meta II. Biophys. J. 51,345-350. Sakmar, T. P., Franke, R. R., & Khorana, H. G. (1989). Glutamic acid-ll3 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc. Natl. Acad. Sci. USA 86, 8309-8313. Salem, N., Jr. (1989). Omega-3 fatty acids: Molecular and biochemical aspects. In: New Protective Roles for Selected Nutrients (Spiller, G. A., & Scala, J., eds.), pp. 109-228, Alan R. Liss Inc., New York, NY. Schertler, G. F., Villa, C., & Henderson, R. (1993). Projection structure of rhodopsin. Nature 362, 770-772. Schick, G. A., Cooper, T. M., Holloway, R. A., Murray, L. P., & Birge, R. R. (1987). Energy storage in the primary photochemical events of rhodopsin and isorhodopsin. Biochemistry 26, 2556-2562.


31

Shichi, H., & Shelton, E. (1974). Assessment of physiological integrity of sonicated retinal rod membranes. J. Supramol. Struct. 2, 7-16. Singer, S. J., & Nicholson, G. L. (1972). The fluid mosaic model of cell membranes. Science 175, 720-731. Smith, H. G., Jr., Stubbs, G. W., & Litman, B. J. (1975). The isolation and purification of osmotically intact discs from retinal rod outer segments. Exp. Eye Res. 20, 211-217. Smith, S. O., Courtin, J., de Groot, H., Gebhard, R., & Lugtenburg, J. (1991). 13C magic-angle spinning NMR studies of bathorhodopsin, the primary photoproduct of rhodopsm. Biochemistry 30, 7409-7415. Stewart, J. G., Baker, B. N., & Williams, T. P. (1977). Evidence for conformeric states of rhodopsm. Biophys. Struc. Mech. 3, 19-29. Stone, W. L., Farnsworth, C. C., & Dratz, E. A. (1979). A reinvestigation of the fatty acid content of bovine. Rat and frog retinal rod outer segments. Exp. Eye Res. 28,387-397. Straume, M., & Litman, B. J. (1987a). Influence of cholesterol on equilibrium and dynamic bilayer structure of unsaturated acyl chain phosphatidylcholine vesicles as determined from higher order analysis of fluorescence anisotropy decay. Biochemistry 26, 5121-5126. Straume, M., & Litman, B. J. (1987b). Equilibrium and dynamic structure of large, unilamellar, unsaturated acyl chain phosphatidylcholine vesicles. Higher order analysis of 1,6-diphenyl- 1,3,5hexatriene and 1-[4-(trimethylammonio)phenyl]- 6-phenyl-l,3,5-hexatriene anisotropy decay. Biochemistry 26, 5113-5120. Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990). Interconversion of metarhodopsins I and II: a branched photointermediate decay model. Biochemistry 29, 9135-9142. Stryer, L. (1991). Visual excitation and recovery. J. Biol. Chem. 266, 10711-10714. Stubbs, G. W., & Litman, B. J. (1976). Microviscosity of the hydrocarbon region of the bovine retinal rod outer segment disk membrane determined by fluorescent probe measurements. Biochemistry 15, 2766-2772. Stubbs, G. W., & Litman, B. J. (1978). Effect of alterations in the amphipathic microenvironment on the conformational stability of bovine opsin. 1. Mechanism of solubilization of disk membranes by the nonionic detergent, octyl glucoside. Biochemistry 17, 215-219. Thomas, D. D., & Stryer, L. (1982). Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes. J. Mol. Biol. 154, 145-157. Thorgeirsson, T. E., Lewis, J. W., Wallace-Williams, S. E., & Kliger, D. S. (1992). Photolysis of rhodopsin results in deprotonation of its retinal Schiff's base prior to formation ofmetarhodopsin II. Photochem. Photobiol. 56, 1135-1144. Thorgeirsson, T. E., Lewis, J. W., Wallace-Williams, S. E., & Kliger, D. S. (1993). Effects of temperature on rhodopsin photointermediates from lumirhodopsin to metarhodopsin II. Biochemistry 32, 13861-13872. Trayhurn, P., Mandel, P., & Virmaux, N. (1974). Removal of a large fragment of rhodopsin without changes in its spectral properties, by proteolysis of retinal rod outer segments. FEBS Lett. 38, 351-353. Wang, J. K., McDowell, J. H., & Hargrave, P. A. (1980). Site of attachment of l l-cis-retinal in bovine rhodopsin. Biochemistry 19, 5111-5117. Weitz, C. J., & Nathans, J. (1992). Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II. Neuron 8, 465-472. Wiedmann, T. S., Pates, R. D., Beach, J. M., Salmon, A., & Brown, M. E (1988). Lipid-protein interactions mediate the photochemical function of rhodopsin. Biochemistry 27, 6469-6474. Wilden, U., Hall, S. W., & Kuhn, H. (1986). Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc. Natl. Acad. Sci. USA 83, 174-1178. Wong, J. K., & Ostroy, S. E. (1973). Hydrogen ion changes of rhodopsin I. Proton uptake during the metarhodopsin 1 478 metarhodopsin II 380 reaction. Arch. Biochem. Biophys 154, 1-7.

32


Yan, M., Manor, D., Weng, G., Chao, H., Rothberg, L., Jedju, T. M., Alfano, R. R., & Callender, R. H. (1991). Ultrafast spectroscopy of the visual pigment rhodopsin. Proc. Natl. Acad. Sci. USA 88, 9809-9812. Zhukovsky, E. A., & Oprian, D. D. (1989). Effect of carboxylic acid side chains on the absorption maximum of visual pigments. Science 246, 928-930. Zvyaga, T. A., Min, K. C., Beck, M., & Sakmar, T. P. (1993). Movement of the retinylidene Schiffbase counterion in rhodopsin by one helix turn reverses the pH dependence of the metarhodopsin I to metarhodopsin II transition. J. Biol. Chem. 268, 4661-4667. Zvyaga, T. A., Fahmy, K., & Sakmar, T. P. (1994). Characterization ofrhodopsin-transducin interaction: a mutant rhodopsin photoproduct with a protonated Schiffbase activates transducin. Biochemistry 33, 9753--9761.

CHARACTERIZATION OF THE PRIMARY PHOTOCHEMICAL EVENTS IN BACTERIORHODOPSIN AN D RHODOPSIN

Jeffrey A. Stuart and Robert R. Birge

I.

II.

III.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Bacteriorhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Rhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Bacteriorhodopsin and Rhodopsin Active Sites . . . . . . . . . . . . . . . A. The Bacteriorhodopsin Active Site . . . . . . . . . . . . . . . . . . . . . . B. Chromophore Orientation and Location . . . . . . . . . . . . . . . . . . . C. Characterization o f the Opsin Shift . . . . . . . . . . . . . . . . . . . . . . D. The Rhodopsin Active Site . . . . . . . . . . . . . . . . . . . . . . . . . . The Primary Event in Bacteriorhodopsin . . . . . . . . . . . . . . . . . . . . . A. The J Intermediate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. C. D.

The K Intermediate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34 34 37 41 42 43 45 56 64 66 66

Ultrafast Spectroscopic Studies . . . . . . . . . . . . . . . . . . . . . . . Quantum Efficiency o f the Primary Event . . . . . . . . . . . . . . . . . .

70 72

Biomembranes Volume 2A, pages 33-139 Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-659-2 33

JEFFREY A. STUART and ROBERT R. BIRGE

34

IV.

V.

VI. VIII.

E. Photoelectric Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 F. Molecular Dynamics of the Primary Event . . . . . . . . . . . . . . . . . 75 G. Energy Storage in the Primary Event . . . . . . . . . . . . . . . . . . . . 78 The Primary Event in Rhodopsin . . . . . . . . . . . . . . . . . . . . . . . . 80 A. Photorhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 B. Bathorhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 C. Ultrafast Spectroscopic Studies . . . . . . . . . . . . . . . . . . . . . . . 88 D. Quantum Efficiency of the Primary Event . . . . . . . . . . . . . . . . . 96 E. Molecular Dynamics of the Primary Event . . . . . . . . . . . . . . . . . 98 F. Energy Storage in the Primary Event . . . . . . . . . . . . . . . . . . . . 103 G. Molecular Origins of Photoreceptor Noise . . . . . . . . . . . . . . . . . 103 H. Parallels between Rhodopsin and Bacteriorhodopsin Photochemistry . . . 114 Response of the Protein During the Primary Event . . . . . . . . . . . . . . . 115 A. Bacteriorh0dopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 B. Rhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Protein Control of Isomerization . . . . . . . . . . . . . . . . . . . . . . . . . 118 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 I.

INTRODUCTION A. Bacteriorhodopsin

Bacteriorhodopsin is a 26 kD (248 amino acids) molecular weight protein contained within the cell membrane of Halobacterium salinarium (informally called Halobacterium halobium and reclassified in accordance with (Tindall, 1992)), a halophilic archaebacterium that grows and prospers in salt marshes and lakes where the concentration of NaC1 can exceed 4 M; roughly six times that of sea water (-0.6, M). Its primary and approximate secondary structure is shown in Figure 1. As the name implies, this unique protein possesses many similarities to the visual protein rhodopsin, although the physiological functions of the two proteins are quite different. Rhodopsin acts as the primary photoreceptor that enables dim light vision for most vertebrate animals. Bacteriorhodopsin (BR) enables the bacteria to act as a facultative anaerobe when the oxygen tension of its environment is low. The protein accomplishes this function by acting as a light driven proton pump which causes the formation of an electrochemical gradient that can be harnessed by the bacterial cell to do work (thus converting sun light into chemical energy). Absorption of light by the retinal cofactor in this protein initiates a primary photochemical event followed by a series of thermal relaxations back to the initial state. Each of the intermediates is characterized by a different absorption maximum, and decays with time constants ranging from picoseconds to milliseconds (Figure 2). The formation of the earliest well defined intermediate, K, involves a trans to cis isomerization about the C13=C14 bond (Figure 3). The primary work done by the

Cytoplasmic Surface

(INSIDE)

35

(OUTS1 DE) Surface N I

figure 1. Amino acid sequences and putative membrane spanning regions of bacteriorhodopsin. Amino acid abbreviations are as follows: (alanine (Ala, A); arginine (Arg, R); Asparagine (Asn, N);Aspartic Acid (Asp, D); Cysteine (Cys, C); Glutarnate (Glu, E); Glutamine (Gln, 0);Glycine (Gly, G); Histidine (His, H); lsoleucine (Ile, I);Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); Valine (Val, V). Amino acids which under nominal conditions carry a charge are shown in thickened circles (positively charged) or boxes (negatively charged).

36

JEFFREYA. STUART and ROBERT R. BIRGE

kv/~(620)~

~

(5 Sis (>150K)]

I hV

\ ,~

-7ms

~V

hv

.umirhod~opsi (497)) n( lOOns (>I30K____)I t

,

a

200 300 400

3000 6000

~

~

I

I

I

I

I

I

I

500

550

600

650

Wavelength (nm)

Figure 11. Differential absorption spectra of rhodopsin at various time delays of the probe pulse after excitation with a 35-fs pump pulse at 500 nm. The breaks in the curves near 570 nm indicate where the spectra from blue (490-570 nm) and red (570-670 nm) 10 fs probe pulses were joined. (Reproduced with permission from Peteanu et al., 1993).

along the potential energy surface away from the Franck-Condon region, as evidenced by the disappearance of the S 1--->Sn absorption feature by 100 fs, at which point a strong bleach is found in the same location. Indeed, resonance Raman intensity analysis supports torsional departure from the Franck-Condon region on this time-scale (Loppnow et al., 1992). It should be noted that there was very little absorption in the red spectral region due to the SI-~S n transition. As the bleach evolves, a concomitant photoproduct absorption band appears between 550 and 650 nm reaches its maximum by 200 fs, and remains largely unchanged for subsequent measurements out to 6 ps. The ~maxof this feature is about 570 nm and is consistent with that of what is widely recognized as the primary photoproduct, photorhodopsin. The following changes were seen between 200 fs and 6 ps (Peteanu et al., 1993):

92


(i) A slight loss in intensity was seen at the red edge of the photoproduct band, between 610 and 630 nm, as well as a shift of the photoproduct maximum from 575-565 nm, and a concomitant increase in absorption at 535 nm. (ii) The isosbestic point shifted from 540 nm at 200 fs to 520 nm at 6 ps, and (iii), after 200 fs the hole due to the bleaching of the reactant had recovered. The gain in absorbance at 535 nm is consistent with the formation of bathorhodopsin. These results contradict those of the Yan and coworkers, and are inconsistent with their interpretation (see above) for the following reasons (according to Peteanu et al. (1993)). The absorption at 620 nm seen in their study (which appeared within the 300 fs time resolution of their experiment) (Yen et al., 1991, 1993) is apparently part of a larger absorption band due to the photoproduct, which is not fully developed until 200 fs; the time resolution of their work did not allow for the observation of the evolution of this signal. Moreover, the signal in the 580--620 nm range purportedly due to an excited state with a 90 ° twist along the polyene chain is expected to be associated with the evolution of a spectral signal, which is not observed between 510-600 nm (Peteanu et al., 1993). The last reason cited by Peteanu and coworkers for the incongruence in interpretation is ascribed to the observation that there was no notable signal evolution seen in their data between 200 fs and 6 ps; one would expect some spectral signature attributable to the 3 ps decay of Rh*(90 °) into the more planar all-trans ground state (Schoenlein et al., 1991; Peteanu et al., 1993). As pointed out in their work, the theoretical predictions of Tallent et al. (1992) indicated observable spectral differences in oscillator strength, bandwidth, and absorption maximum between the twisted excited-state species and the ground-state photoproduct. Photochemical hole-burning studies on both rhodopsin and bacteriorhodopsin by Loppnow and collaborators lend further support for the observed ultrafast isomerization times, based upon the observation of extremely broad homogeneous line widths (1300 cm-1 at 1.5 K) for both proteins; the absence of narrow components in spectra burned and probed at 2 cm-1 resolution is consistent with chromophore isomerization times of approximately 200-500 fs on the excited-state potential surface (Loppnow et al., 1992). Furthermore, the broad and diffuse nature of the holes is consistent with the --25 fs optical relaxation times predicted from resonance Raman intensity analyses (Loppnow et al., 1992). In a recent study on the primary event in octopus rhodopsin, the photoproduct, and therefore the isomerization, was reported to have formed within 400 fs after leaving the excited-state (Taiji et al., 1992). Femtosecond spectroscopy with a range of400-1000 nm revealed an induced absorption between 680-730 nm and640--660 nm that appeared within 100 fs and decayed with time constants of 140+70 fs and 360+180 fs respectively, followed by another induced absorption in the 540--600 nm region that had a rise time of 200-400 fs. The appearance of this signal coincided with the decay of the absorptions in the 640-730 nm region, and was attributed to photoproduct. The initial signal was due to excited-state absorption. After 100 ps, the spectrum matched that of the relaxed all-trans octopus bathorhodopsin (Taiji et al., 1992). The spectral properties of the transient species formed by 400 fs were

Characterization of the Primary Photochemical Events

93

found to be consistent with those ofphotorhodopsin (denoted as "primerhodopsin" by the authors), indicating that the photoisomerization has already taken place (Taiji et al., 1992). Femtosecond studies have also revealed the dynamics of isorhodopsin isomerization, which takes place at the C9~C10bond as opposed to the Cl1~C12bond (Figure 12) (Schoenlein et al., 1993). After excitation with the 500 nm probe pulse, an induced absorption (480-570 nm) appeared within 50 fs that lasted for 100-150 fs, and was attributed to excited state absorption (S~-->Sn). A bleach of the reactant followed, attaining its maximum by 300 fs; recovery of this bleach was found to

O3

"O

-ATFF=1%

-150 fs +33 fs + 100 fs +200 fs +400 fs +600 fs

I

I

+900 fs +3 ps

!

I

1

+6 ps 450

500

550

600

650

700

wavelength (nm)

Figure 12. Differential absorption spectra of isorhodopsin at various time delays of the probe pulse after excitation with a 40-fs pump pulse at 500 nm. Measurements from 450-570 nm were obtained by using a 10 fs probe pulse at 500 nm. Measurements from 570-670 were obtained by using a 10 fs probe pulse at 620 nm. (Reproduced with permission from Schoenlein et al., 1993).

94


be biphasic with time constants of several hundred femtoseconds and 1.5 ps. The biphasic behavior was attributed to two processes, including the return to the ground state of nonisomerized reactant with a consequential redistribution of vibrational energy on a picosecond time-scale, and dynamics of the photoproduct. The former of the two should account for about 80% of the recovery, due to the low isomerization quantum yield ofisorhodopsin (0.22) (Schoenlein et al., 1993). Photoproduct absorption was seen in the 570-640 nm region. At early times (0-150 fs) an emission process was identified as the S 1-~S 0 transition, the result of the excitedstate wave packet leaving the Franck-Condon region (by 150 fs). As this feature decayed, an absorption signal appeared (150-200 fs) in the same region which exhibited a wavelength-dependent rise time, reaching maximum absorbance at earlier times for longer wavelengths (500 fs at 570 nm, 300 fs at 620 nm). Subsequent photoproduct relaxation (after several hundred femtoseconds) was also found to be wavelength dependent in a similar manner (1.3-0.6 ps); this behavior is consistent with vibrational cooling and conformational relaxation (Schoenlein et al., 1993). From 200 to 600 fs, the photoproduct absorption band narrowed and shifted to the blue, as evidenced by an isosbestic point shift from 525 to 515 nm (see below). By 6 ps, the difference spectrum was consistent with that ofbathorhodopsin (Schoenlein et al., 1993). By comparison of the results from the rhodopsin studies, it is apparent that although both proteins share qualitatively similar behavior, the isorhodopsin isomerization occurs approximately three times more slowly, consistent with its lower quantum yield (0.22). Whereas a distinct stimulated emission was observed for isorhodopsin, none was evident in the rhodopsin dynamics, indicating that the initial velocity of the wave packet out of the Franck-Condon region occurs on a slower time-scale (for isorhodopsin) (Schoenlein et al., 1993). Because the photoproduct band is likely to have had components from vibrationally hot species, including contributions (approximately 80%) from non-isomerized reactant molecules (with--20,000 cm-1 excess vibrational energy from the absorbed photon), it became necessary to model the effects of these species on the absorption spectrum (Schoenlein et al., 1993). After deconvolution of the different contributions, an approximate isomerization time of 600 fs was found. The vibrational coherence of the rhodopsin photoisomerization was not evident in isorhodopsin, again consistem with a slower isomerization. There are several conceivable explanations cited by the authors to account for the observed differences between the proteins. Perhaps the most substantial is the difference in the interactions of the chromophore, namely the lack of an interaction between the 13-methyl group and the 10-hydrogen in isorhodopsin. In rhodopsin this steric interaction is thought to result in a torsionally distorted Cl1~C12bond that may aid in driving the isomerization forward (thus helping to account for the unusually high quantum yield of 0.67). No equivalent steric interaction exists in isorhodopsin; however, this does not rule out the existence of other protein-chromophore interactions in the binding site, which are certainly different from those of rhodopsin. Indeed, such interactions have been


95

suggested to account for the temperature and wavelength dependence of the isorhodopsin quantum yield (Hurley et al., 1977; Birge, 1990a; Schoenlein et al., 1993). A small activation barrier of 0.2 kcal/mol has been calculated to be associated with the 9-cis ~-~ 9-trans excited-state torsional surface. This barrier is small enough to be unimportant at ambient temperatures, but is comparable to thermal energy at low temperatures (at 77 K, kT ~ 0.15 kcal/mol vs. 0.6 kcal/mol at ambient), which would therefore have the effect of lowering the quantum yield (see Figure 14) (Birge et al., 1988; Birge, 1990a). It is important to consider the implications of the ultrafast spectroscopic studies upon the nature of the primary intermediate, photorhodopsin, and its transition into the better defined batho-intermediate. As previously mentioned, it is a matter of some controversy as to whether photorhodopsin can be accurately described as a true intermediate due to its inherent inability to be isolated at low (liquid helium) temperatures. The evidence that the primary event, that is, the photoisomerization, has already occurred by the formation ofphotorhodopsin is conclusive, based upon the results of the spectroscopic and chromophore analog studies described above; the photorhodopsin chromophore has an all-trans configuration, albeit highly distorted. Generally speaking, the ultrafast studies indicate a fast femtosecondisomerization, followed by a slight blue-shift on a picosecond time-scale. This shift is most likely the result of thermal cooling of the chromophore, such that photorhodopsin can be described as a thermally "hot" bathorhodopsin. The transition from photo- to bathorhodopsin is then simply a "cooling off'' step, involving the redistribution and dissipation of vibrational energy modes between the chromophore and the protein over several picoseconds. At room temperature, this transition is probably barrierless on a nonadiabatic potential surface (Taiji et al., 1992; Peteanu et al., 1993), and corresponds to a structural relaxation from a highly distorted to a more relaxed planar all-trans configuration. As noted above, bathorhodopsin has a formation time reported to be on a picosecond time-scale, consistent with conformational relaxation times measure by time-resolved vibrational spectroscopy for the bacteriorhodopsin chromophore (van den Berg et al., 1990; Doig et al., 1991). The results cited above by Peteanu et al. (1993) for the temporal period between 200 fs and 6 ps, namely the shift in photoproduct absorption and simultaneous gain at 535 nm, the blue shift in the isosbestic point, and the recovery of the reactant bleach, are all consistent with vibrational cooling and conformational relaxation processes (Peteanu et al., 1993). Observations of these types of behavior were also made in the octopus studies, where the authors described primerhodopsin (syn. photorhodopsin) as "quasi-thermal" bathorhodopsin, which then "thermalizes" with a time constant of 20+10 ps to form the fully relaxed bathorhodopsin (Taiji et al., 1992). Additional support for the vibrationally hot nature ofphotorhodopsin was reported in the studies on isorhodopsin described above. The shift in the isosbestic point concomitant with the blue-shift and narrowing of the photoproduct absorption band, as well as the wavelength-dependent partial relaxation of the same band within several hundred femtoseconds yielding spectral features consis-

96


tent with bathorhodopsin, both indicate vibrational cooling and conformational relaxation processes of the chromophore to a less distorted all-trans configuration (Peteanu et al., 1993).

D. Quantum Efficiency of the Primary Event The quantum yields for the photoconversions involving rhodopsin, bathorhodopsin and isorhodopsin are shown in Figures 13 and 14 and the salient assignments are given below:

''''''"1'"''''"1'""''"1

0.9 0.8

0.7 0.6

0.5

0.3 1

450

470

490

510

Wavelength (nm)

--.d)2(R-->R~ --.

,,,,,,,,I,,,,,,,,,I,,,,,,,,,I,,,,,,,,,I,,,~

530

450

" .... '1' ...... "1'"'""'1" ....... I'"'_:

0.14 0.12 0.10

¢I)3(B_.__)I )

..... ''"1'"~

~

470

490

510

Wavelength (nm)

' 0.16

--

530 I......

. . . . . .

0.14

*0.08

0.040.06

I

T

T~

I

I

~

T'~

I _

,,,,,,,,I,,,,,,,,,I~,,,,,,,,I,,,,,,,,,I,,,

450

470

490

510

Wavelength (nm)

530

(I)0.12 0.080"10 1

(D41][-)B) "~""~1.

~-.

