Progress in Nucleic Acid Research and Molecular Biology, Volume 65

Some Articles Planned for Future Volumes

Proceedings

of the Workshop

on “Base Excision

Repair 2000”

SANKAKMITRA AND R. STEPHENLLOYD Exoribonucleases

and Their Multiple Roles in RNA Metabolism

MURRAYDEUTSCHER A Unique Combination of Thyroid

of Transcription

Factors Controls

Differentiation

Cells

ROBERTODI LAURO, G. DAMANTE,AND L. PELLIZARI ATP Synthase:

The Missing

Link

STANLEYD. DUNN, D. T. MCLACHLIN, AND M. J. RE~INCTON Molecular

Characterization

of Cation-Chloride

Cotransporters

BLISSFORBUSHAND JOHNPAYNE Functional Analysis

of MUC 1, a Carcinoma-Associated

Mucin

SANDRAJ. GENDLER Manipulation

of Aminoacylation

and Combinational

Properties

of tRNAs

by Structured-Based

in Vitro Approaches

RICHARDGIEGE AND JOEMPUETZ The Role of Acid B-Glucosidase

and Saponins

in Glycosphingolipid

Metabolism

and Disorders

GREGORYA. GRABOWSKI Understanding

Nuclear

to the interphase

Receptor Function: From DNA to Chromatin

Nucleus

GORDON HAGER Regulation of Yeast Glycolytic

Gene Expression

MICHAEL HOLLAND AND JOHNJ. KING Branched Chain Aminotransferases

SUSANM. HUTSON,NIMBE TORRES,AND ARMANDOTOVAR Molecular

Mechanisms

for the Interaction of LDL with the LDL Receptor

T~IOMASL. INNERARITYAND JANBOREN Control of Metallothionine

SAMSONT. JACOB

Gene Expression

SOMEARTICLESPLANNEDFORFUTUREVOLUMES

X Specificity

and Diversity

in DNA

Recognition by E. co/i Cyclic AMP

Receptor Protein

JAMES CLEE Molecular

Mechanisms

of Error-Prone

DNA

Repair

ZVI LIVNEH Translation

Initiation Factors in Eukaryotic

Protein Biosynthesis

UMADASMAITRA Regulation and Function of the Cyclic Nucleotide Phosphodiesterases

(PDE)3

Family

VINCENTC.MANG.~NIELLOANDEVADEGERMAN DNA

Polymerase

III Holoenzyme,

a Prototypical

Replicative Complex

CHARLESMCHENRY Multiple

Routes from the Ribosome to and across the Membrane

MATHIASMULLER,H.-G.KOCH,K.BECK,ANDU.SCHAFER Distinct Regulatory and Phosphatase

Properties

of Pyruvate Dehydrogenase

Kinase

lsozymes

THOMASROCHE Complexity Hormone

of Transcriptional

Regulation Associated

Biosynthesis

MICHAELR.WATERMANANDLARRYJ.BISCHOF The Expanding

World

of DNA Triplet Repeats

ROBERTD.WELLSANDRICHARDP.BOWATER

with Steroid

Cyclic

Nucleotide

Phosphodiesterases: Structure

Relating

and Function SHARR~N

H. FRANCIS,

ILLARION

V. TURKO,

AND JACKIE

D. CORBIN

Department of Molecular Physiology and Biophysics Vanderbilt University School of Medicine Nashville, Tennessee 37232 I. II. III. IV.

.

2 3 3 5 7 8 8 9 9 11 14 16 18 20 20 23 24 26 29 33 37 38 39 40 40 41 41

Introduction .................................................. Background .................................................. Classification ................................................. Structural Features of PDEs .................................... .......................................... A. CatalyticDomain B. Regulatory Domains ........................................ Features of Catalysis ........................................... ....................................... A. CatalyticMechanism B. Determinants of Nucleotide Specificity ........................ C. Structural Determinants for PDE Catalytic Activity .............. D. Metal Requirements ........................................ E. Inhibitors .................................................