I= ,,h,,,,,,,,I,,,,,,,,,h,,,,,,,,t,,,,,,,,~h,,,,,,,,h,,,,,,,,h .... ,,,,h,,,

420 440 460 480 500 520 540 560 Wavelength (nm)

Figure 13. Wavelength dependence of the quantum yields for the photoreactions

involving rhodopsin (R), bathorhodopsin (B) and isorhodopsin (I) determined based on photostationary state data collected at 77K (solid circles) and 70K (open circles) (Birge and Callender, 1987; Birge et al., 1988). These graphs support the assumption of wavelength independent values for @1 (R-~B) = 0.67, @2 (B-->R) = 0.49 and @3 (B~I) = 0.076 and the horizontal lines indicate the assigned wavelength independent values (Birge and Callender, 1987). However, the graph for @4 (I-->B) indicates a wavelength dependence that is characteristic of a small barrier in the isorhodopsin --> bathorhodopsin excited state potential surface (Birge et al., 1988). The dotted and solid lines display two models for the quantum yield for the latter photoisomerization, both of which assume a barrier of less than ~70 cm -1. The barrier is so small that the wavelength dependence of the @4 (I-->B) is not observable at ambient temperature [@4 (300 K) = 0.22, (Hurley et al., 1977)].

Characterization of the Primary Photochemical Events •

= 0.67

rhodopsin (R) ~

97

el)_ = 0.073

bathorhodopsin (B) ~

O 2 = 0.49

isorhodopsin (I)

(I)4 (L,T)

where the values cI)1 = 0.67 + 0.02, cI)2 = 0.49 + 0.03, cI)3 = 0.076 + 0.006 are independent with respect to both temperature and excitation wavelength within the error ranges specified (Birge and Callender, 1987). In sharp contrast, (I) 4 is observed to be both temperature and wavelength dependent (Rosenfeld et al., 1977a; Schick et al., 1987; Birge et al., 1988). A summary of the experimental results are shown in Figure 13. This latter observation has interesting implications which have been

-85 %

~15 %

I i

>95%

'

(0.06) "

!

0.49

~, /

~

04) I

~

{(o.6)1"},

•

{(o.2)$}

I

I I

Bathorhodopsin

(0.33)

I I I I

0.073

[0.2251

I I

0.67

~40 %

! ! ! I

-27 kcal/moi -32 kcal/mol

I Isorhodopsin

--5 k c a l / m o l .

.

.

.

.

.

.

.

.

.

.

.

.

. . . . .

. . . . . .

. . . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Rhodopsin Figure 14. Schematic representation of the ground and first excited singlet state surfaces connecting rhodopsin, bathorhodopsin and isorhodopsin using a simplified (linearized) reaction coordinate. The shapes of the ground and excited state surfaces are based on all-valence electron molecular orbital calculations including single and double configuration interaction (Birge et al., 1988). Ground state enthalpies are taken from the experimental measurements of Cooper (1979a,b) and Schick et al. (1987). Absolute quantum yields of photoisomerization are displayed at the tips of the arrows indicating the processes (Birge and Callender, 1987; Birge et al., 1988). Values given in parentheses (o) are predicted by using semiempirical molecular dynamics theory to calculate the reverse/forward yield ratios and multiplying these values by the experimental forward yields (shown without parentheses). Values listed in brackets {o} are ambient temperature quantum yields which display temperature dependence. The arrows indicate the effect that lowering the temperature will have on these values, such as, {0.225} indicates that at lower temperatures, the quantum yield will be lower than 0.22 (Adapted from Birge et al., 1988.)

98


discussed earlier. At ambient temperature, however, this quantum efficiency is equal to 0.22, and is independent of both temperature and wavelength (Hurley et al., 1977). A slight dependence of the isorhodopsin quantum yield upon laser pulse photon density has also been reported (Kandofi et al., 1988); the ratio relative to rhodopsin was measured to be 0.37 in steady actinic light and 0.39 with a weak picosecond laser pulse. Use of laser pulses of higher energy gave ratios > 0.39, indicating saturation effects (Kandori et al., 1988). The quantum yield of the primary event was originally assigned by Dartnall (1972) and subsequent studies have yielded values in agreement within experimental error (Hurley et al., 1977; Rosenfeld et al., 1977a; Birge and Callender, 1987). The observation that O)1 of rhodopsin is larger by a factor of at least two compared to the photoisomefization quantum efficiency of the 11-cis retinyl protonated Schiffbase (RPSB) in solution (Freedman and Becker, 1986; Freedman et al., 1986) is one indication that the protein has a binding site optimized for 11-cis --~ 11-trans photoisomerization. The fact that the primary event also stores --32 kcal mo1-1 (Cooper, 1979b; Boucher and Leblanc, 1985; Schick et al., 1987), whereas the photoisomerization of the 11-cis RPSB in solution generates a more stable species, provides further evidence that the protein is modifying both the ground and the excited state potential surfaces. The observation that (I)~ + (I)2 add up to a number larger than unity (1.16+0.05) indicates that while a common excited state intermediate may be populated during photochemistry, coupling into the ground state is a trajectory dependent (i.e., dynamic) process (see below). A key conclusion is that the excited state Cl1=C~2 torsional surface is barrierless (Honig et al., 1979; Birge and Hubbard, 1980, 1981; Birge, 1981, 1982; Honig, 1982; Doukas et al., 1985; Birge and Callender, 1987; Birge et al., 1988) (Figures 9 and 14). As discussed above, it appears that the C9=C10 excited state torsional surface is not barrierless (Birge et al., 1988) (Figure 14). A study by G~irtner et al. (1991) recently incorporated insect retinals into bovine opsin and determined the quantum yields. Many insects utilize retinals that are hydroxylated at C 3 or Ca; such additions result in chromophores that are more polar and capable of hydrogen bonding to functional groups of the protein, although it is not well understood as of yet what, if any, physiological role these interactions play in the insect rhodopsins. It was found that the analog rhodopsin formed with 3-hydroxy-retinal had a 20% larger quantum yield than the bovine pigment (G~irtner et al., 1991). It was postulated that the higher quantum yield may have been due to steric forces in the binding site that would favor the photoproduct. Slow regeneration times were indeed observed for bovine opsin with 3-hydroxy-retinal, indicating steric restrictions in the bovine opsin binding site which had to be overcome on formation of the active synthetic pigment (G~irtner et al., 1991).

E. Molecular Dynamics of the Primary Event Tallent et al. (1992) recently carried out molecular dynamic simulations of the primary event in rhodopsin based on the binding site model shown in Figure 6. The


99

ground state and excited state surfaces connecting rhodopsin (R) and bathorhodopsin (B) along the ~1~,~2dihedral reaction path were partially adiabatically mapped. The ground state surface was generated by using MNDO/AM 1 procedures and the excited state surface was generated by using INDO-PSDCI procedures including both single and double configuration interaction. The resulting surfaces are shown in Figure 9. The first excited singlet state exhibits a barrierless reaction path for Cl1=C12 dihedral torsion with a local minimum (activated complex) centered at ~11,12 - 90°- Semiempirical molecular dynamics procedures were used to simulate the forward and reverse photochemistry, and the forward dynamics are shown in Figure 9. In the following sections we overview some of the more important observations of this theoretical study as it relates to various experimental observables and recent time-resolved studies of the primary event.

Origin of the Barrierless Excited State Surface We know from experimental studies that the excited state potential surfaces connecting rhodopsin and bathorhodopsin are barrierless (see above). In this section we provide a quantum mechanical perspective on this important characteristic. Many of the comments also apply to bacteriorhodopsin, but the lack of an accurate electrostatic map of the counterion environment in the bR binding site precludes definitive analysis. One of the key aspects of the binding site responsible for the generation of a barrier-less excited state potential surface is associated with the atomic charges on the chromophore in the ground and lowest excited singlet states. The imine nitrogen atom is negatively charged in protonated Schiff bases. The positive charge on the chromophore is highly delocalized, and the most positively charged atoms are C15, C13 and the imine proton (Tallem et al., 1992). Excitation of rhodopsin into the lowest-lying Franck-Condon excited state generates a large redistribution of charge resulting in the transfer of~-0.27 electron units of negative charge into the C13..-N16 portion of the polyene chain. The net charge on the C13--C14--C15--N moiety changes from +0.120 to-0.153 upon excitation (Tallem et al., 1992). This charge reorganization alters the electrostatic interaction with the counterion from a ground state stabilization into an excited state destabilization and forces the chromophore away from the counterion. Torsion about the C l l = C 12bond is the path of minimum energy, and thus the photoisomerization is initiated with a negative barrier. Torsion about the C~l=C12 bond is calculated to mix the second excited ,,1Ag•. state . into . the . lowest-lying . . ~Bu+,, state. In the torsional region 75--105 ° (90 ° = orthogonal), the lowest excited state has considerable ,,lAg,-,, character. This generates a local minimum in the excited state potential surface which is referred to as the "activated complex." The combination of charge reorganization within the chromophore and the partial switch from ionic to covalent character near orthogonality combine to generate a barrierless excited state potential surface for 11-c/s --~ 11-trans photoisomerization.

100


Kinetics of the Photoisomerization Recent experimental studies of the kinetics of the rhodopsin primary event were discussed in detail in Section IV-C. The femtoseconds experiments of Schoenlein et al. (1993) and Peteanu et al. (1991) predict the formation ofbathorhodopsin in ~200 fs. In contrast, Yan et al. (1991, 1993)predict the formation of an excited state complex in --200 fs with relaxation imo ground state bathorhodopsin in -3 ps. Regardless of which model is used, it is clear that the kinetic simulation of the excited state dynamics is overestimating the time necessary to reach the activated complex ((I)11_12 " 9 0 ° ) . We calculate ~375 fs (Figure 9) but the observed value is 200 fs or less. Thus, we are underestimating the steepness of the potential surface, overestimating the inertial component of the chromophore motion, or both. In addition, the simulations start the molecule from a rest position which eliminates thermal assistance of motion in the excited state. Because the center of mass of the ]3-ionylidene ring was fixed during the dynamics, it is likely that the translation of the center of mass of the chromophore during photoisomerization is overestimated. It is difficult to quantitatively analyze the other potential sources of error. Nevertheless, the above observations provide some insight into the two experimentally based models of the primary event. The theoretical dynamics shown in Figure 9 predict a 1/e time for bathorhodopsin formation of 886 fs (see Table III in Tallent et al., 1992). This number should be compared to the 200 fs formation time ofbathorhodopsin predicted by Peteanu et al. (1993) and Schoenlein et al. (1991). Again, the calculated value is too large, but that observation is consistent with our previous observation that the simulations overestimate the time to reach the activated complex. The excited state spectra that were calculated are, in general, consistent with the interpretations advanced by Schoenlein et al. (1991) and Peteanu et al. (1993) in analyzing the transient spectra shown in Figure 11. A key complication is the theoretical prediction that the principal absorption band in the 400-700 nm region is very similar in intensity and position to the ground state absorption spectrum of bathorhodopsin (see Figure 9 ofTallent et al., 1992). However, the calculated spectra display a doublet structure when the ensemble enters the activated complex, and there is an additional absorption band in the 650-850 nm region (see Figure 9 of Tallent et al., 1992). As the ensemble relaxes in the activated complex, the red band decreases in intensity and blue shifts. If one compares the spectra of Figure 9 and 10 from Tallent et al., 1992 to those shown in Figure 11, one could reasonably interpret the spectral features to indicate the following sequence:

R(ll-cis)

hv

> R(FC)*

~ 150)fs

R(Zll)

--300fs

~ Bhot(ll_transoid ) ~__~s B(ll-trans)

where FC refers to the Franck-Condon vertically excited level, x lx refers to the Cl1~C12 orthogonal activated complex, and the subscript "hot" describes a vibrationally activated ground state. The above kinetic assignments are based on an


101

analysis of the weak bands that appear above 600 nm and a comparison of these bands with the calculated S 1---~Sn transitions under the assumption that the experimentally observed bands are indicative of excited state occupation. This assumption is consistent with the observation that the reformation time of rhodopsin lies somewhere between 2 and 6 ps. Our excited state absorption calculations are also consistent with the model of the rhodopsin dynamics proposed by Yan et al. (1991, 1993). However, our dynamics simulations conflict with the ratio of bathorhodopsin formation versus activated complex formation. The model proposed by Yan et al. (1991, 1993) can be described by the following series: hv

R(ll-cis) ~

R(FC)*

"~Fs

,

R0:ll) 2 ~

B(ll-trans)

We make the assumption that the 3 ps time constant for batho formation represents the time leading to formati'on of the relaxed bathorhodopsin product. This assignment yields a ratio of B/R(z11)* ~ 15. The dynamics shown in Figure 9 yield the following times: hv

R(ll-cis) ---> R(FC)* 3]~s R(z11), 8_~s Bhot(ll_transoid ) l:~_~SB(ll_trans ) Thus, the calculated ratio of B/R(Zll)* is 3.6. This is a rather significant difference which indicates an intrinsic flaw in either the calculated dynamics or the model used to interpret the time-resolved data. Yan and coworkers are currently carrying out additional time resolved experiments to help address some of the issues discussed above as well as the significant differences between the two experimental models. These experiments hope to observe some of the red-shifted absorption bands that are predicted to populate the excited state absorption spectra in the 700-900 nm region. The theoretical simulations predict that these features will be highly diagnostic of the excited state torsional angle (see Figures 9 and 10 in Tallent et al., 1992).

Quantum Yield of the Photoisomerization Weiss and Warshel (1979) have proposed that nonadiabatic coupling is the primary contributor to the transfer of rhodopsin from the excited singlet state surface into the ground state manifold. Their analysis, using a pair oforthogonalized LOwdin P7 atomic orbitals, yielded Pna ~-0.5, where Pna is the probability of crossing associated with nonidiabatic coupling as the trajectory passes through the orthogonal region. This value yields excellent agreement with the observed rhodopsin --~ bathorhodopsin quantum yield assuming that this probability is identical on each pass through ~ ) 1 1 , 1 2 " - 90°: 1 tI) 1 =

-

2

-

- Pna

1 --

2 - 0.5

--

0.67

102


There are two features of the Weiss and Warshel model, however, that deserve fiLrther examination. First, if Pna ~ 0.5 for all trajectory passes, then we may conclude that • 2 v_ O1, and thus the bathorhodopsin ~ rhodopsin quantum yield calculated by using the Warshel-Weiss model will be significantly overestimated unless bathorhodopsin has an unusually large non-dynamic decay mode into the grotmd state. The latter possibility is unlikely given the large excited state gradient that is calculated for bathorhodopsin. Second, extrapolation of integral calculations based on two orthogonalized L6wdin Pz atomic orbitals to a polyene with six double bonds will likely overestimate the integrals. However, previous simulations based on purely dynamic (semiclassical S-matrix) coupling consistently underestimated the quantum yields (Birge and Hubbard, 1980, 1981). Tallent et al. (1992) investigated the contributions of nonadiabatic coupling to the quantum efficiencies associated with the dynamic simulations shown in Figure 9. The nonadiabatic probability was explicitly calculated by reference to the phased ground and excited state wavefunctions, and improved the calculated values from OI(R-~B ) = 0.62 and O2'(B--+R ) = 0.48 to • 1 = 0.70 and O 2' -" 0.52, where (I) 2' represents the quantum yield for the B--~R photochemistry when the path to isorhodopsin has been ignored. The experimental values are • 1 = 0.67 +_ 0.02 and • 2' - 0.53 _+0.03. Bathorhodopsin photoconverts to both rhodopsin (0 2 -- 0.49 _+0.03) and isorhodopsin (9-cis-chromophore) (0 3 - 0.076 +_0.006) (see Section IV-D). Thus the adjusted (isorhodopsin pathway excluded) value is: 02 02'-1-0 3

0.49 + 0.02 = 0.53 + 0.03 1-0.076+0.006

When nonadiabatic coupling is included, the forward (O1) quantum yield is overestimated by--4% and the reverse is underestimated by 2%. Given the level of approximation inherent in the theoretical procedures, this represents good agreement with experiment. The nonadiabatic coupling term, when properly phased, changes sign at *11,12 ~ 92 ° . Thus, coupling into the ground state is enhanced preferentially for the forward (R -~ B) trajectory relative to the reverse (B -~ R) trajectory through the (~11,12 -90 ° crossing point. Thus, the contribution of nonadiabatic coupling to the probability of crossing into the ground state is partitioned into trajectory dependent contributions which preferentially enhances • 1 relative to • 2. Nonadiabatic coupling also increases the overall efficiency of coupling into the ground state and decreases the product formation time for both the forward and reverse photochemistry (Tallent et al., 1992). The lower quantum yield of the bathorhodopsin -~ rhodopsin photoisomerization is due to the above partitioning of the nonadiabatic coupling as well as the rapid arrival of the trajectory into the activated complex. The latter precludes equilibration of the excited state prior to arrival at the activated complex and lowers the dynamic coupling term.


103

F. Energy Storage in the Primary Event The first photocalorimetric measurement of the energy stored in the primary photochemical event of rhodopsin was carried out by Alan Cooper in 1979, and his measurement of AHRB - 34.7+_2.2 kcal mo1-1(Cooper, 1979b) prompted considerable interest in the mechanistic origins. First, this value indicates that-%0% of the absorbed photon energy is converted into stored energy, an efficiency that seems unrealistically high given the concomitant high quantum efficiency of 0.67 (net system efficiency ~ 40%). Second, models of the primary event published during the same year predicted much lower values: (e.g., 26 kcal mo1-1 (Birge and Hubbard, 1980) and 14-28 kcal mol-l (Honig et al., 1979). A subsequent experimental study using a different technique (pulsed laser photocalorimetry) and a range of excitation wavelengths yielded AHRB= 32.2+_0.9 kcal mo1-1 (Schick et al., 1987) in good agreement with Cooper's measurement. This study also measured the energy stored in the isorhodopsin -~ bathorhodopsin phototransformation and observed AHm = 27.1_+3.2 kcal mo1-1 (Schick et al., 1987). This value of AHIB indicates that the bathorhodopsins formed from rhodopsin and from isorhodopsin are energetically equivalent, because isorhodopsin has an enthalpy -~5 kcal mo1-1 higher than rhodopsin (Cooper, 1979a). This observation, combined with optical spectroscopic studies, confirms the fact that the bathorhodopsins formed from rhodopsin and isorhodopsin are energetically identical though minor spectroscopic differences can be noted (Monger et al., 1979; Mao et al., 1980; Einterz et al., 1987a,b; Hug et al., 1988). A schematic diagram showing the ground and excited state energetics is presented in Figure 14. The molecular origins of energy storage in bathorhodopsin remain a subject of debate. Some of the early models emphasized energy storage due to charge separation (Honig et al., 1979) while other early models emphasized energy storage due to conformational distortion (Birge and Hubbard, 1980, 1981). Virtually all models, both past and present, recognize that both mechanisms contribute, and discussions center on the extent to which one mechanism dominates the other. An experimental and theoretical study of energy storage in bathorhodopsin yielded the following partitioning: charge separation (-~12 kcal mol-1), intrachromophore-lysine conformational distortion (-~10 kcal mol-l), and chromophore-protein conformational distortion (-10 kcal mo1-1) (Birge et al., 1988). Palings et al. (1989) note the contributions of electrostatic interactions in determining the energy storage based on the observation of a perturbed hydrogen out-of-plane wagging and C-C stretching vibrations associated with the C10-C1~=C~2-C13 region of the chromophore.

G. Molecular Origins of Photoreceptor Noise The human visual system can detect faint stars at night and distinguish objects in direct sunlight for an effective operating range of about 10 log units of light

104


intensity. At high light intensities the key limitation of visual function is the bleaching of photoreceptor pigments. At low light levels the key limiting factor is noise in the photoreceptors. We can reliably detect pulses of light that send roughly 100 photons through the pupil and activate approximately 10-20 rhodopsin molecules in as many rod photoreceptors. We see such dim flashes of light against a background of noise caused by each photoreceptor eliciting false signals ("noise") which are indistinguishable from the signals triggered by single photon absorptions (Baylor et al., 1980). The origin of this photoreceptor noise remains a subject of debate and a number of recent articles have examined this subject from diverse perspectives (Cornsweet, 1970; Baylor et al., 1980; Aho et al., 1988; Barlow, 1988; Barlow, Jr. and Kaplan, 1989; Barlow, Jr. and Silbaugh, 1989; Birge, 1990a, 1993; Fahmy and Sakmar, 1993; Barlow, Jr. et al., 1993; Birge and Barlow, 1994). In this section we examine the origin of photoreceptor noise and demonstrate that the available evidence supports a two-step molecular process inside the protein binding site of rhodopsin as the primary source. We also discuss how nature has optimized the active site of rhodopsin to minimize the thermal reactions that are responsible for generating the false signals.

Rhodopsin is the Source of Photoreceptor Noise Although the origin of photoreceptor thermal noise may remain a subject of debate, investigators agree that thermal and light activated photoreceptor signals are identical with respect to intensity and temporal profile (Baylor et al., 1980, 1984; Aho et al., 1988; Barlow, 1988; Barlow, Jr. and Kaplan, 1989; Barlow, Jr. and Silbaugh, 1989; Barlow, Jr. et al., 1993; Birge and Barlow, 1994). Thus, it is important to understand the nature of the light activation and amplification process. The thermal noise phenomenon must involve either a side reaction or corruption of this process prior to amplification, or a side reaction that undergoes nearly identical amplification. Following the primary event, a series of dark reactions occur which ultimately deprotonate the chromophore and activate the protein. The activated protein initiates a complex biochemical process that hyperpolarizes the plasma membrane of the rod cell in the retina (Liebman et al., 1987; Stryer, 1986, 1987). An analysis of the amplification mechanism coupled with the observation that the intensity and duration of photoreceptor noise signals are identical to light-induced signals leads to the conclusion that if the source of the noise involves a thermal corruption of the above process, rhodopsin must be the source of the noise. That is, activated rhodopsin is being generated by a thermal process, and the resulting R** (i.e., R~hermal)is identical to R* (i.e., Rhv) with respect to the catalytic amplification process described above. There is, however, one other possibility that needs to be considered. The connections among the neural elements in the retina are complex and are responsible for extensive signal processing prior to transferring the photoreceptor signals to the brain. Because the bipolar cells that mediate the photoreceptor signals prior to transfer to the ganglion network have a signal leveling effect, the observation that dark signals have the same shape and intensity


105

may indicate that the bipolar cells have mediated the signal. Thus, the origin of photoreceptor noise may be within the neural network rather than the photoreceptor system. There are two observations that argue against this interpretation. First, the nature of the photoreceptor noise in humans is invariably perceived as point source noise (Cornsweet, 1970). This observation supports a model in which thermal activation involves individual photoreceptors rather than mediating neural elements. Perhaps the strongest argument in favor ofrhodopsin is the observation that there is a near linear proportionality between dark noise and the rhodopsin content of photoreceptors in a range of animals (Figure 15). Because the animals analyzed in Figure 15 have significantly different neural architectures, the observed linear relationship suggests that photoreceptors and not neural elements are responsible. The data of Figure 15 coupled with the observed invariance between light-induced and thermal photoreceptor signals points to rhodopsin as the source. There are many possible mechanisms that could be responsible for the thermal activation of rhodopsin. In the following section, we examine and compare the various possibilities with the goal of eliminating those which are inconsistent with thermodynamic, kinetic, or spectroscopic observation.