VI. Mechanisms Utilized for Regulation of PDEs VII. PDEFamilies ................................................. A. PDElFamily .............................................. B. PDE2 Family .............................................. ............................................ C. PDE3Family.. D. PDE4 Family .............................................. E. PDE5 Family .............................................. E PDEGFamily .............................................. G. PDE7 Family .............................................. ............................................ H. PDE8Family.. I. PDE9 Family .............................................. ............................................. J. PDElOFamily K. PDEll Family .............................................

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

VIII. Concluding Remarks .......................................... References ....................................................

Cyclic nucleotide phosphodiesterases allophosphohydrolases of CAMP and/or

that specifically

cGMP

critical determinants Progress in Nucleic Acid Research and Molecular Biology, Vol. 65

to produce

for modulation

(PDEs) comprise a superfamily of metcleave the 3’,5’-cyclic

the corresponding

phosphate

5’-nucleotide.

moiety

PDEs are

of cellular levels of CAMP and/or cGMP by

1

Copylight 0 2001 by Academic Prrs. All rights ofreproduction m any form reservrd. 0079.6fiwnl $1.500

2

SHARRON many stimuli. Eleven cGMP

families

of PDEs

have been identified in mammalian

with varying

H. FRANCIS

selectivities

ET AL.

for CAMP

or

tissues. Within these families, multiple

isoforms are expressed either as products of different

genes or as products of the

same gene through alternative splicing. Regulation

of PDEs is important for con-

trolling

the visual response,

myriad

physiological

muscle relaxation, and cardiic

platelet

functions, aggregation,

contractility. PDEs

lular CAMP and cGMP by a panoply

including

such as cGMP

immune

are critically involved in feedback phosphorylation

or phosphatidic

association with specific protein partners. a major target for pharmacological tant maladies.

fluid homeostasis,

levels. Activities of the various PDEs

of processes,

small molecules

including

intervention

control of cel-

are highly regulated

events, interaction

acid, subcellular

The PDE

smooth

responses,

superfamily

localization,

with and

continues to be

in a number of medically impor-

0 2000Academic Press.

I. Introduction Cellular levels of cyclic adenosine 3’,5’-monophosphate (CAMP) and guanosine 3’,5’-monophosphate (cGMP) are determined by the relative activities of adenylyl and guanylyl cyclases, which catalyze their synthesis, and cyclic nucleotide phosphodiesterases (PDEs), which hydrolyze them to the respective 5’-nucleoside monophosphates. The sensitivity of physiological processes to cAMP/cGMP signals requires that their levels be precisely maintained within a relatively narrow range in order to provide for optimal responsiveness in a cell. Agents acting through CAMP or cGMP typically produce maximum responses in tissues with only a transient two- to three-fold increase in cyclic nucleotide. Decline of cyclic nucleotide levels often occurs despite the continued presence of hormone. Several mechanisms are likely to contribute to the rapid decline, which involves increased PDE activity. Cyclic nucleotide PDEs provide the major pathway for eliminating the cyclic nucleotide signal from the cell. Cellular levels of CAMP or cGMP rarely achieve the Km values that have been determined for the PDEs in vitro, so that increased cyclic nucleotide synthesis will undoubtedly be accompanied by an increased rate of hydrolysis. Thus, according to the rule of mass action, cyclic nucleotide accumulation is dampened when synthesis increases. In addition, PDEs are highly regulated by inputs from many pathways; the integration of these signals modulates the catalytic activities of the PDEs and plays a major role in determining the intensity and duration of the cellular response to an external stimulus. PDEs are regulated by intracellular CAMP and cGMP concentrations, binding of Ca2+/calmodulin, phosphorylation events, interaction with regulatory proteins, subcellular localization, and alterations in protein level. This article focuses on some of the major advances that have been made in our understanding of the physical and kinetic char-

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

3

acteristics of PDEs, and the roles that PDEs play in modulating intracellular cyclic nucleotide levels. Numerous other reviews provide excellent resource materials for information pertaining to PDEs (1-24).