Possible Mechanisms of Thermal Activation of Rhodopsin Baylor and coworkers have carried out a detailed analysis of electrical dark noise in toad retinal rod outer segments, and assigned the thermodynamic properties of the thermally activated dark processes: (E a = 21.9+1.6 kcal moV l, AG* = 31.9+0.13 kcal mo1-1, AH* = 21.6+1.6 kcal mo1-1, AS* =-35.3+5.6 e.u.) (Baylor et al., 1980). The activation energies measured for the horseshoe crab (Limulus) agree within experimental error (E a = 26.3+7.8 kcal tool-1 (day), 27.9+6.5 kcal roof 1 (night), 26.5+7.5 kcal mo1-1 (in vitro)) (Barlow, Jr. and Kaplan, 1989; Barlow Jr. and Silbaugh, 1989; Barlow, Jr. et al., 1993). We will discuss the Limulus data in more detail below, because this animal is capable of dramatically decreasing photoreceptor noise at night (see Figure 15). A comparison of these data with denaturation activation energies measured by Hubbard for cattle rhodopsin (E a = ---100kcal mol-l), frog rhodopsin (E a = --'45 kcal mo1-1) and squid rhodopsin (E a = -72 kcal moV 1) indicates that protein denaturation is not the origin of the dark signal (Hubbard, 1958). Measurements of thermal isomerization of 11-cis retinal, however, appear to offer a much more compatible set of thermodynamic properties (E a = 22.4 kcal mo1-1, AG* = 29.3 kcal mo1-1, AH* = 21.7 kcal mo1-1, AS* = - 2 1 . 4 e.u.) (1-propanol solution) (Hubbard, 1966). Comparison of the latter measurements on 11-cis retinal with those observed by Baylor on rod segments has prompted some investigators to propose that thermal isomerization of the chromophore is responsible for dark activation of rhodopsin (Baylor et al., 1980; Aho et al., 1988; Barlow, 1988). However, this hypothesis is not consistent with the energetics of ground state isomerization of the protein bound chromophore (Birge, 1990a). The protein bound chromophore is not 11-cis retinal, but the protonated Schiff base of l l-cis retinal. The ground state barrier to

106


LIMULUS • (day) r.~

r.~

¢

>

0.1

m m mm m mm

o~

m

BULLFROG

Z ell

TOAD

HUMAN

~, 0.01

m m

"

m

m

MONKEY eJ

mu mm m n m

,4,,,I

mm

DOGFISH

m

LIMULUS (night) "

LOCUST

0.001

I

,

m

m

,m,,,l

I

I iIiiim

10 0.1 1 Rhodopsin Content (10 9 molecules cell "1)

Figure 15. Dark noise and rhodopsin content of photoreceptors. Dark noise for these various visual systems is approximately proportional to rhodopsin content (slope of dashed line = 1) supporting the concept that the rate constants for noise are about equal and that rhodopsin is the source of the thermal noise (Adapted from Birge and Barlow, 1994). isomerization of the protein bound chromophore is estimated to be AH$ = 45+3 kcal mo1-1 (Birge, 1990a). We can establish a lower limit ofAH $>_42+3 kcal mo1-1 based on the relative enthalpy ofbathorhodopsin (AHRB = 32.2+0.9 kcal mo1-1) (Schick et al., 1987) plus the activation enthalpy of the bathorhodopsin --->lumirhodopsin dark reaction (AH~ = 10+_2 kcal mo1-1) (Grellmann, et al., 1962) and assuming additive errors. In contrast, the activation energies for thermal activation are all less than 36 kcal/mol, and a majority are less than 30 kcal/mol. Thus, thermal (ground state) isomerization of the native (protonated) chromophore cannot be responsible for thermal activation of the protein.


107

Rhodopsin might have sufficient conformational flexibility to undergo a conformational change (R --%R**) that is interpreted (incorrectly) by transducin (T,~C GDP) to represent photochemically activated rhodopsin (R*). Thus the initial step in the amplification process takes place involving this thermally activated rhodopsin (TaI3cGDP + R** ~ R** - T@cGDP + GTP --~ R**-Ta~cGTP + GDP). The key problem with this mechanism, however, involves the observation that the thermal noise signals have intensities identical to the light activated signals. Thus, R** (thermally activated rhodopsin) must have a lifetime nearly identical to R* (photochemically activated rhodopsin). This observation precludes enthusiasm for spontaneous conformational distortion, because the spontaneous process would either generate a very short-lived R**, or would require denaturation of the protein. Another possibility is that there is an equilibrium within the rhodopsin binding site coupling protonated versus unprotonated chromophores. The experiments of Longstaff and Rando have demonstrated that deprotonation of the Schiff base of retinal is obligate for rhodopsin activation (Longstaff et al., 1986; Longstaff and Rando, 1987). This mechanism assumes that deprotonation of the Schiff base is sufficient for activation. More precisely, deprotonation generates a form of rhodopsin that is interpreted by transducin as activated (i.e., R*), and the cascade is initiated. (Deprotonation would break the above mentioned salt bridge.) The observation that the thermal noise signals have intensities identical to the light activated signals requires that the rhodopsin molecule with an unprotonated chromophore have a lifetime identical to R*. This is unlikely due to the instability of the R d species. Recent site directed mutagenesis studies by Fahmy and Sakmar (1993) provide more explicit evidence that deprotonation is not sufficient for activation. These investigators replaced the primary counterion of rhodopsin, the glutamate (E) residue at position 113, with glutamine (Q) and regenerated the mutant opsin with 11-cis retinal. The E 113Q substitution dramatically decreases the pK a of the Schiffbase proton and generates a pigment containing a mixture of protonated and unprotonated retinyl chromophores with absorption maxima at 490 and 380 nm, respectively. The key observation is that the unprotonated (380 nm) species does not activate transducin which indicates that deprotonation is not sufficient to generate R**. The above observations coupled with the theoretical and experimental studies described below led us to conclude that the most likely mechanism for thermal activation ofrhodopsin is a two step process (Birge, 1990a, 1993; Barlow, Jr. et al., 1993). The first step is deprotonation of the 11-cis protonated Schiffbase chromophore. The second step is thermal 11-cis to 11-trans isomerization of the chromophore. This mechanism is schematically diagrammed in Figure 16 and the adiabatic potential surfaces generated by using MNDO/AM 1 and INDO-PSDCI molecular orbital theory are shown in Figure 17. The calculated adiabatic activation energies from Figure 17 are given below:

01%

~ .,

H~,~ ""

-,', 15-fold retarded compared to H. salinariurn SR-I). Also a gene 71% identical to sopI was recently identified in a Red Sea isolate Halobacterium sp. strain SGI (Soppa et al., 1993). Studies of the growing family of sensory rhodopsin variants can be expected to provide information regarding their evolution and structure/function relationships.

I!!. EVOLUTIONARY AND MECHANISTIC RELATEDNESS OF MICROBIAL RHODOPSINS AND VISUAL PIGMENTS The evolutionary relationships among archaeal rhodopsins, microbial eukaryotic rhodopsins, and higher animal visual pigments may be difficult to determine given

Microbial SensoryRhodopsins

209

the extreme phylogenetic differences. Sequence comparisons between the archaeal family and visual pigments (and related 7-transmembrane receptors) reveal no obvious primary structure homology (Henderson and Schertler, 1990; Soppa et al., 1993), however the secondary and tertiary structural motifs are quite similar and may be indicative of a progenitor protein. Several protein structure/function relationships in the two best characterized sensory retinylidene proteins, SR-I and bovine rhodopsin, are remarkably similar (compare for example Ganter et al., 1989 and Yan et al., 199 l a; Hofmann, 1995 and Spudich and Spudich, 1993). If the mechanistic similarities derive from convergent evolution they evidently reflect fundamental constraints on the design of retinal-containing sensory receptors. This is especially significant when the nonsensory 2 retinylidene proteins BR and HR differ in a particular property from SR-I and rhodopsin. In all of these proteins retinal binds in a protonated Schiffbase linkage to a lysine residue placed in the middle of the C-terminal (7th) transmembrane helix. Although the photoisomerization occurs across different double bonds in SR-I and rhodopsin (13-trans to 13-cis and 11-cis to 11-trans, respectively), retinal/apoprotein interactions in the sensory proteins are similar in that: (1) unlike in BR thermal isomerization of retinal is restricted, that is, only one configuration of the isomerizing double bond is tolerated in the pocket in the nonphotoactivated protein in the dark (13-trans in SR-I and 11-cis in rhodopsin); and (2) unlike in BR, formation of functional photoproducts requires steric interactions between retinal polyene chain methyl groups and protein residues; specifically apoprotein interaction with a methyl group near the photo-isomerizing double bond (the 13-methyl in SR-I and the 9-methyl in rhodopsin); and (3) formation of the signaling conformations of SR-I and rhodopsin ($373 and Metarhodopsin-II38 o, respectively) each require Schiffbase deprotonation, and, moreover both couple with membrane anchored transducers (HtrI and transducin) which influence the Schiffbase protontransfer reactions. Further understanding of similarities and differences between the SR-I/HtrI and rhodopsin/transducin signal relay mechanisms may well reveal fundamental principles in the design of retinylidene receptors and more generally, of 7-transmembrane receptors.

IV. SENSORY RHODOPSINS OF UNICELLULAR EUKARYOTES A. Chlamydomonasreinhardtii Introduction C. reinhardtii is a motile, unicellular alga that exhibits two behavioral responses to light which allow the cells to access photosynthetically optimal environments. One, phototaxis, is the orientation of the cells' swimming direction along the axis of a light beam. 1 The second light induced behavioral change is called the photo-

210

JOHN L. SPUDICH and DAVID N. ZACKS

phobic response (a.k.a. "stop" or "shock" response) and consists of a brief cessation in forward swimming on temporal changes in light intensity. Early photobiologists and microscopists qualitatively described these phenomena (Jennings, 1915; Buder, 1919), but did not define the receptors controlling these behaviors, and the field of Chlamydomonas phototaxis progressed slowly for almost half a century. Then, in the late 1960s interest renewed, with laboratories investigating the physiology of these photoresponses, primarily the calcium dependence of photoinduced behavioral changes. This literature reviewed by Feinleib (1980) and Nultsch (1983). The nature of the receptor for phototaxis was finally defined in 1984 by Foster and coworkers who provided evidence for the existence of a Chlamydomonas rhodopsin. This set off another wave of research, this time concentrating on the physical characteristics of the photoreceptor itself. Currently, we are at the threshold of understanding the receptor and transduction events involved in Chlamydomonas reinhardtii responses to light. Harz et al. (1992) and Witman (1993) review the phototransduction cascade. In this section we will review the physical properties of the photoreceptors controlling these two behavioral responses, the behavioral relationship between the two responses, and clarify several points which have caused a certain amount of confusion in the field.

Receptor Identification The first clues imo the nature of the photoreceptor came in 1971 when Nultsch and coworkers measured a phototaxis action spectrum from fluence response curves (responses to different intensities, or doses, of light) obtained at different wavelengths. Movements of cell populations were monitored in a "phototaxigraph" which records the cells' absorption of non-actinic, infrared light in two parts of a cuvette. Directional actinic light causes the cells to accumulate towards either end of the cuvette. This asymmetric cell distribution between the different parts of the cuvette results in a differential absorbance of the monitoring beams that is proportional to the cells' phototaxis response. The action spectrum thus obtained showed a major peak at 503 nm with a secondary peak at 443 nm. The shape of this spectrum was consistent with the presence ofa retinylidene photoreceptor, but did not exclude the possibility of a ravin and/or pterin based pigment. Foster and Smyth (1980), in their review of algal ultrastructure as it relates to phototaxis, suggested a new approach to measuring action spectra based on the threshold for receptor activation, rather than the more traditional finite-response or criterion-response action spectra used by Nultsch's group. The latter type of action spectra is based on the light intensity, at various wavelengths, required to elicit a criterion response. The threshold action spectrum, however, extrapolates the minimum light intensity at each wavelength required to elicit a just discemable response. The advantage of this technique is that the threshold intensity is a more accurate measure of the photoreceptor absorption, eliminating complications presented by the absorbance of screening pigments present in the cell, particularly the eyespot. They recalculated the action spectrum for phototaxis by extrapolating the fluence

Microbial Sensory Rhodopsins

211

response curves of Nultsch et al. (1971) to zero light intensity, and by doing so eliminated the minor peak at 443 nm observed in the finite-criterion action spectrum. This new threshold action spectrum fit well the absorption spectrum of bovine rhodopsin, suggesting a retinal-based photoreceptor for phototaxis. A test of the hypothesis that Chlamydomonas contains a rhodopsin came from the same group of Foster and coworkers (1984) using the general pigment-deficient mutant strain FN68 that lacks both the phototaxis and photophobic responses. The cells' ability to undergo phototaxis was shown to be restored upon the addition of retinal to the cell suspension, and the phototaxis threshold action spectrum maxima shifted depending on the isomer or analog used to reconstitute the response. These two facts: that the cells' maximum wavelength sensitivity depends on the analog used, and that the maxima are red shifted compared to pure retinal's absorption maximum, are consistent with the characteristics of all other known rhodopsins (reviewed by Balogh-Nair and Nakanishi, 1990), strongly indicating that the retinal was reconstituting a photoreceptor, and not effecting the response by some secondary mechanism. Further evidence supporting the existence of a rhodopsin in Chlamydomonas came from the hydroxylamine bleaching experiments of Hegemann et al. (1988). Hydroxylamine is known to remove the retinal moiety from archaeal and eukaryotic rhodopsins. Hegemann and coworkers showed that incubation of either wild-type cells or retinal-reconstituted FN68 cells with hydroxylamine followed by exposure to light resulted in a decrease ofphotosensitivity. This reduction in sensitivity could be reversed by washing out the hydroxylamine, and in the case of the FN68 cells, also adding back retinal, suggesting that the hydroxylamine only affected the interaction of the chromophore with the opsin. These findings are consistent with hydroxylamine bleaching experiments done in other rhodopsins (Oesterhelt et al., 1974). The experiments of Foster and coworkers described earlier used a petri-dish population migration assay to measure and quantify the phototaxis response. In this assay, actinic light is delivered from the side onto a uniform distribution of cells spread over a petri dish. If the cells accumulate towards either end of the dish, then they produce a clear zone. The degree of cleating on the side facing the light minus the degree of clearing on the side opposite the light is taken as an index of phototaxis. This index taken over time gives the phototactic rate of the population. This assay provides an easy method for determining general phototaxis capabilities of cell populations, but cannot provide quantitative measurements of cell motility or individual cell responses. These limitations must be considered when evaluating the conclusions of these papers regarding the chromophore properties discussed in the next section.

ChromophoreProperties In Foster and coworkers' experiments (1984), restoration of the phototaxis response sensitivity with the different natural isomers of retinal: 9-cis, 11-cis, and

212


all-trans was maximal with 11-cis retinal. The suggestion was made that the native chromophore for the phototaxis photoreceptor in Chlamydomonas is 11-cis retinal, similar to the chromophore for visual rhodopsin (Wald, 1968) but unlike the archaeal rhodopsins which have all-trans retinal as their chromophore (Ottolenghi and Sheves, 1989). In 1987, Foster and coworkers reported evidence from reconstitution studies with analogs that the retinal chromophore is in a planar conformation across the C6-C7 single bond which links the retinal's polyene chain to its ionone ring. This is the conformation of the archaeal rhodopsins' chromophores (Bogomolni and Spudich, 1991), but not of the visual rhodopsins' chromophores (Birge, 1990). The primary transduction event in known retinylidene proteins is the isomerization of the chromophore across one of the double bonds in the polyene chain. In visual rhodopsins 11-cis retinal isomerizes to all-trans retinal (Birge, 1990) and in the archaeal rhodopsins all-trans retinal isomerizes to 13-cis retinal (Bogomolni and Spudich, 1991). Foster et al. (1988b, 1989) incorporated over 20 analogs of retinal and reported that they all restored phototaxis in Chlamydomonas reinhardtii, as measured with the petri-dish assay. They concluded that no retinal isomerization was necessary since analogs which were prevented from isomerizing across any or all of the chromophore's double bonds restored phototaxis. Even hexanal--which does not have any carbon-carbon double bonds----restored light sensitivity. The authors reported shifted action spectra peaks, indicating that the analogs were binding to the opsin apoprotein and forming a functional photoreceptor. These findings were extended in later work from the same group (Nakanishi et al., 1988), where they put forth the hypothesis that charge redistribution and not isomerization was the mechanism of light energy transduction in the phototaxis photoreceptor of C. reinhardtii (this hypothesis is discussed in greater detail in Foster et al., 1991). The hypothesis and the evidence supporting it are in direct contrast to the dogma in the field of visual science that chromophore isomerization is necessary for photosensitivity, and indicated, therefore, a novel mechanism of photosensory transduction. However, there are several arguments based on further information and more highly resolving phototaxis assays which suggest reevaluation of this conclusion. The mutant strain FN68 exhibits a property which suggests an alternative explanation for some of the results. Light exposure alone, without the addition of exogenous retinal, restores the phototaxis response (Foster et al., 1988a). The mechanism by which this effect occurs is unknown, but it is not inhibited by the protein synthesis inhibitors cycloheximide and/or chloramphenicol (Foster et al., 1988a). Foster's group concluded that light itself induces the synthesis of retinal, and that a retinylidene protein is the photoreceptor responsible for the auto-induction of photosensitivity since the action spectrum of the light induced photosensitivity shifted to the same extent as that of phototaxis when a retinal analog was added. The petri-dish phototaxis assay used in all these experiments exposes the cells to 10 minutes of light, an amount of time sufficient to restore considerable phototaxis sensitivity in the cells (Beckmann and Hegemann, 1991). Thus, the possibility


213

arises that the non-isomerizing analogs found to restore phototaxis actually serve as the chromophore for photoinduction of phototaxis, and the observed shifts in phototaxis action spectra maxima actually correspond to the photoinduction receptor. An alternative mechanism of energy transduction that does not involve chromophore isomerization may occur with the photoinduction phenomenon (an interesting possibility in and of itself) but isomerization may still be required for photobehavior. In the mutant strain FN68 both the photoinduction of photosensitivity and photobehavior are restored by retinal addition, making independent analysis of each photoresponse more difficult. Recently, several groups have reexamined the chromophore properties of the photoreceptors using assays with higher resolution than that provided by the petri-dish test. One technique employed is a computerized videomicrographic motion analysis system for monitoring photophobic responses in Chlamydomonas. In this assay individual cells are monitored by infrared, dark field microscopy and their swimming behavior is digitally recorded and analyzed as a function of time by computer. This type of assay was first developed for monitoring light induced reversals in the archaeon H. salinarium (Takahashi and Kobatake, 1982; Sundberg et al., 1986), and applied to the description of Chlamydomonas photophobic responses by Hegemann and Bruck (1989). The primary advantage this type of assay has over a population assay is that the behavioral responses of individual cells to light stimuli are measured. Using a computerized system allows for the analysis of motility and swimming parameters of large numbers of cells, thus providing more accurate population behavioral data as a function of individual cell motion. Lawson et al. (1991) used computerized cell tracking and motion analysis to study the photophobic responses in a second pigment-deficient mutant CC-2359. This mutant was shown to be insensitive to photophobic stimuli over a wide range of light intensities, and did not show regeneration of sensitivity upon light exposure. The photophobic stimuli used were 5 to 20 msec pulses of 500 nm light, as opposed to 10 min exposure periods used in the petri-dish assay, greatly reducing possible light effects on the retinal isomers and analogs themselves. The group tested the ability of various retinal isomers and analogs to reconstitute the photophobic response. Their experiments showed that all-trans retinal reconstituted the photophobic response most efficiently, followed, in decreasing order of efficacy, by the 13-cis, 11-cis and 9-cis isomers. This indicated that the polyene chain configuration of the natural chromophore for the photophobic receptor is all-trans, in contrast to the conclusions of Foster et al. (1984) for phototaxis. An analog with the C6-C7 bond "locked" in a trans configuration was able to reconstitute photosensitivity, whereas an analog with the same bond "locked" in a cis configuration was not. These data strongly suggested that the polyene chain is planar and in a trans conformation with respect to the ionone ring. As mentioned before, a central dogma of visual science is that signal transduction by rhodopsins is initiated by the retinal chromophore's isomerization on light stimulation. Lawson et al. (1991) tested the ability of 13-trans locked and 13-cis

214


locked retinal analogs to reconstitute photophobic responses in CC-2359. Neither of these analogs were able to restore sensitivity to photophobic stimuli, and both these analogs competitively inhibited all-trans retinal from reconstituting the response. Inhibition occurred regardless of whether the locked retinal analog had already been added to the cells and their response reconstituted, or whether the analogs were preincubated with the cells before retinal addition. These experiments solidified the idea that the retinal was binding to a receptor protein, in the same manner that a ligand binds to receptor, and suggested that the retinal/apoprotein interaction (presumably including a Schiff base linkage) is more dynamic than previously thought for retinylidene proteins. Dissociation and inhibition constants were determined for the all-trans retinal, 13-trans locked retinal, and the 13-cis locked retinal of 2.5 x 10-11 M, 5.2 x 10-:° M, and 5.4 x 10-9 M, respectively. The authors state, however., that these numbers represent only apparent in vivo dissociation and inhibition constants, and that these values may well be scaled by some factor, but that the relative order of affinities should not be affected. The two major conclusions from this workman all-trans chromophore, and 13-14 double bond photoisomerization--distinctly differ from the conclusions of Foster and coworkers, as described above, and suggested that the chromophore of the photophobic response receptor is more similar to the chromophores of the archaeal rhodopsins than to the chromophores of other known eukaryotic rhodopsins. These two points have been independently confirmed by other groups. Hegemann et al. (1991) reconstituted the same strain CC-2359 with several different retinal isomers and analogs, and monitored phototaxis in a taxigraph (similar in principle to Nultsch's apparatus described above) and in a light scattering apparatus. The light scattering assay is an indirect method for examining whole cell population responses to light stimuli, which correlates well with the photophobic and phototaxis responses, depending on the stimulus intensity (Uhl and Hegemann, 1990). Their results also show greatest reconstitution efficiency with the all-trans isomer of retinal, followed by the 13-cis, 11-cis, 9-cis and 7-cis isomers. Analogs lacking the ionone ring, but with at least 3 conjugated double bonds in the polyene chain also restored photosensitivity, but at much lower efficiencies, suggesting a "minimal" polyene chain chromophore structure capable of reconstituting phototaxis. Analog molecules prevented from isomerizing around any of the polyene chain double bonds, by the introduction of a phenyl ring into the polyene system, do not reconstitute responses, providing further support for the role ofisomerization in phototransduction. Hegemann et al. (1991) also investigated the role of each of the methyl groups at the C9 and C 13 positions using analogs lacking them. Chromophores lacking the C9 methyl group were capable of reconstituting photosensitivity, but not chromophores lacking the C 13 methyl group. Bovine rhodopsin requires the C9 methyl group for activation (Ganter et al., 1989), whereas SR-I requires the C 13 methyl group (Yan et al., 1991a). Hence, the C 13 methyl requirement further indicates the chromophore environment of the Chlamydomonas photoreceptors more closely resembles the archaeal chromophore environment than that of visual pigments.


215

Takahashi et al. ( 1991) developed a motion analysis system for the simultaneous study of photophobic and phototaxis responses. Their reconstitution experiments also showed that both responses were more efficiently restored by all-trans retinal than by 11-cis retinal, in strain CC-2359, and that analogs prevented from isomerizing around the C 13-C 14 double bond were ineffective in reconstituting photosensitivity. Further work by this group (Takahashi et al., 1992) used analogs known to slow the photocycle of the archaeal SR-I and SR-II, and found that they caused an inversion of the sign of phototaxis in strain FN68. That is to say, FN68 cells, which are normally only negatively phototactic when reconstituted with all-trans retinal, became only positively phototactic when these analogs were incorporated into the photoreceptor. This is the first evidence, albeit indirect, that there is a photocycle in the photoreceptor, and that the period of this cycle may be working in conjunction with the cell's rotation and eyespot shading to control the direction of phototaxis. If one of these factors is shifted out of phase, then the sign of the response changes accordingly. Chromophore extraction experiments (Derguini et al., 1991; Beckmann and Hegemann, 1991) have shown the presence of all-trans retinal but not of 11-cis retinal. These studies together provide very convincing evidence that the photophobic and phototaxis receptors contain chromophores that are in an all-trans configuration, 6-s-trans conformation, and that isomerization across the C13-C14 double bond is required for behavioral responses to occur. The differences between these conclusions and those made by Foster's group have been usually explained away as due to strain differences, different types of assays employed, and different responses examined. To help resolve these discrepancies direct comparison of the chromophore properties of the photophobic and phototaxis receptors in strain FN68 was undertaken (Zacks et al., 1993). Analogs and isomers of retinal were compared in their ability to reconstitute both photoresponses in the same population of cells. Behavior was analyzed with a computerized videomicroscopic motion analysis system similar to the one used by Lawson et al. (1991) for photophobic response analysis and modified to also allow a direct examination of large number of individual cells' phototaxis responses. Both photoresponses are most effectively reconstituted by all-trans retinal, followed by the other isomers in the same order as described in Lawson et al. ( 1991) and Hegemann et al. (1991). Neither response in this strain is reconstituted by the analogs prevented from C13-C14 cis/trans isomerization, consistent with the results in strain CC-2359. A difference between the chromophore properties of the two responses appears, however, in the inhibition of all-trans retinal reconstitution by the nonisomerizable 13 trans-locked analog. The photophobic response reconstitution is inhibited, as is the case in strain CC-2359 (Lawson et al., 1991). The phototaxis response however, does not decrease even at very high concentrations of this analog. A second difference found between the two responses is in their desensitization to repetitive light stimuli. Cells become desensitized to photophobic stimuli after repetitive flashes of light (Hegemann and Bruck, 1989) or after several seconds exposure to continuous light (Zacks et al., 1993), whereas the phototaxis response remains fully sensitive.

216


Relationship Between the Photophobic and Phototaxis Responses High speed cinematographic analysis of Chlamydomonas swimming behavior shows that as the cells swim they rotate around their longitudinal axis forming a helical path (Kamiya and Witman, 1984; Ruffer and Nultsch, 1985). The cell's eyespot region, which is thought to contain the photoreceptors for phototaxis (Melkonian and Robenek, 1980; Foster and Smyth, 1980), normally faces the outside of the helix (Kamiya and Witman, 1984; Ruffer and Nultsch, 1985). Ruffer and Nultsch (1991) concluded that the helical swimming pattern is important for phototaxis orientation because this type of movement generates periodic illumination and shading of the photoreceptor region by the eyespot. Their conclusion was strongly supported by their demonstration that increases and decreases in light intensity cause asymmetric changes in the two flagella's beat pattems which allow the cell to orient relative to a light beam. It is not known whether phototaxis and photophobic responses are mediated by the same photoreceptor. One possibility is that each response has its own receptor and transduction mechanism. The function of the photophobic desensitization in this case would be simply to prevent photophobic reactions from interfering with phototaxis. A second possibility, however, is that the two light induced behaviors are controlled by the same photoreceptor. This would be possible if the flagellar beat changes which cause photophobic orientation are the residual of the photophobic responses after desensitization (i.e., "miniphotophobic" reactions which cause brief reorienting motions rather than full stops). Photophobic desensitization, in this case, would need to cause a precise grading of the photoreceptor output to yield at different light intensities the asymmetric flagellar beat patterns observed by Ruffer and Nultsch (1991) instead of full-fledged stop responses. Supporting the notion of a graded output, a continuum in the response to photophobic stimuli was observed by Lawson et al. ( 1991) who reported that low intensity flashes cause cell tuming, whereas higher intensity flashes generate stops. Zacks and Spudich (1994) report three specific experimental observations which support the graded output model, which are: (1) The desensitization of the photophobic response obeys Weber's Law (i.e., at each light intensity (I) in the cells' dynamic range responses to photophobic stimuli are desensitized to a point where the relative contrast (AI/I) generated by the eyespot yields a constam photophobic signal regardless of the signal expected from the absolute contrast (AI) at the eyespot; (2) Cells exhibiting faster rates of desensitization of photophobic responses have a faster onset of phototaxis; and (3) Cells orienting to a light beam exhibit a loss of orientation when the light is suddenly shifted to a lower intensity, due to the more highly desensitized state of the cells relative to the lower intensity, and the consequent deviation of the relative contrast from its constant value. These data are consistent with there being only one receptor for both photobehaviors and with the photophobic response providing the elemental course corrections causing phototaxis orientation.


217

Pigment Localization Ultrastructural examination of the Chlamydomonas cells has suggested an intimate relationship between the eyespot structure and the photoreceptor. The eyespot has been prolSosed by Foster and Smyth (1980) to function as a quarter-wave plate interference reflector. In their model, the eyespot serves to reflect and concentrate light onto a plane, in which the receptor is presumably located, parallel to the eyespot surface. Recently, Kreimer and Melkonian (1990) have tested the reflection hypothesis in different algal species using laser confocal scanning microscopy, and confirm increased reflection intensity caused by the eyespot. This group has extended their study (Kreimer et al., 1992) to the eyespot reflectance in wild-type and eyespot deficient mutants of C. reinhardtii, again confirming the eyespot's role as a reflector. Electron microscopy of the FN68 and CC-2359 mutants reveals an eyespot lacking pigment granules in the former and no detectable eyespot structure in the latter (M. Lawson and E Satir, personal communication). Since both strains exhibit photophobic and phototaxis responses, a fully developed eyespot structure is not essential for either behavior, although it probably increases photodetection efficiency and contrast. In C. reinhardtii the outer chloroplast membrane and the cell membrane are positioned in very close proximity to the eyespot and overlay the structure in parallel planes. According to the theory of Foster and Smyth (1980) the eyespot increases the light intensity at these membranes. Melkonian and Robenek (1980) observed structural specializations in these membranes, and suggested that they may represent the photoreceptors and/or transduction apparatus involved in phototaxis. In vivo microspectrophotometric measurements of this region in wild-type cells show an absorbance peak at 500 nm with a bandwidth that fits standard absorption spectra of rhodopsins (Crescitelli et al., 1992).

Pigment Enrichment In vitro studies on Chlamydomonas photoreceptors have proven difficult due to the lack of convenient enrichment procedures. To date, only two reports on pigment enrichment have been published. The first report, at a meeting poster session (Starace and Foster, 1989), describes reconstitution of light bleachable eyespot absorbance at 498 nm after addition of 11-cis or 9-cis retinal but not after addition ofall-trans retinal. Beckmann and Hegemann (1991) report.in detail the enrichment of membrane fractions from wild-type cells that show absorbance at 495 nm, with a shoulder at 465 nm. This absorbance is bleached by light, and restored by addition of all-trans retinal. The shoulder may derive from fine structure of the pigment spectrum, as is the case of the archaeal SR-II (Takahashi et al., 1990), or may be due to the presence of multiple pigments or pigment species. Radioactive retinal labeling of this fraction showed only one labeled band on an SDS-PAGE gel, with an apparent molecular mass of 32,000 kDa.

218


A genetic approach toward rhodopsin identification has been reported by Martin et al. (1986), where bovine opsin and Drosophila opsin cDNA probes were used to label C. reinhardtii genomic DNA. Several fragments of DNA were labelled, but rhodopsin-encoding genes in these fragments have not been reported.

B. Euglena gracilis Euglena gracilis is another motile, unicellular green alga that exhibits phototaxis and photophobic responses. The swimming motion of this organism is different than that of Chlamydomonas, because of a different flagellar architecture, and this results in a different manifestation of the light induced responses. Temporal changes in light intensity result in a type of tumbling behavior that decays much more slowly than the stop response of Chlamydomonas. One consequence of this response is that when cells move across a light dark boundary a ttmable is induced, causing them to accumulate in the area. The receptors responsible for these photoresponses have been the focus of several studies, and much of the evidence points to a flavin and/or pterin chromophore (Doughty and Diehn, 1980; Lenci et al., 1983; Ghetti et al., 1985, Schmidt et al., 1990). Recently, however, there have been some indications that a rhodopsin may be present in Euglena, and may serve as the photoreceptor for at least one of these photoresponses. Evidence for a retinylidene photoreceptor first appeared in 1989 when Gualtieri and coworkers reported a technique for isolating the paraflagellar swelling of Euglena gracilis. The absorption spectrum of these swellings taken with a microspectrophotometer showed a maximum at 500 nm with a bandwidth of approximately 80 nm. The absorbance is distinct from the absorbance of the eyespots and of the chloroplasts, and the authors suggested that this absorbance could derive from a rhodopsin molecule. Further work from this group (Barsanti et al., 1992) showed the elimination of paraflagellar swellings when the cells were grown up in medium containing nicotine. Nicotine is known to inhibit the biosynthetic pathway of carotenoids. Nicotine-treated Euglena cells did not photoaccumulate in an illuminated region of a petri dish, suggesting that perhaps the nicotine inhibited the production of a carotenoid chromophore for the photoreceptor. Carotenoids have been known to exist in Euglena for many years (Krinsky and Goldsmith, 1960). Batra and Tollin (1964) developed a method for enriching eyespot fractions from Euglena, and extracted the carotenoids lutein, cryptoxanthin, and 13-carotene. Suspensions of these eyespot fractions showed absorption maxima at 490,462, and 436 nm, which corresponded to the action spectrum profile for phototaxis. The suggestion was made that perhaps the eyespots contain the photoreceptor, but no further evidence was available to support this conjecture. In 1992, Gualtieri and coworkers extracted all-trans retinal from Euglena cells, and suggested that this chromophore derives from a rhodopsin-like photoreceptor. Unfortunately, retinal-deficient mutants of Euglena are not available, precluding the type of retinal reconstitution experiments that were done with Chlamydomonas.

Microbgal Sensory Rhodopsins

219

C. Spermatozopsis similis A third green alga, Spermatozops& simil&, has been studied spectroscopically, and evidence was found for a retinylidene pigment. Kreimer et al. (1991a,b) reported a technique for the enrichment ofeyespots and associated membranes. The absorption spectrum of this enriched sample is complex, due to the variety of carotenoids present in the eyespot, but a rapidly photobleachable peak at approximately 550 nm is resolved by difference absorption spectroscopy. Absorbance difference spectra between bleached versus unbleached samples show a shape characteristic of rhodopsins, with a peak at approximately 460 nm. Reconstitution of a bleached sample with 9-c& retinal yielded a species with a maximal absorbance at 540 nm. Both bleaching and reconstitution spectra taken over time form single isosbestic points at 400 and 480 nm, respectively. Isolated, non-bleached eyespot fractions contain extractable 11-c& and all-trans retinal, suggesting that retinal is the chromophore for this photoreceptor. These data strongly suggest the presence of a retinylidene protein, but further work needs to be done to correlate these findings to behavioral responses in the organism.

V. EUBACTERIAL RHODOPSIN-LIKE PHOTOACTIVITY: THE CASE OF PHOTOACTIVE YELLOW PIGMENT So far we have seen that retinylidene proteins exist in the archaea and in eukaryotes, both multi- and unicellular, but what about eubacteria? Retinylidene 7-transmembrane receptors have not been demonstrated in eubacteria, but a pigment implicated in phototaxis reception by the halophilic, phototropic eubacterium Ectothiorhodospira halophila exhibits an SR-I-like photoreaction. A search for soluble cytochromes and ferrodoxins in E. halophila uncovered the presence of large quantities ofa photoactive yellow pigment (PYP) (Meyer, 1985). This pigment is a protein of 15.3 kDa, with a maximal absorbance at 446 nm and an extinction coefficient of 48,000 M-lcm-1. PYP exhibits photocycling, with kinetics similar to sensory rhodopsin I ofH. salinarium (Meyer et al., 1987, 1989, 1991). The function of this pigment in photosensory reception has not yet been proven, but recent work on the motility behavior of this organism shows a repellent response wavelength-dependence with a peak matching the absorbance maximum of PYP (Sprenger et al., 1993). McRee and coworkers (1986, 1989) reported the crystallization and X-ray diffraction of PYP. The structure is totally different from that of rhodopsins (Henderson et al., 1990), and is characterized by a "13-clam" structure. This structure consists of two layers ofl3-pleated sheets, forming a pocket into which the chromophore inserts, as opposed to the 7 c~-helical structure of rhodopsins, but similar to the structure of known soluble carotenoid and lipid binding proteins. The exact chromophore species was not determined, but is believed to be similar to all-trans retinal (McRee et al., 1989), but probably not identical (Hoff, Van Beeumer, and Hellingwerf, unpublished results, as cited in Sprenger et al., 1993). Although PYP differs greatly in structure from rhodopsins,

220


the structural basis o f its SR-I-like p h o t o c y l e m a y p r o v i d e insight into the p h o t o t r a n s f o r m a t i o n s in the r h o d o p s i n family.

ACKNOWLEDGMENTS The authors were supported by National Institutes of Health grant GM27750 (JLS), National Science Foundation grant MCB-9219854 (JLS), and NIH training grant T32 BM07288 from the National Institute of General Medical Sciences (DNZ).

NOTES 1. In keeping with common usage the term"phototaxis" is used in two different senses in this review. For discussion of archaeal behavior the term is used in its general sense as any motility behavior in response to light. For discussion of algae where a finer distinction is important, we use the more restrictive definition ofphototaxis as orientation of swimming direction with respect to a beam of light. 2. Photoexcitation of BR has been shown to cause an attractant response.in energy-depleted cells (Bibikov et al., 1991, 1993: Yan et al., 1992). This appears to be a secondary consequence of membrane electrical potential changes due to proton pumping rather than to primary sensory signaling, such as that mediated by sensory rhodopsins.

REFERENCES Alam, M., & Hazelbauer, G. L. (1991). Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction. J. Bact. 173, 58375842. Alam, M., Lebert, M., Oesterhelt, D., & Hazelbauer, G. L. (1989). Methyl-accepting taxis proteins in Halobacterium halobium. EMBO J. 8, 631-639. Applebury, M. L., & Hargrave, P. A. (1986). Molecular biology of the visual pigments. Vision Res. 26, 1881-1895. Balogh-Nair, V., & Nakanishi, K. (1990). Visual pigment and bacteriorhodopsin analogs. In Chemistry and Biology of Synthetic Retinoids. Eds: Dawson, M., & Okamura, W., pp. 147-176. CRC Press, Boca Raton, Florida. Barsanti, L., Passarelli, V., Lenzi, P., & Gualtieri, P. (1992). Elimination of photoreceptor (paraflagellar swelling) and photoreception in Euglena gracilis by means of the carotenoid biosynthesis inhibitor nicotine. J. Photochem. Photobiol. 13, 135-144. Batra, P., & Tollin, G. (1964). Phototaxis in Euglena. I. Isolation of the eyespot granules and identification of the eyespot pigments. Biochim. Biophys. Acta. 79, 371-378. Beckmann, M., & Hegemann, P. (1991). In vitro identification of rhodopsin in the green alga Chlamydomonas. Biochemistry 30, 3692-3697. Bibikov, S. I., Gfishanin, R. N., Kaulen, A. D., Marwan, W., Oesterhelt, D., & Skulachez, V. P. (1993). Bacteriorhodopsin is involved in halobacterial photoreception. Proc. Natl. Acad. Sci. USA 90, 9446-9450. Bibikov, S. I., Grishanin, R. N., Marwan, W., Oesterhelt, D., & Skulachev, V. P. (1991). The proton pump bacteriorhodopsin is a photoreceptor for signal transduction in Halobacterium halobium. FEBS Lett. 295, 223-226. Birge, R. (1990). Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin. Biochim. Biophys. Acta 1016, 293-327. Bivin, D. B., & Stoeckenius, W. (1986). Photoactive retinal pigments in haloalkaliphilic bacteria. J. Gen. Micrbiol. 132, 2167-2177.


221

Blanck, A., Oesterhelt, D., Ferrando, E., Schegk, E. S., & Lottspeich, E (1989). Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor. EMBO J. 8, 3963-3971. Bogomolni, R. A., & Spudich, J. L. (1982). Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proc. Natl. Acad. Sci. USA 79, 6250-6254. Bogomolni, R. A., & Spudich, J. L. (1991). Archaebacterial rhodopsins: Sensory and energy transducing membrane proteins. In Sensory Receptors and Signal Transduction. Modem Cell Biology. Vol. 10. Ed. J. L. Spudich and B. Satir. pp. 227-249. Bogomolni, R. A., Stoeckenius, W., Szundi, I., Perozo, E., Olson, K. D., & Spudich, J. L. (1994). Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I. Proc. Natl. Acad. Sci. USA 91, 10188-10192. Bourret, R. B., Borkovich, K. A., & Simon, M. I. (1991). Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu. Rev. Biochem. 60, 401-441. Buder, J. (1919). Zur kennmis der phototaktischen Richtungsbewegungen. Jahrb. Wiss. Bot. 58, 102-220. Crescitelli, F., James, T. W., Erikson, J. M., Loew, E. R., & McFarland, W.N. (1992). The eyespot of Chlamydomonas reinhardtii: A comparative microspectrophotometric study. Vision Res. 32(9), 1593-1600. Derguini, F., Mazer, P., & Nakanishi, K. (1991). All-trans retinal is the chromophore bound to the rhodopsin of the alga Chlamydomonas reinhardtii. Photochem. Photobiol. 54(6), 1017-1021. Doughty, M., & Diehn, B. (1980). Flavins as photoreceptor pigments for behavioral responses in motile microorganisms, especially in flagellated alga Euglena sp. Struct. Bond. 41, 45-70. Ehrlich, B. E., Schen, C. R., & Spudich, J. L. (1984). Bacterial rhodopsins monitored with fluorescent dyes in vesicles and in vivo. J. Memb. Biol. 82, 89-94. Feinleib, M. E. (1980). Photomotile responses in flagellates. In Phoreception and Sensory Transduction in Aneural Organisms. Ed. F. Lenci & G. Colombetti, pp. 45-68. Plenum Press. New York. Ferrando-May, E., Brustmann, B., & Oesterhelt, D. (1993a). A C-terminal truncation results in high-level expression of the functional photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium. Mol. Microbiol. 9, 943-953. Ferrando-May, E., Krah, M., Marwan, W., & Oesterhelt, D. (1993b). The methyl-accepting transducer protein HtrI is functionally associated with the photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium. The EMBO Journal 12, 2999-3005. Fodor, S. E A., Gebhard, R., Lugtenburg, J., Bogomolni, R. A., & Mathies, R. A. (1989). Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy. J. Biol. Chem. 264, 18280-18283. Foster, K., & Smyth, R. (1980). Light antennas in phototactic algae. Microbiol. Rev. 44, 572-630. Foster, K., Saranak, J., Patel, N., Zarrilli, G., Okabe, M., Kline, T., & Nakanishi, K. (1984). A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas. Nature. 311,756-759. Foster, K., Saranak, J., van der Steen, R., & Lugtenburg, J. (1987). Retinal in Chlamydomonas rhodopsin is in a planar 6-s-trans conformation as shown by in vivo incorporation of 6-7 locked retinal analogs. Invest. Optham. Vis. Sci. 28(supplement), $253. Foster, K., Saranak, J., & Zarrilli, G. (1988a). Autoregulation ofrhodopsin synthesis in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 85, 6379-6383. Foster, K., Saranak, J., Derguini, E, Rao, J., Zarrilli, G., Okabe, M., Fang, J., Shimizu, N., & Nakanishi, K. (1988b). Rhodopsin activation: A novel view suggested by in vivo Chlamydomonas experiments. J. Amer. Chem. Soc. 110, 6588. Foster, K., Saranak, J., Derguini, F., Zarrilli, G., Johnson, R., Okabe, M., & Nakanishi, K. (1989). Activation of Chlamydomonas rhodopsin in vivo does not require isomerization of retinal. Biochemistry 28, 819-824. Foster, K., Saranak, J., & Dowben, P. (1991). Spectral sensitivity, structure and activation of eukaryotic rhodopsins: activation spectroscopy of rhodopsin analogs in Chlamydomonas. J. Photochem. Photobiol. 8, 385-408.

222


Ganter, U. N., Schmid, E. D., Perez-Sala, D., Rando, R. R., & Seibert, E (1989). Removal ofthe 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation. Biochemistry 28, 5954-5962. Ghetti, F., Colombetti, G., Lenci, E, Campani, E., Polacco, E., & Quaglia, M. (1985). Fluorescence of Euglena gracilis photoreceptor pigment: An in vivo microspectrophotometric study. Photochem. Photobiol. 42(1), 29-33. Gualtieri, P., Barsanti, L., & Passarelli, V. (1989). Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis. Biochim. Biophys. Acta. 993,293-296. Gualtieri, P., Pelosi, P., Passarelli, V., & Barsanti, L. (1992). Identification of a rhodopsin photoreceptor in Euglena gracilis. Biochim. Biophys Acta. 1117(1), 55-59. Harz, H., Nonnengasser, C., & Hegemann, P. (1992). The photoreceptor current of the green alga Chlamydomonas. Phil. Trans. Royal Soc.: Biol. Sci.(B). 338(1238), 39-52. Hasselbacher, C. A., Spudich, J. L., & Dewey, T. G. (1988). Circular dichroism of halorhodopsin: Comparison with bacteriorhodopsin and sensory rhodopsin I. Biochemistry 27, 2540-2546. Hegemann, E, & Bruck, B. (1989). Light-induced response in Chlamydomonas reinhardtii: Occurrence and adaptation phenomena. Cell Mot. Cytoskel. 14, 501-515. Hegemann, P., Gartner, W., & Uhl, R. (1991). All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin. Biophys. J. 60, 1477-1489. Hegemann, P., & Harz, H. (1993). Photoreception in Chlamydomonas. In Signal Transduction, Prokaryotic and Simple Eukaryotic Systems. J. Kurjan & Talor, B. L., Eds. Academic Press: New York, 1993, pp. 279-307. Hegemann, E, Hegemann, U., & Foster, K. (1988). Reversible bleaching of Chlamydomonas reinhardtii rhodopsin in vivo. Photochem. Photobiol. 48, 123-128. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, E, Beckmann, E., & Downing, K. H. (1990). Model for the structure ofbacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 213,899-929. Henderson, R., & Schertler, G. (1990). The structure ofbacteriorhodopsin and its relevance for the visual opsins and other seven-helix G-protein coupled receptors. Philos. Trans. Roy. Soc. London 326, 379-389. Hirayamal J., Imamoto, Y., Shichida, Y., Kamo, N., Tomioka, H., & Yoshizawa, T. (1992). Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry. Biochemistry 31, 2093-2098. Hofmann, K. P., & Heck, M. (1996)-Chapter 3. Imamoto, Y., Shichida, Y., Hirayama, J., Tomioka, H., Kamo, N., & Yoshizawa, T. (1992). Chromophore configuration ofpharaonis phoborhodopsin and its isomerization on photon absorption. Biochemistry 31, 2523-2528. Imamoto, Y., Schichida, Y., Yoshizawa, T., Tomioka, H., Takahashi, T., Fujikawa, K., Kamo, N., & Kobatake, Y. (1991). Photoreaction cycle ofphoborhodopsin studied by low-temperature spectrophotometry. Biochemistry. 30, 7416-7424. Jennings, H. S. (1915). Behavior of the Lower Organisms. Columbia Univ. Press. New York. Kamiya, R., & Witman, G. B. (1984). Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas. J. Cell Biol. 98, 97-107. Krebs, M. P., Spudich, E. N., Khorana, H. G., & Spudich, J. L. (1993). Synthesis ofa gene for sensory rhodopsin I and its functional expression in Halobacterium halobium. Proc. Natl. Acad. Sci. USA 90, 3486-3490. Kreimer, G., & Melkonian, M. (1990). Reflection confocal laser scanning microscopy of eyespots in flagellated green algae. Eur. J. Cell Biol. 53, 101-111. Kreimer, G., Brohsonn, U., & Melkonian, M. (1991a). Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga. Eur. J. Cell Biol. 55, 318-327. Kreimer, G., Mamer, F., Brohsonn, U., & Melkonian, M. (1991b). Identification of 11-cis and all-trans retinal in the photoreceptive organelle of a flagellate green alga. FEBS. Lett. 293(1, 2), 49-52.


223

Kreimer, G., Overlander, C., Sineshchekov, O. A., Stolzis, H., Nultsch, W., & Melkonian, M. (1992). Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey627, mt. Planta 188, 513--521. Krinsky, N., & Goldsmith, T. (1960). The carotenoids of the flagellated alga, Euglena gracilis. Arch. Biochem. Biophys. 91, 271-279. Lawson, M., Zacks, D. N., Derguini, F., Nakanishi, K., & Spudich, J. L. (1991). Retinal analog restoration ofphotophobic responses in a blind Chlamydomonas reinhardtii mutant: Evidence for an archaebacterial-like chromophore in a eukaryotic rhodopsin. Biophys. J. 601,490-1498. Lenci, F., Colombetti, G., & H/ider, D. (1983). Role of flavin quenchers and inhibitors in the sensory transduction of the negative phototaxis in the flagellate, Euglena gracilis. Curr. Microbiol. 9, 285-290. Martin, R. L., Wood, C., Baehr, W., & Applebury, M. L. (1986). Visual pigment homologies revealed by DNA hybridization. Science 232, 1266-1269. Marwan, W., & Oesterhelt, D. (1990). Quantitation of photochromism of sensory rhodopsin-I by computerized tracking of Halobacterium halobium cells. J. Mol. Biol. 215, 277-285. Marwan, W., & Oesterhelt, D. (1987). Signal formation in the halobacterial photophobic response mediated by a fourth retinal protein (P480). J. Mol. Biol. 195, 333--342. Melkonian, M., & Robenek, M. (1980). Eyespot membranes of Chlamydomonas reinhardtii: A freezefracture study. J. Ultrastruct. Res. 72, 90-102. Meyer, T. E. (1985). Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila. Biochim. Biophys. Acta 806, 175-183. Meyer, T. E., Tollin, G., Hazzard, J. H., & Cusanovich, M. A. (1989). Photoactive yellow protein from the purple phototrophic bacterium, Ectothiorhodospira halophila. Quantum yield ofphotobleaching and effects of temperature, alcohols, glycerol, and sucrose on kinetics of photobleaching and recovery. Biophys. J. 56, 559-564. Meyer, T. E., Yakali, E., Cusanovich, M. A., & Tollin, G. (1987). Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin. Biochemistry 26, 418--423. Meyer, T. E., Tollin, G., Causgrove, T. P., Cheng, P., & Blankenship, R. E. (1991). Picosecond decay kinetics and quantum yield of fluorescence of the photoactive yellow protein from the halophilic purple phototrophic bacterium, Ectothiorhodospira halophila. Biophys. J. 59, 988-991. McRee, D. E., Meyers, T. E., Cusanovich, M. A., Parge, H. E., & Getzoff, E. D. (1986). Crystallographic characterization ofa photoactive yellow protein with photochemistry similar to sensory rhodopsin. J. Biol. Chem. 261(29), 13850-13851. McRee, D. E., Tainer, J. A., Meyer, T. E., van Beeumen, J., Cusanovich, M. A., & Getzoff, E. D. (1989). Crystallographic structure of a photoreceptor protein at 2.4 A resolution. Proc. Natl. Acad. Sci. USA 86, 6533-6537. Muccio, D., & Cassim, J. Y. (1979). Interpretation of the absorption and circular dichroic spectra of oriented purple membrane. Biophys. J. 26, 427-440. Nakanishi, K., Derguini, F., Rao, J., Zarrilli, G., Okabe, M., Lien, T., Johnson, R., Foster, K., & Saranak, J. (1989). Theory of rhodopsin activation: Probable charge redistribution of excited state chromophore. Pure and Appl. Chem. 61, 361-364. Nultsch, W. (1983). The photocontrol of movement of Chlamydomonas. In The Biology of Photoreception. Ed. D. Cosens and D. Vince-Prue. Soc. Exp. Biol. Symp. XXXVI. pp. 521-539. Nultsch, W., & H/ider, D.-P. (1988). Photomovement in motile microorganisms-II. Photochem. Photobiol. 47, 837-869. Nultsch, W., Throm, G., & Rimscha, I. (1971). Phototaktische Untersuchungen an Chlamydomonas reinhardtii dangeard in homokontinuierlicher Kultur. Arch. Mikrobiol. 80, 351-369. Oesterhelt, D., & Marwan, W. (1990). Signal transduction in Halobacterium halobium. Symp. Soc. Gen. Microbiol. 46, 21 9-239.

224


Oesterhelt, D., Schuhmann, L., & Gruber, H. (1974). Light-dependent reaction of bacteriorhodopsin with hydroxylamine in cell suspensions of Halobacterium halobium: Demonstration of an apo-membrane. FEBS. Lett. 44(3), 257-261. Oesterhelt D., & Stoeckenius, W. (1971). Rhodopsin-like protein from the purple membrane of Halobacteium halobium. Nature[New Biol.] 233, 149-152. Olson, K. D., Deval, P., & Spudich, J. L. (1992). Absorption and photochemistry of sensory rhodopsi.n-I: pH effects. Photochem. Photobiol. 56, 1181-1187. Olson, K. D., & Spudich, J. L. (1993). Removal of the transducer protein from sensory rhodopsin I exposes sites of proton release and uptake during the receptor photocycle. Biophysical. J. 65, 2578-2586. Otomo, J., Tomioka, H., & Sasabe, H. (1992). Bacterial rhodopsins of newly isolated halobacteria. J. Gen. Microbiol. 138, 1027-1037. Ottolenghi, M., & Sheves, M. (1989). Synthetic retinals as probes for the binding site and photoreactions in rhodopsins. J. Membrane Biol. 112, 193-212. Rath, P., Olson, K. D., Spudich, J. L., & Rothschild, K. J. (1994). The Schiff base counterion in bacteriorhodopsin is protonated in sensory rhodopsin I: Spectroscopic and functional characterization of the mutated proteins D76N and D76A. Biochemistry 33, 5600-5606. Riaffer, U., & Nultsch, W. (1985). High-speed cinematographic analysis of the movement of Chlamydomonas. Cell. Motil. 5, 251-263. Riffler, U., & Nultsch, W. (1991). Flagellar photoresponses of Chlamydomonas cells held on micropipettes II: change in flagellar beat pattern. Cell Motil. Cytoskeleton 18, 269-278. Scharf, B.. Hess, B., & Engelhard, M. (1992a). Chromophore of sensory rhodopsin II from Halobacterium halobium. Biochemistry 31, 12486-12492. Scharf, B., Pevec, B., Hess, B., & Engelhard, M. (1992b). Biochemical and photochemical properties of the photophobic receptors from Halobacterium halobium and Natronobacterium pharaonis. Eur. J. Biochem. 206, 359-366. Scherrer, P., McGinnis, K., & Bogomolni, R. A. (1987). Biochemical and spectroscopic characterization of the blue-green photoreceptor in Halobacterium halobium. Proc. Natl. Acad. Sci. USA 84, 402-406. Schmidt, W., Galland, P., Senger, H., & Furuya, M. (1990). Microspectro-photometry of Euglena gracilis. Planta 182, 375-381. Schobert, B., & Lanyi, J. K. (1982). Halorhodopsin is a light driven chloride pump. J. Biol. Chem. 257, 10306-10313. Soppa, J., Duschl, J., & Oesterhelt, D. (1993). Bacterioopsin, haloopsin, sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG 1: three new members of a growing family. J. Bact. 175, 2720-2726. Sprenger, W. W., Hoff, W. D., Armitage, J. P., & Hellingwerf, K. J. (1993). The eubacteriumEctothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein. J. Bact. 175, 3096-3104. Spudich, E. N., Hasselbacher, C. A., & Spudich, J. L. (1988). A methyl-accepting protein associated with bacterial sensory rhodopsin I. J. Bact. 170, 4280-4285. Spudich, E. N., Takahashi, T., & Spudich, J. L. (1989). Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis. Proc. Natl. Acad. Sci. USA 20, 7746--7750. Spudich, E. N., & Spudich, J. L. (1982). Control of transmembrane ion fluxes to select halorhodopsindeficient and other energy transduction mutants of Halobacterium halobium. Proc. Natl. Acad. Sci. USA 79, 4308-4312. Spudich, E. N., & Spudich, J. L. (1993). The photochemical reactions of sensory rhodopsin I are altered by its transducer. J. Biol. Chem. 268, 16095-16097. Spudich, J. L. (1993). Color sensing in the Archaea: a eukaryotic-like receptor coupled to a prokaryotic transducer. J. Bact. 175, 7755-7761.


225

Spudich, J. L., & Bogomolni, R. A. (1984). The mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature 312, 509--513. Spudich, J. L., & Bogomolni, R. A. (1988). Sensory rhodopsins of halobacteria. Annu. Rev. Biophys. Chem. 17, 193--215. Spudich, J. L., & Stoeckenius, W. (1979). Photosensory and chemosensory behavior of Halobacterium halobium. J. Photobiochem. Photobiophys. 1, 43-53. Spudich, J. L., McCain, D. A., Nakanishi, K., Okabe, M., Shimizu, N., Rodman, H., Honig, B., & Bogomolni, R. A. (1986). Chromophore/protein interaction in bacterial sensory rhodopsin and bacteriorhodopsin. Biophys. J. 49, 479-483. Starace, D., & Foster. K. (1989). Absorption spectroscopy of the rhodopsin of Chlamydomonas reinhardtii. Biophys. J. 55, 379a. Stock, J. B., Stock, A. M., & Mottonen, J. M. (1990). Signal transduction in bacteria. Nature 344, 395-400. Sundberg, S. A., Alam, M., Lebert, M., Spudich, J. L., 0esterhelt, D., & Hazelbauer, G. L. (1990). Characterization of mutants of Halobacterium halobium defective in taxis. J. Bacteriology 172, 2328-2335. Sundberg, S., Alam, M., & Spudich, J. L. (1986). Excitation signal processing times in Halobacterium halobilum phototaxis. Biophys. J. 50, 895-900. Sundberg, S.A., Bogomolni, R. A., & Spudich, J. L. (1985). Selection and properties of phototaxis-deficient mutants of Halobacterium halobium. J. Bact. 164, 282-287. Takahashi, T., & Kobatake, Y. (1982). Computer-linked automated method for measurement of the reversal frequency in phototaxis of Halobacterium halobium. Cell Struct. Funct. 7, 183-192. Takahashi, T., Mochizuki, Y., Kamo, N., & Kobatake, Y. (1985a). Evidence that the long-lifetime photointermediate of S-Rhodopsin is a receptor for negative phototaxis in Halobacterium halobium. Biochem. Biophys. Res. Commun. 127, 99-105. Takahashi, T., Tomioka H., Kamo, N., & Kobatake, Y. (1985b). A photosystem other than PS370 also mediates the negative phototaxis of Halobacterium halobium. FEMS Microbiol. Lett. 28, 161164. Takahashi, T., Yan, B., Mazur, P., Derguini, F., Nakanishi, K., & Spudich, J.L. (1990). Color regulation in the archaebacterial phototaxis receptor phoborhodopsin (sensory rhodopsin II). Biochemistry 29, 8467--8474. Takahashi, T., Yoshihara, K., Watanabe, M., Kubota, M., Johnson, R., Derguini, F., & Nakanishi, K. (1991). Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: Simultaneous measurements of phototactic and photophobic responses. Biochem. Biophys. Biol. Res. Comm. 178(3), 1273-1279. Takahashi, T., Kubota, M., Watanabe, M., Yoshihara, K., Derguini, F., & Nakanishi, K. (1992). Diversion of the sign ofphototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs. FEBS Lett. 314(3), 275-279. Tomioka, H., Takahashi, T., Kamo, N., & Kobatake, Y. (1986a). Flash spectrophotometric identification of a fourth rhodopsin-like pigment in Halobacterium halobium. Biochem. Biophys. Res. Commun. 139, 389-395. Tomioka H., Takahashi, T., Kamo, N., & Kobatake, Y. (1986b). Action spectrum of the photoattractant response of Halobacterium halobium in early logarithmic growth phase and the role of sensory rhodopsin. Biochim. Biophys. Acta. 884, 578-584. Uhl, R., & Hegemann, P. (1990). Probing visual transduction in a plant cell. Optical recordings of rhodopsin-induced structural changes from Chlamydomonas reinhardtii. Biophys. J. 58, 1295-1302. Wald, G. (1968). The molecular basis of visual excitation. Nature 219, 800--807. Witman, G. B. (1993). Chlamydomonas phototaxis. Trends Cell Biol. 3,403--408. Wolff, E. K., Bogomolni, R. A., Scherrer, P., Hess, B., & Stoeckenius, W. (1986). Color discrimination in halobacteria: Spectroscopic characterization of a second sensory receptor coveting the bluegreen region of the spectrum. Proc. Natl. Acad. Sci. USA 83, 7272-7276.

226


Yan, B., Cline, S. W., Doolittle, W. E, & Spudich, J. L. (1992). Transformation ofa BOP-HOP-SOP-I -SOP-IF Halobacterium halobium mutant to BOP+: Effectsofbacteriorhodopsin photoactivation on cellular proton fluxes and swimmingbehavior. Photochem. Photobiol. 56, 553-561. Yan, B., Nakanishi, K., & Spudich, J. L. (199l a). Mechanism of activation of sensory rhodopsin-I: Evidence for a steric trigger. Proc. Natl. Acad. Sci. USA 88, 9412-9416. Yan, B., Takahashi, T., & Spudich, J. L. (1991b). Identification of signaling states of a sensory receptor by modulation of lifetimes of stimulus-induced conformations: The case of sensory rhodopsin II. Biochemistry 30, 10686-10692. Yan, B., & Spudich, J. L. (1991). Evidence the repellent receptor form of sensory rhodopsin I is an attractant signaling state. Photochem. Photobiol. 54, 1023-1026. Yan, B., Takahashi,T., Johnson, R., Derguini, E, Nakanishi, K., & Spudich, J. L. (1990). All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in ttalobacterium halobium. Biophys. J. 57, 807-814. Yao, V. J., Spudich, E. N., & Spudich, J. L. (1994). Identification of distinct domains for signaling and receptor interaction of the sensory rhodopsin I transducer, Htrl. J. Bacteriology 176, 6931-6935. Yao, V. J., & Spudich, J. L. (1992). Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. Proc. Natl. Acad. Sci. USA 89, 11915-11919. Zacks, D. N., Derguini, E, Nakanishi, K., & Spudich, J. L. (1993). Comparativestudy ofphototactic and photophobic receptor chromophore properties in Chlamydomonas reinhardtii. Biophys. J. 65, 508-518. Zacks, D. N., & Spudich, J. L. (1994). Gain setting in Chlamydomonas reinhardtii: Mechanism of phototaxis and the role of the photophobic response. Cell Motil. Cytoskeleton 29, 225-230.

NOTE ADDED IN PROOF Several advances have been made since the preparation of this review. These include the cloning and sequencing of SR-II and HtrII-encoding genes from Natronobacterium pharaonis and Haloarcula vallismortis [Seidel et al. (1995) Proc. Natl. Acad. Sci. USA 92, 3036-3040] and of a histidine kinase gene (cheA) required for taxis by Halobacterium salinarium [Rudolph and Oesterhelt (1995) EMBO J. 14, 667-673]. An SR-I residue (aspartyl-201) critical for attractant signaling has been identified [Olson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 3185-3189] and further progress has been reported on the control of proton transfers in SR-I by Htri [Spudich (1994) Cell 79, 747-750, and references therein]. An in vitro approach has been established for study of SR-I/HtrI interaction based on rapid high-yield purification and liposome reconstitution of polyhistidinetagged SR-I [Krebs et al. (1995) Protein Expression and Purification 6, 780-788]. Also the chromophore of PYP has been demonstrated to be a thio ester linked p-coumaric acid [Hoff et al. (1994) Biochemistry 33, 13959-13962; Baca et al. (1994) Biochemistry 33, 14369-14377] and the PYP crystal structure has been redetermined and shows the protein has an tx/13 fold, resembling eukaryotic proteins involved in signal transduction [Borgstahl et al. (1995) Biochemistry 34, 6278-6287]. For discussion of these and other recent advances, the reader is referred to Spudich, J.L., Zacks, D.N., and Bogomolni, R.A. (1996) Microbial Sensory Rhodopsins: Photochemistry and Function, Israel Journal of Chemistry, in press.

ALPHA-ADREN ERG IC RECEPTORS

David B. Bylund

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Alpha Adrenergic Receptors . . . . . . . . . . . . . . . . . Alpha- 1 Adrenergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . A. Subtypes of Alpha-1 Adrenergic Receptors . . . . . . . . . . . . . . . . B. Alpha-1 Adrenergic Receptor Functions . . . . . . . . . . . . . . . . . . IV. Alpha-2 Adrenergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . A. Subtypes of Alpha-2 Adrenergic Receptors . . . . . . . . . . . . . . . . B. Peripheral Alpha-2 Adrenergic Receptor Functions . . . . . . . . . . . . C. Central Alpha-2 Adrenergic Receptor Functions . . . . . . . . . . . . . . V. Correlation of Receptor Binding and Function . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

II. III.

!.

227 228 229 229 230 231 231 234 236 237 238 238

INTRODUCTION

Many tissues and organ systems have marked responses to norepinephrine released from sympathetic nerves and to epinephrine, a circulating hormone. The effects of these catecholamines are mediated through adrenergic receptors. Historically adrenergic receptors have been divided into two major types, alpha and beta (Ahlquist,

Biomembranes Volume 2A, pages 227-244 Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-659-2

227

228

DAVID B. BYLUND

1948). Subsequently, both the beta and alpha types were further subdivided into beta-l, beta-2, alpha-1 and alpha-2 subtypes (for a more complete historical perspective, see Bylund, 1988b). More recently, it has become clear that a more useful classification scheme is based on three major types---alpha-1, alpha-2 and beta--each of which is further divided imo three or more subtypes (Bylund, 1988a). The rationale for this current classification scheme of three major adrenergic receptor types is based on three lines of evidence. First, the difference in affinity of drugs that show selectivity is on the order of 1,000- to 10,000-fold among the three major receptor types (alpha-1, alpha-2, and beta), whereas the difference in affinities among subtypes (e.g., alpha-lA, alpha-lB, and alpha-lC) is generally 10- to 100-fold. Second, the family of guanine nucleotide binding proteins (G-proteins) which couples the receptor to the second messenger systems is different for each of the major receptor types: Gq couples to alpha-l; Gi to alpha-2; and G s to beta. Finally, the predicted amino acid sequence of the adrenergic receptors indicate that structurally alpha-1 and alpha-2 receptors are no more closely related to each other than they are to the beta receptor. The alpha-1 and beta adrenergic receptors both have short third cytoplasmic loops and long cytoplasmic C-terminals, whereas the alpha-2 adrenergic receptors (along with the muscarinic cholinergic receptors) have long third cytoplasmic loops and short cytoplasmic C-terminals. Thus, from a structural point of view the alpha- 1 receptor may be more closely related to the beta receptor than it is to the alpha-2 receptor (Bylund, 1992). In any case, in spite of their names, the alpha- 1 and alpha-2 are not more closely related to each other than either is to the beta receptor. Therefore, alpha-l, alpha-2 and beta should be considered as three separate, major types of adrenergic receptors. It is the purpose of this paper to review selected aspects of the alpha- 1 and alpha-2 adrenergic receptors. For these two major adrenergic receptor types there are several reviews and books available to the interested reader including those on alpha adrenergic receptors (Harrison et al., 1991; Lomasney et al., 1991a; Ruffolo et al., 1995; Hieble et al., 1995; Ruffolo et al., 1991; Bylund, 1992; Summers and McMartin, 1993; Bylund et al., 1994), on alpha-1 adrenergic receptors (Ruffolo, 1987; Minneman, 1988; Bylund et al., 1995) and on alpha-2 adrenergic receptors (Limbird, 1988; Byltmd, 1988a; Maze and Tranquilli, 1991).

II. CLASSIFICATION OF ALPHA ADRENERGIC RECEPTORS The idea that there may be more than one type of alpha adrenergic receptor dates from the mid 1960s and was based on the finding that the pharmacological characteristics, as defined by both agonists and antagonists, of alpha adrenergic receptors in the vas deferens and rabbit intestine were different (Rossum, 1965). However, nearly 10 years went by before the terms alpha-1 and alpha-2 were used to differentiate among various alpha adrenergic receptors (Delbarre and Schmitt, 1973; Langer, 1974). For a while it was thought that alpha receptors could be differentiated on the basis of their localization, that is whether they were pre- or

Alpha Adrenergic Receptors

229

postsynaptic. However, a major conceptual advance was made in 1977 by Pettinger who realized that the anatomical localization of a receptor was not necessarily a useful criteria for classifying receptors and suggested instead that there were at least two pharmacologically distinct types of alpha adrenergic receptors (Berthelson and Pettinger, 1977). This pharmacological definition of the alpha-1 and alpha-2 adrenergic receptors has proven to be very useful and has been supported by both second messenger and molecular cloning data.

III. ALPHA-1 ADRENERGIC RECEPTORS A. Subtypesof Alpha-1 Adrenergic Receptors Building on an initial report that both phentolamine and WB 4101 inhibited [3H]prazosin binding in rat frontal cortex in a manner consistent with more than a single type of receptor (Battaglia et al., 1983), Morrow and Creese (1986) formally proposed a definition of alpha- 1A and alpha- 1B receptor binding sites. The essential features of this classification scheme were that [3H]prazosin had similar affinities for the two receptor subtypes whereas the antagonists WB 4101 and phentolamine were approximately 40- to 20-fold more potent at the alpha-lA site as opposed to the alpha- 1B site (Morrow and Creese, 1986). At about the same time Johnson and Minneman (1987) observed that the alkylating agent chlorethylclonidine potentially and selectively inactivated only half of the [t25I]HEAT binding to alpha-1 adrenergic receptors in rat cerebral cortex, whereas other alkylating agems inactivated all of the binding sites. Additional evidence indicated that the sites inactivated by chlorethylclonidine represented the alpha-1B adrenergic receptor subtype (Han et al., 1987), the same subtype having low affinity for WB 4101 (Minneman et al., 1988; Wilson and Minneman, 1989). More recemly it has been shown that 5-methylurapidil and (+)-nigoldapine differentiate two alpha-1 adrenergic receptor subtypes that correspond to the alpha-lA and alpha-1B subtypes (Hanft and Gross, 1989; Gross et al., 1989). To date only these two alpha-1 adrenergic receptor subtypes have been clearly identified in tissues by pharmacological means. Three alpha-1 adrenergic receptor subtypes have been identified by molecular cloning. Since there is not yet good agreement between the pharmacologically defined subtypes, and the molecularly defined subtypes, the pharmacologically defined subtypes will be designated by uppercase letters, whereas molecularly defined subtypes will have lowercase letters. The alpha- 1b adrenergic receptor from the DDT cell (hamster smooth muscle) was cloned first (Cotecchia et al., 1988), followed by a novel alpha- 1 adrenergic receptor from a bovine brain cDNA library which has been named the alpha- 1c subtype (Schwinn et al., 1990). Recent evidence suggests that this clone corresponds to the pharmacological alpha- 1A subtype (Ford et al., 1994). More recently an alpha- 1 adrenergic receptor subtype has been cloned from rat cerebral cortex cDNA libraries (Lomasney et al., 1991b; Perez et al., 1991). Although this was initially said to represent the alpha-la adrenergic receptor

230

DAVID B. BYLUND

subtype, a more careful evaluation of its pharmacological characteristics has revealed that it is not the pharmacologically defined alpha-1A adrenergic receptor subtype and has thus been called the alpha-1 d. Accordingly, it appears that there are three alpha-1 receptors: alpha-lA, -1B, and -1D, but not an alpha-lC (Ford et al., 1994; Hieble et al., 1995). In addition, a prazosin insensitive alpha-1 subtype may also exist (Muramatsu et al., 1995). Binding studies have also indicated the existence of additional subtypes. For example, the rat parotid gland acinar cell appears to have two alpha-1 receptors, one of which is an atypical alpha-1 subtype that differs from subtypes previously characterized in the rat (Porter et al., 1992). In addition to an alpha-lA site, which has high affinity for the subtype-selective antagonists 5-methylurapidil and WB 4101, radioligand binding studies revealed an additional site which has low affinities for these antagonists. This site, however, is not CEC sensitive, and thus can not be classified as either an alpha-lB nor alpha-lc subtype. The species orthologues of these three receptor subtypes have been cloned from" the human: the alpha-ld (Bruno et al., 1991); the alpha-lb (Ramarao et al., 1992); and the alpha-1 c (Hirasawa et al., 1993). In contradistinction to the other alpha adrenergic receptors, and to most other G-protein coupled receptors, both the human and rat alpha-lb receptors contain a single large intron of at least 16 kilobases (Ramarao et al., 1992; Gao and Kunos, 1993). The organization of the genes for the alpha- 1b receptor in the two species is well conserved in that the intron interrupts the coding sequence at the same place in the putative sixth transmembrane domain, both genes display certain features of housekeeping genes, and both genes are present as a single copy in the genome.

B. Alpha-1 Adrenergic Receptor Functions A major function of the sympathetic nervous system is to regulate blood pressure under a variety of conditions, and the alpha-1 adrenergic receptors are responsible for mediating much of this regulation. In nearly all vascular beds, postsynaptic alpha-1 adrenergic receptors mediate vasoconstriction. These alpha-1 adrenergic receptors are located postjunctionally and respond primarily to neuronally released norepinephrine. In most, but not all cases, the response to alpha-1 adrenergic receptor stimulation is more prominent than the response to activation of postsynaptic alpha-2 adrenergic receptors. Several exceptions to this general rule include the canine saphenous vein (Fowler et al., 1984; Alabaster et al., 1985; Ruffolo and Zeid, 1985), and perhaps the pulmonary vein (Shebuski et al., 1986). In peripheral tissues other than the vasculature, the role of alpha-1 adrenergic receptors is less well understood. In the heart, for example, the predominant postjunctional adrenergic receptor is the beta adrenergic receptor which mediates both the inotropic and the chronotropic responses. Alpha-1 adrenergic receptors exist in cardiac tissue but their physiological role is unknown at present (Terzic et al., 1993). It is possible that myocardial alpha-1 adrenergic receptor may serve as


231

a reserve mechanism to maintain myocardial responsiveness to catecholamine under conditions where beta adrenergic receptor function is compromised (Ask et al., 1987). Myocardial alpha- 1 adrenergic receptors may also play an important role in the induction of ventricular arrhythmias (Benfey, 1982), particularly during ischemia or reprefusion (Culling et al., 1987). The kidney receives dense noradrenergic innervation which extends to all portions ofthe nephron to the vasculature. An increase in sympathetic activity produces an alpha-1 adrenergic receptor-mediated antinatriuretic response in dogs (Osborn et al., 1983) and rabbits (Hesse and Johns, 1984). Results from experiments with reverse transcription combined with polymerase chain reaction, as well as radioligand binding data, indicate that the alpha-1A, alpha-lB and alpha-1 d subtypes are differentially expressed in rat nephron segments (Feng et al., 1993). In the central nervous system alpha-1 adrenergic receptors appear to be widely distributed in the brain and spinal cord. However, precise roles for alpha-1 adrenergic receptors have not yet been defined. The major electrophysiological response to activation of alpha-1 adrenergic receptors is a decrease in potassium conductants which produces a slowly developing depolarization (McCormick and Prince, 1988). Activation of cortical alpha-1 adrenergic receptors produces an excitatory effect on spontaneous neuronal activity (Szabadi and Bradshaw, 1987).

IV. ALPHA-2 ADRENERGIC RECEPTORS A. Subtypesof Alpha-2 Adrenergic Receptors The potential for alpha-2 adrenergic receptor subtypes has been evident for many years. However, a consistent and useful definition of alpha-2 adrenergic receptor subtypes has only more recently been developed based on pharmacological characteristics as defined mainly by radioligand binding studies. Several alpha-2 adrenergic antagonists including prazosin, oxymetazoline, and ARC-239 were found to have significantly different affinities for inhibiting [3H]yohimbine binding in tissues such as neonatal rat lung and rat kidney (subsequently defined as alpha-2B) as compared to the human platelet (classified as alpha-2A). Cell lines in continuous culture were then identified that had pharmacological characteristics of either the alpha-2A (HT29 cells) or alpha-2B (NG 108-15 cells) receptors (Bylund et al., 1988). Additional studies using the attenuation of the inhibition of cyclic AMP production as a functional assay confirmed the radioligand binding studies (Bylund and Ray-Prenger, 1989). The characteristics of the alpha-2A subtype include a high affinity for rauwolscine and oxymetazoline and a low affinity for prazosin and ARC-239. The pharmacological characteristics for the alpha-2B receptor include a low affinity for oxymetazoline and a relatively high affinity for prazosin and spiroxatrine. The high affinity for prazosin was interpreted originally by some investigators to indicate that the alpha-2B receptor might be an alpha-1

232

DAVID B. BYLUND

adrenergic receptor. However, the affinity of prazosin at the alpha-1 adrenergic receptor is approximately 50- to 100-fold higher than it is at the alpha-2B receptor. Subsequently a third pharmacological subtype, the alpha-2C, was identified by radioligand binding studies in an opossum kidney (OK) cell line (Blaxall et al., 1991; Murphy and Bylund, 1988). The alpha-2C subtype is characterized b y a 5-fold higher affinity for rauwolscine as compared to the alpha-2A and alpha-2B subtypes and a relatively high affinity for prazosin and ARC-239. It is best differentiated from the alpha-2B receptor by a 15-fold higher affinity for BAM 1303 and a 10-fold higher affinity for WB 4101. The Y79 human retinoblastoma cell line also appears to express the alpha-2C adrenergic receptor (Gleason and Hieble, 1992). Some workers unfortunately have used the high affinity for prazosin as the sole or major criterion for identifying the alpha-2B adrenergic receptor subtype. This has led to some considerable confusion in the literature in that in some articles the alpha-2C receptor is identified as the alpha-2B subtype. This confusion illustrates the principle that it is inappropriate to classify receptors based on one or even two drugs. The alpha-2D adrenergic receptor was originally identified in the rat "submaxillary" gland on the basis of the unique pharmacological profile (Michel et al., 1989), although it was not given a name at that time. The alpha-2 adrenergic receptor in the bovine pineal gland was also shown to have a pharmacological profile that was significantly different from that for the alpha-2A, alpha-2B, and alpha-2C subtypes, but was similar to that in the rat "submaxillary" gland. This receptor in the bovine pineal was identified as the alpha-2D subtype (Simonneaux et al., 1991). It has a low affinity for rauwolscine, 15- to 20-fold lower than the alpha-2A and alpha-2B and 50-fold lower than the alpha-2C, as well as a modestly lower affinity for SKF104078. In addition to rauwolscine, BAM 1303 and the mianserin isomers clearly differentiate it from the alpha-2A and alpha-2D receptor subtypes. The previous report that adult rat submandibular glands do not contain a detectable level of alpha-2 adrenergic receptor binding (Bylund and Martinez, 1980) is at odds with the report of alpha-2 receptors in the "submaxillary" gland. Michel et al. (1989) report that they obtained their "submaxillary" gland from Pelfreeze. On inquiry we discovered that Pelfreeze does not separate the sublingual from the submandibular gland. Using an antagonist radioligand we were unable to detect any alpha-2 adrenergic receptor binding in the rat submandibular gland obtained from Pelfreeze after removal of the sublingual gland (unpublished data). By contrast, we found a high level of alpha-2 adrenergic receptor binding in the sublingual gland using [3H]rauwolscine as the radioligand (Bmax = 380 fmol/mg/ protein; K D= 15 nM). Further investigations of the pharmacological characteristics of the alpha-2 adrenergic receptor in the sublingual gland were consistent with the conclusion that the rat sublingual gland receptor can be classified as an alpha-2D adrenergic receptor. Thus, the results obtained in the study of Michel et al. (1989) in the "submaxillary" gland were due to the alpha-2 adrenergic receptors in the sublingual gland and not in the submandibular gland.


233

The cloning, sequencing, and expression of the gene coding for the human platelet alpha-2 adrenergic receptor was first reported in 1987 (Kobilka et al., 1987). The gene coding for the human platelet alpha-2 adrenergic receptor was found to be localized to chromosome 10 and thus the cloned receptor was named alpha-2C 10. The pharmacological characterization of this expressed receptor was consistent with the pharmacology of the alpha-2 receptor found in the human platelet. Because the platelet is one of the prototypic tissues for the alpha-2A receptor, the alpha-2-C10 clearly corresponds to the alpha-2A subtype. An alpha-2 receptor having a high sequence similarity to the alpha-2-C 10 receptor has been isolated from the pig (Guyer et al., 1990). This clone appears to express pharmacological characteristics of an alpha-2A receptor, although only a limited pharmacological profile was reported. Molecular cloning has also identified receptors with high sequence similarity to the human alpha-2-C 10 receptor in the rat (Chalberg et al., 1990; Lanier et al., 1991) and mouse (Link et al., 1992). The pharmacological characteristics of the rat and mouse clones are significantly different from the human and pig clones but are similar to those in the rat salivary gland and the bovine pineal. Thus they have been identified as containing the alpha-2D rather than the alpha-2A subtype. However, the rodent clones are apparently species orthologues of the human and pig clones. The human alpha-2-C 10 and the porcine orthologues have cysteine at position 201 whereas the rat, mouse and bovine orthologues have serine at position 201. Link and colleagues (1992) showed that a serine to cysteine mutation at position 201 may be responsible for the low affinity binding of the alpha-2 adrenergic receptor antagonist yohimbine, one of the characteristics of alpha-2D pharmacology. However, the affinity of other antagonists including rauwolscine, WB 4101 and SKF104078 is not altered by the mutation at residue 201 (Bylund, unpublished data). The chicken pineal alpha-2 adrenergic receptor has alpha-2A subtype pharmacology (Bylund et al., 1988) however it has a serine at position 201 (Blaxall et al., 1993). This suggests that other amino acids may be responsible for the differences between alpha-2A and alpha-2D pharmacology. A decision as to whether or not the alpha-2D should be identified as a subtype separate from the alpha-2A remains controversial. Whereas it is clearly pharmacologically different, it is also genetically very similar. Furthermore, immunoprecipitation data indicates a high degree of similarity between the human alpha-2-C 10 adrenergic receptor and the alpha-2 adrenergic receptors expressed in rat submaxillary gland and bovine pineal (Kurose et al., 1993). Referring to the rodent and bovine clones as "alpha-2A/D" would emphasize both the differences in the pharmacological characteristics as well as the similarities in the genetic sequence. The alpha-2B adrenergic receptor has been cloned from the human (Weinshank et al., 1990; Lomasney et al., 1990), the rat (Zeng et al., 1990) and mouse (Chruscinski et al., 1992). The human clone has been designated as alpha-2-C2, reflecting its localization to chromosome 2, and the rat clone has been designated

234

DAVID B. BYLUND

as RNG alpha-2, reflecting the lack of N-linked glycosylation in the amino terminal portion of the receptor. The alpha-2C adrenergic receptor has been cloned from the human (Regan et al., 1988), the rat (Lanier et al., 1991; Voigt et al., 1991; Flordellis et al., 1991), the mouse (Link et al., 1992) and the opossum (Blaxall et al., 1994). Unfortunately, in one report the clone was misidentified as an alpha-2B adrenergic receptor (Flordellis et al., 1991). The opossum OK cell alpha-2C adrenergic receptor shows only a 64% identity to the human alpha-2-C4, whereas the human and rat orthologues have a 90% identity. Nevertheless, the pharmacological characteristics of the human and opossum receptors are indistinguishable. This may be due to the fact that within the transmembrane regions, the opossum receptor has an 89% identity to human alpha-2-C4, and if conservative substitutions are included, the overall similarity becomes 99%. The amino terminus and third cytoplasmic loop are the least-conserved regions. The lower sequence identity of the OK cell alpha-2C adrenergic receptor to the human, rat, and mouse orthologues may be a reflection of the greater evolutionary separation between metatherian (marsupial) and eutherian (placental) mammals. The opossum parathyroid hormone receptor (Jiappner et al., 1991) has 78% identity to the rat parathyroid receptor (Abou-Samra et al., 1992) and the opossum serotonin 5-HT1B receptor has 82% identity to the human receptor (Cerutis et al., 1994). Many workers in the field doubt that additional alpha-2 adrenergic receptor subtypes will be found beyond the three genetic (or four pharmacological) subtypes that have been identified to date. However, there is some evidence suggesting that additional subtypes and/or heterogeneity do exist (Hieble et al., 1991; Uhlen and Wikberg, 1991; Uhlen et al., 1992). The molecular basis for this additional apparent heterogeneity is not yet known. Several of the widely used alpha-2 adrenergic drugs including clonidine and idazoxan also bind with high affinities to nonadrenergic sites which have been designated as imidazoline binding sites (Ernsberger et al., 1987). These binding sites are not part of the alpha-2 adrenergic receptor family because of their very low aff'mity for the catecholamines, epinephrine and norepinephrine. The 11 imidazoline sites have high affinity for clonidine, moxonidine and oxymetazoline whereas the 12 imidazoline sites have high affinity for idazoxan, cirazoline and guanabenz (Lanier et al., 1993; MacKinnon et al., 1993; Piletz and Sletten, 1993; Molderings et al., 1993). The I2 sites appear to be related to MAO (Raddatz et al., 1995).

B. Peripheral Alpha-2 Adrenergic Receptor Functions Essentially all sympathetically innervated tissues possess presynaptic alpha-2 adrenergic receptors which mediate the inhibition of norepinephrine release. In these tissues alpha-2 adrenergic receptor antagonists may produce effects due to the enhancement of neurotransmitter release. In addition, many tissues also have postsynaptic alpha-2 receptors.


235

The vasoconstriction caused by neuronally released norepinephrine as well as circulating epinephrine has been classically ascribed to mediation through the alpha-1 adrenergic receptor. More recently, however, it has become clear that in many vascular beds postsynaptic alpha-2 adrenergic receptors also may play an important role in mediating vasoconstriction. Indeed, in some vessels such as the dog saphenous vein, the alpha-2 adrenergic-mediated vasoconstriction may be more important than the alpha-1 adrenergic receptor-mediated vasoconstriction (Fowler et al., 1984; Alabaster et al., 1985; Ruffolo and Zeid, 1985). As a general rule it appears that alpha-1 receptors are located postjunctionally whereas the postsynaptic alpha-2 adrenergic receptors are localized extrajunctionally. Postsynaptic vascular alpha-2 adrenergic receptors are thought to reside extrajunctionally because they are probably not activated by norepinephrine released from sympathetic nerve, but can be stimulated by administered epinephrine or norepinephrine (Langer and Shepperson, 1982; Wilffert et al., 1982). The physiological role of extrajunctional alpha-2 adrenergic receptors is not fully understood. It has been suggested, however, that the importance of the alpha-2 receptor may lie in responses to stress and/or in certain pathological states such as hypertension and possibly congestive heart failure (Bolli et al., 1984; Jie et al., 1986; Brodde and Michel, 1992). Further support for the concept that alpha-2 adrenergic receptors may be involved in certain disease state comes from studies in the coronary arterial circulation (Heusch and Deussen, 1983; Seitelberger et al., 1988), the pulmonary arterial circulation (Hyman and Kadowitz, 1986; Shebuski et al., 1986). An exception to the general rule that alpha-1 adrenergic receptors are more prominent than alpha-2 adrenergic receptors is the cutaneous circulation, where it appears that alpha-2 adrenergic receptors play a predominant role in thermal regulation (Flavahan et al., 1987a). In several tissues the alpha-2 adrenergic receptors appear to be localizedjunctionally rather than extrajunctionally. These include the hepatic portal system (Segstro and Greenway, 1986) and the saphenous vein (Flavahan et al., 1987b). There is a relatively high density of alpha-2 adrenergic receptors in the kidney (twice as high as alpha-1 adrenergic receptors in the rat) (Sanchez and Pettinger, 1981), but the physiological function of these alpha-2 adrenergic receptors in renal function is not yet clear. In the gastrointestinal tract, alpha-2 adrenergic receptors mediate several responses at different levels, such as regulation of gastric and intestinal mobility and secretions, some of which may be centrally mediated (Nagata and Osumi, 1993). Stimulation of alpha-2 adrenergic receptors promotes net sodium and chloride absorption (Field and McColl, 1973). This results in a net water absorption and thus clonidine produces an inhibition of watery diarrhea (McArthur et al., 1982). In the pancreas alpha-2 adrenergic receptor antagonists increase glucose-stimulated insulin release (Nakaki et al., 1980). Thus it appears that insulin secretion is under tonic inhibition of pancreatic alpha-2 adrenergic receptors, but the precise role of these receptors is unclear. Both circulating epinephrine released from the

236

DAVID B. BYLUND

adrenal medulla and sympathetic release of norepinephrine may contribute to the alpha-2-mediated tonic suppression of insulin release. Lower urinary tract function is under control of the autonomic nervous system and both alpha- 1 and alpha-2 adrenergic receptors appear to mediate the contractile response. Although functional studies originally suggested that only alpha-1 adrenergic receptor mediate the contractile response (Tsujimoto et al., 1986; Kunisawa et al., 1985), more recent results suggest a predominance of alpha-2 adrenergic receptor in the female (Latifpour et al., 1990; Yoshida et al., 1991). Functional studies suggest the presence of alpha-2 adrenergic receptors on vascular endothelium which cause a relaxation mediated by an endothelium derived relaxing factor, nitric oxide (Cocks and Angus, 1983; Angus et al., 1986; Bockman et al., 1993; Richard et al., 1990). The recent report that norepinephrine-induced release of nitric oxide is enhanced in mineraiocorticoid hypertension (Bockman et al., 1992), suggests that endothelium alpha-2 adrenergic receptors may play an enhanced role in regulating vascular tone under pathological conditions. In general, however, these studies have not been particularly productive. Alpha-2 adrenergic receptor-mediated lowering of intraocular pressure in animals and humans was demonstrated as early as 1966 (Makabe, 1966; Hasslinger, 1969). This appears to be the result of suppressing aqueous flow (Lee et al., 1984; Gharagozloo et al., 1988; Toris et al., 1995), although whether the receptors responsible for this action are presynaptic or postsynaptic is not yet entirely clear. The ability of epinephrine to induce aggregation of human platelets and to potentiate aggregation induced by other agents such as thrombin and ADP is well known. This function is mediated by alpha-2 adrenergic receptors. In general, however, the plasma concentrations of epinephrine are much lower than those required to induce platelet aggregation in vitro. It is possible that the physiological control of platelet aggregation is mediated by the actions of multiple agems including epinephrine, each of which is present at levels below that which would individually cause platelet aggregation. Radioligand binding assays of human platelet alpha-2 adrenergic receptors has been used in numerous clinical studies in an attempt to understand the role of alpha-2 adrenergic receptors in various pathological and physiological states.

C. Central Alpha-2 Adrenergic Receptor Functions Alpha-2 adrenergic receptors are widely distributed in the central nervous system and appear to be directly involved in several central functions. Stimulation of central alpha-2 adrenergic receptors in the ventrolateral medulla induces a reduction in sympathetic outflow to the periphery and a resulting reduction in arterial blood pressure. The hypotensive action of alpha-2 adrenergic receptor agonists appears to be due to stimulation of postsynaptic alpha-2 receptors rather than presynaptic receptors. The imidazoline receptors also may participate in the mediation of the antihypertensive effect of many alpha-2 adrenergic receptor drugs.


237

For nearly two decades alpha-2 adrenergic receptor agonists have been widely used by veterinarians to achieve dose-dependent sedation, analgesia and muscle relaxation in a variety of domesticated and wild animal species. More recently this is being extended to human medicine (Hayashi and Maze, 1993). The site and mechanism of action for these effects remain controversial. Alpha-2 adrenergic receptors also seem to play a role in the regulation of affect or mood, and some alpha-2 adrenergic antagonists appear to be effective antidepressants. Alpha-2 adrenergic agonists also appear to improve memory under certain conditions. Opiate and alpha-2 adrenergic receptor agonists act through independent receptors within the locus coeruleus but produce similar depressant effects on net cell activity (Aghajanian, 1978). Since opiates and alpha-2 adrenergic receptor agonists do not exhibit cross tolerance, alpha-2 adrenergic agonists have become a mainstay in the management of acute detoxification from opiate addiction (Gold et al., 1978).

V. CORRELATION OF RECEPTOR BINDING A N D FUNCTION One of the more difficult issues facing investigators who study receptors is the correlation of receptor binding with receptor function. Of particular difficulty is establishing that the receptor identified by radioligand binding studies is the same receptor identified in functional studies. Frequently investigators feel it is sufficient to show that the pharmacological characteristics of the receptors are the same. However, studies with the submandibular gland indicated that this approach is not sufficient. We have shown that the alpha-2 adrenergic receptor labeled in radioligand binding studies by [3H]clonidine is localized postsynaptically (Bylund and Martinez, 1981). This conclusion is based on the following evidence. (1) Partial destruction of the presynaptic nerve terminal with 6-hydroxydopamine did not decrease the density of alpha-2 adrenergic receptors following subsequent reserpine administration; (2) duct ligation which results in atrophy of the gland markedly decrease the density of receptors following subsequent reserpine administration; (3) surgical denervation resulted in an appearance of high density of alpha-2 adrenergic receptors; and (4) the changes in alpha-2 adrenergic receptor binding paralleled the changes in the postsynaptic beta adrenergic receptor (Bylund and Martinez, 1981). By contrast functional studies such as those involving the release of [3H]norepinephrine, indicate that the alpha-2 adrenergic receptors which modulate inhibition of [3H]norepinephrine release are localized presynaptically (Filinger et al., 1978). Thus, the alpha-2 receptors which are identified functionally and appear to be localized presynaptically, may not be the same alpha-2 adrenergic receptors identified in this tissue by radioligand binding studies and which are postsynaptically localized. This is in spite of data indicating that the pharmacological characteristics of the submandibular gland presynaptic receptor as identified by functional studies

238

DAVID B. BYLUND

appears to be of the alpha-2D subtype (Smith et al., 1992) the same subtype identified in binding studies (Michel et al., 1989; Bylund, 1992). These results demonstrate that considerable care is needed in attempting to correlate receptor binding and function. In fact, no study has yet clearly identified presynaptic alpha-2 adrenergic receptors based on radioligand binding studies. This may be due to a much lower density of the presynaptic receptor which is beyond the detection limit of current assay systems.

Vi.

CONCLUSION

Adrenergic receptors are classified into three main types, the alpha-1, alpha-2, and beta adrenergic receptor types. Each of these types is further divided into three or more subtypes. For the alpha-2 adrenergic receptor subtypes there is good agreement between the pharmacologically defined subtypes and the subtypes identified by molecular cloning. By contrast, it is not yet clear how the various cloned alpha- 1 adrenergic receptors fit into the existing pharmacological classification scheme. Alpha-1 and alpha-2 adrenergic receptors are known to mediate a multitude of peripheral and central functions, although the particular subtype (i.e., alpha-lA, -1B and alpha-2A, -2B or -2C) involved is generally not known. A determination of this subtype specificity for each of these functions is an important challenge for the rest of this decade.

REFERENCES Abou-Samra, A.-B., JOppner, H., Force, T., Freeman, M. W., Kong, X.-F., Schipani, E.,. Urena, E, Richards, J., Bonventre, J. V., Ports, J. T., Jr., Kronenberg, H. M., & Segre, G. V. (1992). Expression cloning of a commonreceptor for a parathyroidhormone and parathyroidhormone-relatedpeptide from rat osteoblast-likecells: A singlereceptor stimulatesintracellularaccumulationof both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc. Natl. Acad. Sci. USA 89, 2732-2736. Aghajanian, G. K. (1978). Tolerance of locus coeruleus neurons to morphine and suppression of withdrawal response by clonidine. Nature 276, 186-188. Ahlquist, R. P. (1948). A study of adrenotropic receptors. Am. J. Physiol. 153,586-600. Alabaster, V. A., Keir, R. F., & Peters, C. J. (1985). Comparisonofactivity ofalpha-adrenoceptoragonists and antagonists in dog and rabbit saphenous vein. Naunyn-Schmiedeberg'sArch. Pharmacol. 330, 33--36. Angus, J. A., Cocks, T. M., & Satoh, K. (1986). The et-adrenoceptors on endothelial cells. Fed. Proc. 45, 2355-2359. Ask, J. A., Stene-Larsen, G., Helle, K. B., & Besch, F. (1987). Functional ot-adrenoceptors in human atrial preparations in the presence of 13-receptorblockade. Acta Physiol. Scand. 131,439--445. Battaglia, G., Shannon, M., Borgundvaag, B., & Titeler, M. (1983). Properties of 3H-prazosin labeled oq-adrenergic receptors in rat brain and porcine neurointermediate lobe tissue. J. Neurochem. 41, 538-542. Berthelson, S., & Pettinger, W. A. (1977). A functionalbasis for classificationofct-adrenergic receptors. Life Sci. 21,595--606. Benfey, B. G. (1982). Function of myocardial ot-adrenoceptors. Life Sci. 31,101-112.


239

Blaxall, H. S., Cerutis, D. R., Hass, N. A., & Bylund, D. B. (1994). Clonirig and expression of the alpha-2C adrenergic receptor from the OK cell line. Mol. Pharmacol. Blaxall, H. S., Heck, D. A., & Bylund, D. B. (1993). Molecular determinants of the alpha-2D adrenergic receptor subtype. Life Sci. 53, 255-259. Blaxall, H. S., Murphy, T. J., Baker, J. C., Ray, C., & Bylund, D. B. (1991). Characterization of the alpha-2C adrenergic receptor subtype in the opossum kidney and in the OK cell line. J. Pharmacol. Exp. Ther. 259, 323-329. Bockman, C. S., Jeffries, W. B., & Abel, P. W. (1993). Binding and functional characterization of alpha-2 adrenergic receptor subtypes on pig vascular endothelium. J. Pharmacol. Exp. Ther. 267, 11261133. Bockman, C. S., Jeffries, W. B., Pettinger, W. A., & Abel, P. W. (1992). Enhanced release ofendothelium-derived relaxing factor in mineralocorticoid hypertension. Hypertension 20, 304-313. Bolli, P., Eme, P., Block, L. H., Ji, B. H., Kiowski, W., & Buhler, F.R. (1984). Adrenaline induces vasoconstriction through post-junctional u2-adrenoceptors and this response is enhanced in patients with essential hypertension. J. Hypertension 2, 115-118. Brodde, O.-E., & Michel, M. C. (1992). Adrenergic receptors and their signal transduction mechanisms in hypertension. J. Hypertension 10, S 133-S145. Bruno, J. F., Whittaker, J., Song, J., & Berelowitz, M. (1991). Molecular cloning and sequencing of a cDNA encoding a human alA adrenergic receptor. Biochem. Biophys. Res. Comm. 179, 14851490. Bylund, D. B. (1985). Heterogeneity of alpha-2 adrenergic receptors. Pharmacol. Biochem. Behav. 22, 835--843. Bylund, D. B. (1988a). Subtypes of a2-adrenoceptors: pharmacological and molecular biological evidence converge. TIPS 9, 356-361. Bylund, D. B. (1988b). Alpha-2 adrenergic receptors: A historical perspective. In: The Alpha-2 Adrenergic Receptors (L. Limbird, ed.), pp. 1-13 Humana Press, Clifton, N.J. Bylund, D. B. (1992). Subtypes of eq-and Gt2-adrenergic receptors. FASEB J. 6, 832--839. Bylund, D. B., Eikenberg, D. C., Hieble, J. P., Langer, S. Z., Lefkowitz, R. J., Minneman, K. P., Molinoff, P. B., Ruffolo, R. R., Jr., & Trendelenburg, A. U. (1994). IV. International Union of Pharmacology Nomenclature of Adrenoceptors. Pharmacol. Rev. 46, 121-136. Bylund, D. B., & Martinez, J. R. (1980). a2-Adrenergic receptors appear in rat salivary glands after reserpine treatment. Nature 285, 229-230. Bylund, D. B., & Martinez, J. R. (1981). Postsynaptic localization of u2-adrenergic receptors in rat submandibular gland. J. Neurosci. 1, 1003-1007. Bylund, D. B., & Ray-Prenger, C. (1989). Alpha-2A and alpha-2B adrenergic receptor subtypes: attenuation of cyclic AMP production in cell lines containing only one receptor subtype. J. Pharmacol. Exp. Ther. 251,640-644. Bylund, D. B., Ray-Prenger, C., & Murphy, T. J. (1988). Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype. J. Pharmacol. Exp. Ther. 245, 600-607. Bylund, D. B., Regan, J. W., Faber, J. E., Hieble, J. P., Triggle, C. R., & Ruffolo, R. R., Jr. (1995). Vascular a-adrenoceptors: From the gene to the human. Can. J. Physiol. Pharmacol. 73,533-543. Cerutis, D. R., Hass, N. A., Iversen, L. J., & Bylund, D. B. (1994). The cloning and expression of an OK cell cDNA encoding a 5-HT m receptor. Mol. Pharmacol., in press. Chalberg, S. C., Duda, T., Rhine, J. A., & Sharma, R. K. (1990). Molecular cloning, sequencing and expression of an ot2-adrenergic receptor complementary DNA from rat brain. Mol. Cell. Biochem. 97, 161-172. Chruscinski, A. J., Link, R. E., Daunt, D. A., Barsh, G. S., & Kobilka, B. K. (1992). Cloning and expression of the mouse homolog of the human et2-C2 adrenergie receptor. Biochem. Biophys. Res. Comm. 186, 1280-1287.

240

DAVID 13. BYLUND

Cocks, T. M., & Angus, J. A. (1983). Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature 305, 627-630. Cotecchia, S., Schwinn, D. A., Randall, R. R., Lefkowitz, R. J., Caron, M. G., & Kobilka, B. K. (1988). Molecular cloning and expression of the cDNA for the hamster Otl-adrenergic receptor. Proc. Natl. Acad. Sci. USA 85, 7159-7163. Culling, W., Penny, W. J., Cunliffe, G., Flores, N. A., & Sheridan, D. J. (1987). Arrhythmogenic and electrophysiological effects of alpha adrenoceptor stimulation during myocardial ischaemia and reperfusion. J. Mol. Cell. Cardiol. 19, 251-258. Delbarre, B., & Schmitt, H. (1973). A further attempt to characterize sedative receptors activated by clonidine in chickens and mice. Eur. J. Pharmacol. 22, 355-359. Emsberger, P., Meeley, M. P., Mann, J. J., & Reis, D. J. (1987). Clonidine binds to imidazole binding sites as well as alpha2-adrenoceptors in the ventrolateral medullar. Eur. J. Pharmacol. 134, 1-13. Feng, E, Abel, E W., Scofield, M., Liu, F., Wolff, D. W., & Jeffries, W. B. (1993). Heterogeneous expression of ctl-adrenoceptor subtypes among rat nephron segments. Mol. Pharmacol. 44, 926-933. Field, M., & McColl, I. (1973). Ion transport in rabbit ileal mucosa. III. Effects of catecholamines. Am. J. Physiol. 225, 852-857. Filinger, E. J., Langer, S. Z., Perec, C. J., & Stefano, F. J. E. (1978). Evidence for the presynaptic location of the a-adrenoceptors which regulate noradrenaline release in the rat submaxillary gland. Naunyn Schmiedeberg's Arch. Pharmacol. 304, 21-26. Flavahan, N. A., Cooke, J. P., Shepherd, J. T., & Vanhoutte, E M. (1987a). Human postjunctional alpha- 1 and alpha-2 adrenoceptors: differential distribution in arteries of the limbs. J. Pharmacol. Exp. Ther. 241, 361-365. Flavahan, N. A., Miller, V. M., Aarhus, L. L., & Vanhoutte, P. M. (1987b). Denervation augments alpha-2 but not alpha-1 adrenergic responses in canine saphenous veins. J. Pharmacol. Exp. Ther. 240, 589-593. Flordellis, C. S., Handy, D. E., Bresnahan, M. R., Zannis, V. I., & Gavras, H. (1991). Cloning and expression of a rat brain CtEB-adrenergicreceptor. Proc. Natl. Acad. Sci. USA 88, 1019-1023. Ford, A. P. D. W., Williams, T. J., Blue, D. R., & Clarke, D. E. (1994). txl-Adrenoceptor classification: sharpening Occam's razor. Trends Pharmacol. Sci. 15, 167-170. Fowler, P. J., Grous, M., Price. W., & Matthews, W. D. (1984). Pharmacological differentiation of postsynaptic alpha adrenoceptors in the dog saphenous vein. J. Pharmacol. Exp. Ther. 229, 712-718. Gao, B., & Kunos, G. (1993). Isolation and characterization of the gene encoding the rat ala-adrenergic receptor. Gene 131,243-247. Gharagozloo, N. Z., Relf, S. J., & Brubaker, R. F. (1988). Aqueous flow is reduced by the alpha-adrenergic agonist, apraclonidine hydrochloride (ALO 2145). Ophthalmology 95, 1217-1220. Gleason, M. M., & Hieble, J. P. (1992). The ct2-adrenoceptors of the human retinoblastoma cell line (Y79) may represent an additional example of the a2C-adrenoceptor. Br. J. Pharmacol. 107, 222-225. Gold, M. S., Redmond, D. E., Jr., & Kleber, H. D. (1978). Clonidine blocks acute opiate withdrawal symptoms. Lancet 2, 599-602. Gross, G., Hanft, G., & Mehdom, H. M. (1989). Demonstration of tXlA- and otlB-adrenoceptor binding sites in human brain tissue. Eur. J. Pharmacol. 169, 325-328. Guyer, C. A., Horstman, D. A., Wilson, A. L., Clark, J. D., Cragoe, E. J., Jr., & Limbird. L. E. (1990). Cloning, sequencing, and expression of the gene encoding the porcine (x2-adrenergic receptor. Allosteric modulation by Na ÷, H÷, and amiloride analogs. J. Biol. Chem. 265, 17307-17317. Han, C., Abel, E W., & Minneman, K. E (1987). Heterogeneity of ctl-adrenergic receptors revealed by chloroethylclonidine. Mol. Pharmacol. 32, 505-510. Hanft, G., & Gross, G. (1989). Subclassification of al-adrenoceptor recognition sites by urapidil derivatives and other selective antagonists. Br. J. Pharmacol. 97, 691-700.


241

Harrison, J. K., Pearson, W. R., & Lynch, K. R. (1991). Molecular characterization of otl- and ct2-adrenoceptors. TIPS 12, 62--67. Hasslinger, C. (1969). Catapres (2-[2,6-dichlorophenylamino]-2-imidazoline hydrochloride}---a new drug lowering intraocular pressure. Klin. Mbl. Augenheilk. 154, 95-105. Hayashi, Y., & Maze, M. (1993). Alpha2 adrenoceptor agonists and anaesthesia. Brit. J. Anaes. 71, 108-118. Hesse, I. F. A., & Johns, E. J. (1984). The subtype of cz-adrenoceptor involved in the neural control of renal tubular sodium reabsorption in the rabbit. J. Physiol. 352, 527-538. Heusch, G., & Deussen, A. (1983). The effects of cardiac sympathetic nerve stimulation on perfusion of stenotic coronary arteries in the dog. Circ. Res. 53, 8-15. Hieble, J. P., Bondinell, W. E., & Ruffolo, R. R., Jr. (1995). or- and 13-Adrenoceptors: From the gene to the clinic. 1. Molecular biology and adrenoceptor subclassification. J. Med. Chem. 38, 3415-3444. Hieble, J. P., Bylund, D. B., Clarke, D. E., Eikenburg, D. C., Langer, S. Z., Lefkowitz, R. J., Minneman, K. P., & Ruffolo, R. R., Jr. (1995). International Union of Pharmacology. X. Recommendation for nomenclature of czl-adrenoceptors: Consensus update. Pharmacol. Rev. 47, 267-270. Hieble, J. P., Sulpizio, A. C., Edwards, R., Chapman, H., Young, P., Roberts, S. P., Blackburn, T. P., Wood, M. D., Shah, D. H., Demarinis, R. M., & Ruffolo, R. R., Jr. (1991). Additional evidence for functional subclassification of alpha-2 adrenoceptors based on a new selective antagonist, SK&F 104856. J. Pharmacol. Exp. Ther. 259, 643--652. Hirasawa, A., Horie, K., Tanaka, T., Takagaki, K., Mural, M., Yano, J., & Tsujimoto, G. (1993). Cloning, functional expression and tissue distribution of human cDNA for the atlc-adrenergic receptor. Biochem. Biophys. Res. Comm. 195, 902-909. Hyman, A. L., & Kadowitz, P. J. (1986). Enhancement of~t- and 13-adrenoceptorresponses by elevations in vascular tone in pulmonary circulation. Am. J. Physiol. 250, H 1109-H 1116. Jie, K., van Brummelen, P., Vermey, P., Timmermans, P. B. M. W. M., & van Zwieten, P. A. (1986). Alpha 1- and alpha2-adrenoceptor mediated vasoconstriction in the forearm of normotensive and hypertensive subjects. J. Cardiovasc. Pharmacol. 8, 190-196. Johnson, R. D.. & Minneman, K. P. (1987). Differemiation of ¢xl-adrenergic receptors linked to phosphatidylinositol turnover and cyclic AMP accumulation in rat brain. Mol. Pharmacol. 31, 239-246. JOppner, H., Abou-Samra, A.-B., Freeman, M., Kong, X. F., Schipani, E., Richards, J., Kolakowski, L. F., Jr., Hock, J., Ports, J. R., Jr., Kronenberg, H. M., & Segre, G. V. (1991). A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254, 10241026. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J., & Regan, J. W. (1987). Cloning, sequencing, and expression of the gene coding for the human platelet ot2-adrenergic receptor. Science 238, 650-656. Kunisawa, Y., Kawabe, K., Niijima, T., Honda, K., & Takeraka, T. (1985). A pharmacological study of c~-adrenergic subtypes in smooth muscle of human urinary bladder base and prostatic urethra. J. Urol. 134, 396-398. Kurose, H., Arriza, J. L., & Lefkowitz, R. J. (1993). Characterization of et2-adrenergic receptor subtype-specific antibodies. Mol. Pharmacol. 43,444-450. Langer, S. Z. (1974). Presynaptic regulation of catecholamine release. Biochem. Pharmacol. 23, 1793-1800. Langer, S. Z., & Shepperson, N. B. (1982). Postjunctional cq- and c~2-adrenoceptors: preferemial innervation of otl-adrenoceptors and the role of neuronal uptake. J. Cardiovasc. Pharmacol. 4, $8-S13. Lanier, S. M., Downing, S., Duzie, E., &Homcy, C. J. (1991). Isolation of rat genomic clones encoding subtypes of the ot2-adrenergic receptor. Identification of a unique receptor subtype. J. Biol. Chem. 266, 10470-10478.

242

DAVID B. BYLUND

Lanier, S. M., Ivkovic, B., Singh, I., Neumeyer, J. L., & Bakthavachalam, V. (1993). Visualization of multiple imidazoline/guanidinium-receptive sites. J. Biol. Chem. 16047-16051. Latifpour, J., Kondo, S., O'Hollaren, B., Morita, T., & Weiss, R. M. (1990). Autonomic receptors in urinary tract: Sex and age differences. J. Pharmacol. Exp. Ther. 253, 661--667. Lee, D. A., Topper, J. E., & Brubaker, R. E (1984). Effect ofclonidine on aqueous humor flow in normal human eyes. Exp. Eye Res. 38, 239-246. Limbird, L. E. (ed.) (1988). The Alpha-2 Adrenergic Receptors, Humana Press, Clifton. N.J. Link, R., Daunt, D., Barsh, G., Chruscinski, A., & Kobilka, B. (1992). Cloning of two mouse genes encoding a2-adrenergic receptor subtypes and identification of a single amino acid in the mouse a2-C 10 homolog responsible for an interspecies variation in antagonist binding. Mol. Pharmacol. 42, 16-27. Lomasney, J. W., Cotecchia, S., Lefkowitz, R. J., & Caron, M. G. (1991a). Molecular biology of a-adrenergic receptors: implications for receptor classification and for structure-function relationships. Biochim. Biophys. Acta 1095, 127-139. Lomasney, J. W., Cotecchia, S., Lorenz, W., Leung, W.-Y., Schwinn, D. A., Yang-Feng, T. L., Brownstein, M., Lefkowitz, R. J., & Caron, M. G. (1991b). Molecular cloning and expression of the cDNA for the alA-adrenergic receptor. The gene for which is located on human chromosome 5. J. Biol. Chem. 266, 6365-6369. Lomasney, J. W., Lorenz, W., Allen, L. F., King, K., Regan, J. W., Yang-Feng, T. L., Caron, M. G., & Lefkowitz, R. J. (1990). Expansion of the t~z-adrenergic receptor family: Cloning and characterization of a human ct2-adrenergic receptor subtype, the gene for which is located on chromosome 2. Proc. Natl. Acad. Sci. USA 87, 5094-5098. MacKinnon, A. C., Stewart, M., Olverman, H. J., Spedding, M., & Brown, C. M. (1993). [3H]p-Aminoclonidine and [3H]idazoxan label different populations of imidazoline sites on rat kidney. Eur. J. Pharmacol. 232, 79-87. Makabe, R. (1966). Ophthalmological studies with dichlorophenyamine-Imidazolines. Dtsch. Med. Wochenschr. 91, 1686-1696. Maze~ M., & Tranquilli, W. (1991). Alpha-2 adrenoceptor agonists: defining the role in clinical anesthesia. Anesthesiology 74, 581-605. McArthur, K. E., Anderson, D. S., Durbin, T. E., Orloff, M. J., & Dharmsathaphom, K. (1982). Clonidine and lidamidine to inhibit watery diarrhea in a patient with lung cancer. Ann. Intem. Med. 96, 323--325. McCormick, D. A., & Prince, D. A. (1988). Noradrenergic modulation of firing pattern in guinea pig and cat thalamic neurons, in vivo. J. Neurophysiol. 59, 978-996. McGrath, J. C., Brown, C. M., & Wilson, V. G. (1989). Alpha-adrenoceptors: A critical review. Med. Res. Rev. 9, 407-533. Michel, A. D., Loury, D. N., & Whiting, R. L. (1989). Differences between the ct2-adrenoceptor in rat submaxillary gland and the aZA- and a2B-adrenoeeptor subtypes. Br. J. Pharmacol. 98, 890-897. Minneman, K. P. (1988). ot1-Adrenergic receptor subtypes, inositol phosphates, and sources of cell Ca 2+. Pharmacol. Rev. 40, 87-119. Minneman, K. E, Han, C., & Abel, P. W. (1988). Comparison of cq-adrenergic receptor subtypes distinguished by chloroethylclonidine and WB 4101. Mol. Pharmaeol. 33, 509-514. Molderings, G. J., Moura, D., Fink, K., Brnisch, H., & Grthert, M. (1993). Binding of [3H]elonidine to Ii-imidazoline sites in bovine adrenal medullary membranes. Naunyn-Schmiedeberg's Arch. Pharmacol. 348, 70-76. Morrow, A. L., & Creese, I. (1986). Characterization of cq-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB 4101 and [3H]prazosin binding. Mol. Pharmacol. 29, 321-330. Muramatsu, I., Oshita, M., Ohmura, T., Kigoshi, S., Akino, H., Gobara, M., & Okada, K. (1994). Pharmacological characterization of ctl-adrenoceptor subtypes in the human prostate: Functional and binding studies. Br. J. Urol. 74, 572-578.


243

Murphy, T. J., & Bylund, D. B. (1988). Characterization of alpha-2 adrenergic receptors in the OK cell, an opossum kidney cell line. J. Pharmacol. Exp. Ther. 244, 571-578. Nagata, M., & Yoshitsugu, O. (1993). Central ct2-adrenoceptor-mediated inhibition of gastric motility in rats. Japan. J. Pharmacol. 62, 329-330. Nakaki, T., Nakadate, T., & Kato, R. (1980). ct-Adrenoceptors modulating insulin release from isolated pancreatic islet cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 313, 151-153. Osborn, J. L., Holdaas, H., Thames, M. D., & Di Bona, G. F. (1983). Renal adrenoceptor mediation of antinatriuretic and renin secretion responses to low frequency renal nerve stimulation in the dog. Circ. Res. 53,298-305. Perez, D. M., Piascik, M. T., & Graham, R. M. (1991). Solution-phase library screening for the identification of rare clones: isolation of an OqD-adrenergic receptor cDNA. Mol. Pharmacol. 40, 876-883. Piletz, J. E., & Sletten, K. (1993). Nonadrenergic imidazoline binding sites on human platelets. J. Pharmacol. Exp. Ther. 267, 1493-1502. Porter, J. E., Dowd, F. J., & Abel, P. W. (1992). Atypical alpha-1 adrenergic receptors in the rat parotid gland acinar cell. J. Pharmacol. Exp. Ther. 263, 1062-1067. Raddatz, R., Parini, A., & Lanier, S. M. (1995). Imidazoline/guanidinium binding domains on monoamine oxidases---Relationship to subtypes of imidazoline-binding proteins and tissuespecific interaction of imidazoline ligands with monoamine oxidase B. J. Biol. Chem. 270, 27961-27968. Ramarao, C. S., Denker, J. M., Perez, D. M., Gaivin, R. J., Riek, R. P., & Graham, R. M. (1992). Genomic organization and expression of the human OtlB-adrenergic receptor. J. Biol. Chem. 267, 2193621945. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., & Kobilka, B. K. (1988). Cloning and expression of a human kidney cDNA for an ot2-adrenergic receptor subtype. Proc. Natl. Acad. Sci. USA 85, 6301-6305. Richard, V., Tanner, F. C., Tschudi, M., & Luscher, T. F. (1990). Different activation of L-arginine pathway by bradykinin, serotonin, and clonidine in coronary arteries. Am. J. Physiol. 259, H1433--H1439. Rossum, J. M. (1965). Different types of sympathomimetic a-receptors. J. Pharm. Pharmacol. 17, 202-216. Ruffolo, R., Jr. (ed.) (1987). The Alpha-1 Adrenergic Receptors, Humana Press, Clifton, N.J. Ruffolo, R., Jr. (ed.)(1991). ct-Adrenoceptors: molecular biology, biochemistry and pharmacology, S. Karger AG. Switzerland. Ruffolo, R. R., Bondinell, W., & Hieble, J. P. (1995). ct- and 13-adrenoceptors: From the gene to the clinic. 2. Structure-activity relationships and therapeutic applications. J. Med. Chem. 38, 36813716. Ruffolo, R. R., Jr., & Zeid, R. L. (1985). Relationship between ct-adrenoceptor occupancy and response for the ctl-adrenoceptor agonist, cirazoline, and the ot2-adrenoceptor agonist, B-HT 933, in canine saphenous vein. J. Pharmacol. Exp. Ther. 235, 636--643. Ruffolo, R. R., Jr., Nichols, A. J., Stadel, J. M., & Hieble, J. P. (1991). Structure and function of et-adrenoceptors. Pharmacol. Rev. 43, 475--505. Sanchez, A., & Pettinger, W. A. (1981). Dietary sodium regulation of blood pressure and renal ot1- and et2-receptors in WKY and SHR. Life Sci. 29, 2795-2802. Schwinn, D. A., & Lomasney, J. W. (1992). Pharmacologic characterization of cloned otl-adrenoceptor subtypes: selective antagonists suggest the existence of a fourth subtype. Eur. J. Pharmacol. 227, 433-436. Schwinn, D. A., Lomasney, J. W., Lorenz, W., Szklut, P. J., Fremeau, R. T., Jr., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., & Cotecchia, S. (1990). Molecular cloning and expression of the cDNA for a novel eq-adrenergic receptor subtype. J. Biol. Chem. 265, 8183-8189.

244

DAVID B. BYLUND

Segstro, R., & Greenway, C. V. (1986). Alpha receptor subtype mediating sympathetic mobilization of blood from the hepatic venous system in anesthetized cats. J. Pharmacol. Exp. Ther. 236, 224-229. Seitelberger, R., Guth, B. D., Heusch, G., Lee, J. D., Katayama, K., & Ross, J., Jr. (1988). Intracoronary a2-adrenergic receptor blockade attenuates ischemia in conscious dogs during exercise. Circ. Res. 62, 436-442. Shebuski, R. J., Fujita, T., & Ruffolo, R. R., Jr. (1986). Evaluation of alpha-1 and alpha-2 adrenoceptor-mediated vasoconstriction in the in situ, autoperfused, pulmonary circulation of the anesthetized dog. J. Pharmacol. Exp. Ther. 238, 217-223. Simonneaux, V., Ebadi, M., & Bylund, D. B. (1991). Identification and characterization of alpha-2D adrenergic receptors in bovine pineal gland. Mol. Pharmacol. 40, 235-241. Smith K., Connaughton, S., & Docherty, J. R. (1992). Investigations ofprejunctional a2-adrenoceptors in rat atrium, vas deferens and submandibular gland. Eur. J. Pharmacol. 211, 251-256. Summers, R. J., & McMartin, L. R. (1993). Adrenoceptors and their second messenger systems. J. Neurochem. 60, 11-21. Szabadi, E., & Bradshaw, C. M. (1987). Alpha- 1 adrenergic receptors in the central nervous system: In The Alpha- 1 Adrenergic Receptor (Ruffolo, R. R., ed.), pp. 405-433, Humana Press, Clifton, NJ. Terzic, A., Pucrat, M., Vassort, G., & Vogel, S. M. (1993). Cardiac al-adrenoceptors: An overview. Pharmacol. Rev. 45, 147-175. Timmermans, P. B. M. W. M., & van Zwieten, P. A. (1982). a2-Adrenoceptors: classification, localization, mechanisms, and targets for drugs. J. Med. Chem. 25, 1390-1401. Toffs, C. B., Tafoya, M. E., Camras, C. B., & Yablonski, M. E. (1995). Effects of apraclonidine on aqueous humor dynamics in human eyes. Ophthalmology 102, 456-461. Tsujimoto, G., Timmins, P. V., & Hoffman, B. B. (1986). ct-Adrenergic receptors in the rabbit bladder base smooth muscle: al-adrenergic receptor mediate contractile responses. J. Pharmacol. Exp. Ther. 236, 384-389. Uhlen, S., & Wikberg, J. E. S. (1991). Delineation of three pharmacological subtypes of a2-adrenoceptors in the kidney. Br. J. Pharmacol. 104, 657-664. Uhlen, S., Xia, Y., Chhajlani, V., Felder, C. C., & Wikberg, J. E. S. (1992). [3H]-MK-912 binding delineates two a2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d a2-adrenoceptor. Br. J. Pharmacol. 106, 986-995. Voigt, M. M., McCune, S. K., Kanterman, R. Y., & Felder, C. C. (1991). The rat ct2-C4 adrenergic receptor gene encodes a novel pharmacological subtype. FEBS Lett. 278, 45-50. Weinshank, R. L., Zgombick, J. M., Macchi, M., Adham, N., Lichtblau, H., Branchek, T. A., & Hartig, P. R. (1990). Cloning, expression, and pharmacological characterization of a human a2B-adrenergic receptor. Mol. Pharmacol. 38, 681-688. Wilffert, B., Timmermans, P. B. M. W. M., & van Zwieten, P. A. (1982). Extrasynaptic location of alpha-2 and noninnervated beta-2 adrenoceptors in the vascular system of the pithed normotensive rat. J. Pharmacol. Exp. Ther. 221,762-768. Wilson, K. M., & Minneman, K. P. (1989). Regional variations in t~l-adrenergic receptor subtypes in rat brain. J. Neurochem. 43, 1782-1786. Yoshida, M., Latifpour, J., Nishimoto, T., & Weiss, R. M. (1991). Pharmacological characterization of alpha adrenergic receptors in the young and old female rabbit urethra. J. Pharmacol. Exp. Ther. 257, 1100-1108. Zeng, D., Harrison, J. K., D' Angelo, D. D., Barber, C. M., Tucker, A. L., Lu, Z., & Lynch, K. R. (1990). Molecular characterization of a rat Ct2B-adrenergic receptor. Proc. Natl. Acad. Sci. USA 87, 3102-3106.

Biomembranes

A Multi-Volume Treatise Edited by A.G. Lee, Department of Biochemistry, University of Southampton "Progress in understanding the nature of the biological membranes has been very rapid over a broad front, but still pockets of ignorance remain. Application of the techniques of molecular biology has provided the sequences of a very large number of membrane proteins, and led to the discovery of superfamilies of membrane proteins of related structure. In turn, the identification of these supeffamilies has led to new ways of thinking about membrane processes. Many of these processes can now be discussed in molecular terms, and unexpected relationships between apparently unrelated phenomena are bringing a new unity to the study of biological membranes. The quantity of information available about membrane proteins is now too large for any one person to be familiar with anything but a 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." n From the Preface

Volume 1, General Principles 1995, 279 pp. ISBN 1-55938-658-8

$97.50

CONTENTS: Preface, A.G. Lee. The Functional Role of Lipids in Biological Membranes, David B. Fenske, Myrna A. Monck, Michael J. Hope, and lan R. Cullis. Principles of Membrane Protein Structure, M.S.P. Sansom and lan D. Kerr. Fatty Acidand Isoprenoid-Linked Membrane Proteins, Marco Parenti and Anthony I. Magee. The Biosynthesis of Membrane Proteins, David Stephens, Sunita Kulkami, and Brian Austen. Specificity of Lipid-Protein Interactions, Derek Marsh. Effects of Lipid-Protein Interactions on Membrane Functions, A.G. Lee. General Principles of Membrane Transport, lan C. West. Membrane Signaling Systems, C.U.M. Smith.

JAI PRESS INC.

55 Old Post Road No. 2 - P.O. Box 1678 Greenwich, Connecticut 06836-1678 Tel: (203) 661- 7602 Fax: (203) 661-0792

.1 A_

P R E S S

J A l P R E S S

Advances in Developmental Biochemistry Edited by Paul Wassarman, Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Volume 1, 1992, 256 pp. ISBN 1-55938-347-X

$97.50

CONTENTS: Introduction. Organelle Assembly and Function

in the Amphibian Germinal Vesicle, Joseph G. Gall. DNA Replication and the Role of Transcriptional Elements During Animal Development, Melvin L. DePamphilis. Transcriptional Regulation During Early Drosophila Development, K. Prakash, Joanne Topoi. C.R. Dearolf, and Carl S. Parker. Translational Regulation of Maternal Messenger RNA, L. Dennis Smith. Gut Esterase Expression in the Nematode Caenorhabditis elegans, James D. McGhee. Transcriptional Regulation of Crystallin Genes: Cis Elements, Trans-factors and Signal Transduction Systems in the Lens, Joram Piatigorksy and Peggy S. Zelenka. Subject Index. Volume 2, 1993, 237 pp. ISBN 1-55938-609-6

$97.50

CONTENTS: Preface, Paul M. Wassarman. Drosophila Ho-

meobox Genes, William McGinnis. Structural and Functional Aspects of Mammalian HOX Genes, Denis Duboule. Developmental Control Genes in Myogenesis of Vertebrates, Hans Henning-Amold. Mammalian Fertilization: Sperm Receptor Genes and Glycoproteins, Paul M. Wassarman.The Fertilization Calcium Signal and How It Is Triggered, Michael Whitaker. Subject Index. Volume 3, 1994, 188 pp. ISBN 1-55938-853-6

$97.50

CONTENTS: Preface, Paul M. Wassarman. Expression and

Function of Protein Kinases During Mammalian Gametogenesis, Deborah L. Chapman and Debra J. Wolgemuth. Regulation of the Dopa Decarboxylase Gene During Drosophila Development, Martha J. Lundell and Jay Hirsh. Transcription Factors in Mammalian Development: Murine Homeobox Genes, S. Steven Potter. Expression and Function of C-Mos in Mammalian Germ Cells, Geoffrey M. Cooper. Regulation of Pigmentation During Mammalian Development, Friedrich Beermann, Ruth Ganss, and Gunther Schutz. Index.

Advances in Developmental Biology Edited by Paul Wassarman, Department of Cell and Developmental Biology, Roche Institute of Molecular Biology Volume 1, 1992, 192 pp. ISBN 1-55938-348-8

$97.50

CONTENTS: Introduction. Y Chromosome Function in Mammalian Development, Paul S. Burgoyne. A Super Family of Putative Developmental Signalling Molecules Related to the Proto-Oncogene Wnt-1/int-1, Andrew P. McMahon and Kenneth R. Howard. Pattern Formation in Caenorhabditis Elegans, Min Han and Paul W. Stemberg. Gap Junctional Communication During Mouse Development, Norton B. Gilula, Miyuki NishL and Nalin Kumar. Lens Differentiation and Oncogenesis in Transgenic Mice, Heiner WestphaL Subject Index.

Volume 2, 1993, 195 pp. ISBN 1-55938-582-0

$97.50

CONTENTS: Preface, Paul M. Wassarman. The Sry Gene and Sex Determination in Mammals, Blanche Capel and Robin LovelI-Badge. Molecular and Genetic Studies of Human X Chromosome Inactivation, Carolyn J. Brown and Huntington F. Willard. Genomic Imprinting and Regulation of Mammalian Development, Colin L. Stewart. Cell Interactions in Neural Crest Cell Migration, Marianne Bronner-Fraser. Enzymes and Morphogenesis: Alkaline Phosphatase and Control of Cell Migration, Saul L. Zackson. Subject Index.

Volume 3, 1994, 194 pp. ISBN 1-55938-865-X

$97.50

CONTENTS: Preface, Paul M. Wassarman. Mechanisms of Neurogenesis in Drosophila melanogaster, Jose A. CamposOrtega. The Role of Growth Factors in Mammalian Pregastrulation Development, Daniel A. Rappolee and Zena Werb. Retinoid Signaling in Mouse Embryos, Elwood Linney and Anthony-Samuel LaMantia. RNA Localization During Oogenesis in Drosophila, Elizabeth R. Gavis and Ruth Lehmann. Actin as a Tissue-Specific Marker in Studies of Ascidian Development and Evolution, William R. Jeffery. Index.

.1 A [ P R

S S

J A l

Advances In Cell and Molecular Biology of Membranes and Organelles (Previosly published as Advances In Cell and Molecular Biology of Membranes)

Edited by Alan M. Tartakoff, Institute of Pathology, Case Western Reserve University Volume 1, Endosomes and Lysosomes: A Dynamic Relationship 1993, 434 pp. ISBN 1-55938-362-3

$97.50

Edited by Brian Storrie, Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University and Robert F. Murphy, Department of Biological Sciences, Camegie Mellon University

P R E S S

CONTENTS: Preface, Brian Storrie and Robert F. Murphy. Models of Endosome and Lysosome Traffic, Robert F. Murphy. Endocytic Receptors, Michael G. Roth. Functions of the Mannose 6-Phosphate Receptors, Bemard Hoflack and Peter LobeL Chemistry of Lysosomal Cysteine Proteinases, Robert W. Mason and Donna Wilcon. Mechanism and Regulation of Autophagic Degradation of Cellular Proteins, William A. Dunn, Jr.. Cell-Free Systems for Endocytosis, William A. Braell. Genetic Analysis of Membrane Traffic in Mammalian Cells, Penelope A. Colbaugh and Rockford K. Draper. Plasma Membrane Lipid Transport in Cultured Cells: Studies Using Lipid Analogs and Model Systems, Michael KovaL Endosomes, Lysosomes, and Trans-Golgi-Related Systems in Conventional Neurons and the Grof Retina: Shards and Suppositions, Eric Holtzman, Eliene Augenbraun, Robert St. Jules, and Maria Santa-Hernandez. The Role of Endocytosis in Epidermal Growth Factor Signaling, Bryan K. McCune, William R. Huckle, and H. Shelton Earp. Membrane Traffic Through the Late Stages of the Yeast Secretory, Eric A. Whitters, Henry B. Skinner, and Vytas A. Bankaitis. Regulation of Lysosomal Trafficking and Function During Growth and Development of Dictyostelium discoideum, James A. Cardelli. Towards an Understanding of the Inheritance of Mammalian Lysosomes and Yeast Vacuoles, Brian Storrie. Volume 2, Membrane Transport in Protozoa 1993, 483 pp. 2 Part Set Set ISBN 1-55938-628-2 Edited by Helmut Plattner, Fakult~t f(~rBiologie, Universit&t Konstanz

$195.00

PART A - CONTENTS: Preface. Involvement of the TransGolgi Network, Coated Vesicles, Vesicle Fusion and Secretory Product Condensation in the Biogenesis of Pseudomicrothorax Trichocysts, Robert K. Peck, Barbara Swiderski and Anne-Marie TourmeL Early Steps of the Secretory Pathway in Paramecium: Ultra-structural, Immunocyto-Chemical and Genetic Analysis of Trichocyst Biogenesis, Nicole Garreau de Loubresse. Calcium and Trichocyst Exocytosis in Paramecium: Genetic and Physiological Studies, Jean Cohen and Daniel Kerboeuf. Exocytotic Events During Cell Invasion by Apicomplexa, Jean Francois Dubremeta and Roff Entzeroth. Pathways of Lysosomal Enzyme Secretion in Tetrahymena, Amo Tiedtke, Thomas Kiy, Christian VosskEihlerand Leif Rasmussen. Synchronization of Different Steps of the Secretory Cycle in Paramecium Tetraurelia: Trichocyst Exocytosis, Exocytosis-Coupled Endocytosis and Intracellular Transport, Helrout Plattner, Gerd Knoll, and Regina Pape. The Ciliary Membrane and its Engagement In Conjugation, Jason Wolfe. Ciliary and Plasma Membrane Proteins in Paramecium: Description, Localization and Intracellular Transit, Yvonne Capdeville, Ren6e Charret, Claude Anthony, Julienne Delorme, Pierre Nahon and Andre. Adoutte. PART B - - CONTENTS: Endocytosis and Intracellular Trans-

port of Variant Surface Glycoproteins in Trypanosomes, Michael Duszenko and Andreas Seyfang. A Comparative Survey on Phagosome Formation in Protozoa, Klaus Hausmann and Renate Radek. Endosomal Membrance Traffic of Ciliates, Richard D. Allen and Agnes K. Fok. Membrane Flow in the Digestive Cycle in Paramecium, Agnes K. Fok and Richard D. Allen. Signal Coupling During Endocytosis in Amoeba proteus, Robert D. Prusch. Membrane Recycling and Turnover in Large, Free-Living Amoebae, Kwang W. Jeon. Food Uptake and Digestion in Amoebae, Wilhelm Stockem and Melpo Christofidou-Solomidou. The Lysosomal System in Malaria Parasites, Christian Slomianny. Membrane and Microtubule Dynamics in Heliozoa, Toshinobu Suzaki and Yoshinobu Shigenaka. The Host-Symbiont-lnterface in Ciliate-Algae Associations: Inhibition of Membrane Fusion, Wemer Reisser. Lipid Composition of Membranes Involved in Membrane Traffic in Tetrahymena, Shigenobu Umeki and Yoshinori Nozawa. Volume 3 Signal Transduction Through Growth Factor Receptors

1994, 223 pp. ISBN 1-55938-344-5

$97.50

Edited by: Yasuo Kitagawa, BioSciences Center, Laboratory of Organogenesis, Nagoya University and Ryuzo Sasaki, Faculty of Agriculture, Department of Food Science and Technology, Kyoto University

.1 A l P E S S

J A l

CONTENTS: The Hepatocyte Growth Factor/c-MET Signaling Pathway, D. P. Bottario, A. M.-L. Chan, J. S. Rubin, E. Gak, E. Fortney, J. , Schind/er, M. Chedid, and S., A. Aaronson. Insulin Receptor, Y. Ebina, H. Hayashi, F. Kanai, S. Kamohara, and Y. Nishioka. Interleukin-3 Receptor: Structure and Signal Transduction, T. Kitamura, and A. Miyajima. Interleukin-5 Receptor, K. Takatsu. Interleukin-6 Receptor and Signal Transduction, T. Matsuda, T. Nakajima, T. Kaisho, K. Nakajima, and 7. Hirano. Receptor for Granulocyte ColonyStimulating Factor, S. Nagata, & R. Fukunaga. Receptor for Granulocyte/Macrophage Colony-stimulating Factor, K. Kurata, T. Yokota, A. Miyajima, & K. Arai. Perspectives On The Structure And Mechanisms of Signal Transduction by The Erythropoietin Receptor , S. S. Jones. Interleukin-1 Signal Transduction, J. E. Sims, T. A. Bird, J. G. Giri, and K. S. Dower. Volume 4, Protein Export and Membrane Biogenesis 1995, 276 pp. $97.50 ISBN 1-55938-924-9

P R E

S S

Edited by Ross E. Dalbey, Department of Chemistry, The Ohio State University CONTENTS: Introduction to the Series, Alan M. Tartakoff. Preface, Ross E. Dalbey. Membrane Protein Assembly, Paul Whitley and Gunnar yon Heijne. Membrane Insertion of Small Proteins: Evolutionary and Functional Aspects, Dorothee Kiefer and Andreas Kuhn. Protein Translocation Genetics, Koreaki Ito. Biochemical Analyses of Components Comprising the Protein Translocation Machinery of Escherichia coil, Shin-ichi Matsuyama and Shoji Mizushima. Pigment Protein Complex Assembly in Rhodobacter sphaeroides and Rhodobacter capsulatus, Amy R. Vargas and Samuel Kaplan. Identification and Reconstitution of Anion Exchange Mechanisms in Bacteria, Atul Varadhachary and Peter C. Maloney. Helix Packing in the C-Terminal Half of Lactose Permease, H. Ronald Kaback, Kirsten Jung, Heinrich Jung, Jianhua Wu, Gilbert C. Prive, and Kevin Zen. Export and Assembly of Outer Membrane Proteins in E. coil, Jan Tommassen and Hans de Cock. StructureFunction Relationships in the Membrane Channel Porin, Georg E. Schulz. Role of Phospholipids in Escherichia coil Cell Function, William Dowhan. Mechanism of Transmembrahe Signaling in Osmoregulation, Alfaan A. Rampersaud. Index.

JAI PRESS INC.

55 Old Post Road No. 2 - P.O. Box 1678 Greenwich, Connecticut 06836-1678 Tel: (203) 661- 7602 Fax: (203) 661-0792

Rhodopsin and G-Protein Linked Receptors, Part A,

Linked

Linked

Linked

Linked

The Chemokine Receptors (The Receptors)

The Cannabinoid Receptors (The Receptors)

The Dopamine Receptors (The Receptors)

The Cannabinoid Receptors (The Receptors)

The Chemokine Receptors (The Receptors)

The Melanocortin Receptors (The Receptors)

Prostacyclin and Its Receptors

BLyS Ligands and Receptors

Nuclear Receptors: A Practical Approach

Nuclear Receptors: A Practical Approach

PAF Receptors

X-Linked Mental Retardation

Somatostatin Receptors

fMLP Receptors

Peptide Receptors, Part II (Handbook of Chemical Neuroanatomy)

TNF Receptors

Activin Receptors

Neurotransmitter Receptors

TPO Receptors

TGFβ Receptors

Adenosine Receptors and Parkinson's Disease

PNH and the GPI-Linked Proteins

Antibodies and T-Cell Receptors,

Linked By Blood

Insurance Linked Securities

Negative Co-Receptors and Ligands

Rhodopsin and G-Protein Linked Receptors, Part A,

Linked

Linked

Linked

Linked

The Chemokine Receptors (The Receptors)

The Cannabinoid Receptors (The Receptors)

The Dopamine Receptors (The Receptors)

The Cannabinoid Receptors (The Receptors)

The Chemokine Receptors (The Receptors)

The Melanocortin Receptors (The Receptors)

Prostacyclin and Its Receptors

BLyS Ligands and Receptors

Nuclear Receptors: A Practical Approach

Nuclear Receptors: A Practical Approach

PAF Receptors

X-Linked Mental Retardation

Somatostatin Receptors

fMLP Receptors

Peptide Receptors, Part II (Handbook of Chemical Neuroanatomy)

TNF Receptors

Activin Receptors

Neurotransmitter Receptors

TPO Receptors

TGFβ Receptors

Adenosine Receptors and Parkinson's Disease

PNH and the GPI-Linked Proteins

Antibodies and T-Cell Receptors,

Linked By Blood

Insurance Linked Securities

Negative Co-Receptors and Ligands

Recommend Documents