II. Background Early studies by Sutherland and colleagues (25,26) demonstrated that tissue extracts contained a PDE activity that could ablate the biological actions of CAMP. This was subsequently identified as PDE catalytic activity, which was increased in the presence of divalent cations and inhibited by methylxanthines such as caffeine and theophylline (26). In the ensuing four decades, PDEs have been shown to be a large superfamily of diverse enzymes that are highly regulated by multiple signaling pathways. These enzymes are targets for many drugs that are used to treat cardiovascular diseases, asthma, depression, male impotence, and other maladies. In addition to the vital role of PDEs in hydrolyzing CAMP and cGMP, a group of these enzymes may also have functions as intracellular receptors or even sinks for cGMP; these PDEs (PDEB, PDE5, and PDEG) bind cGMP with high specificity and affinity at homologous allosteric sites that are arranged in tandem in their amino-terminal regulatory domains, but the physiological roles of these binding sites are poorly understood.

III. Classification Members of the PDE superfamily differ substantially in their tissue distributions, physicochemical properties, substrate and inhibitor specificities, and regulatory mechanisms. Based on differences in primary structure of known PDEs, they have been subdivided into two major classes, class I and class II (27). To date, class I contains the largest number of PDEs and includes all known mammalian PDEs, four freshwater sponge (Ephydutiajluuiatilis) PDEs (28), a Drosophila PDE (29), two nematode (Cuenorhubditis) PDEs (28), and the Sacchuromyces ceretksiue PDE2 gene product (30); each class I PDE contains a conserved segment of -250-300 amino acids in the carboxyl-terminal portion of the proteins, and this segment has been demonstrated to include the catalytic site of these enzymes (Fig. 1) (27, 31). All known class I PDEs are contained within cells and vary in subcellular distribution, with some being primarily associated with the particulate fraction or the cytoplasmic fraction of the cell, others being more evenly distributed in both compartments. Certain PDEs appear to be expressed selectively in different mammalian tissues, but the tissue distribution of PDEs across a num-

SHARRON

H. FRANCIS

ET AL.

PDE Family Number Regulatory

Regions +

Conserved Catalytic Domain

Calmodulin binding sites 4

1

I

I

Calmodulin-stimulated

. Id-l

cAMPlcGYP

I

’PDE

cGYP-binding sites

I

2

cGMP-stimulated cAMPlcGMP ’ PDE

I

membrane association region

3

I \I/0

cGYP-inhibited cAMPlcGMP PDE

.

I II

I

1

UCR sites

A

4

4,

::.C..: _. _.

cAMP-specltic, Rollpram-inhibited PDE

]

I

cGMP-binding sites

I

)

I

Photoreceptor cGMP-specific PDE

6

I

7

I CAMP-specific ;&pram-insensitive

m

8

I

9

I

I

I

I

CAMP-specific PDE IBMX-insensitive

I

High affinity cAMPIcGMP PDE

I

cGMP-binding sites?

10

cGMP-binding, cGMP-specific PDE

cGMP-binding, cAMP/cGYP PDE

4

I

I

I

I

cGMP-binding site?

11

4 WA

I

I

CAMPI cGMP PDE

FIG. 1. Schematic depiction of characteristic structural features of known mammalian (class I) PDE families. Arrangements of functional domains are shown for a monomer, although all known mammalian PDEs exist as dimeric enzymes. Known sites of phosphorylation (P) are shown; upstreamed conserved regions (UCR) are characteristic for PDE4 family. The cGMPbinding sites denote the two allosteric cyclic nucleotide-binding sites that have relatively high specificity for cGMP; PDEs 10 and 11 have sequence homologous to the cGMP-binding sites, but have not been shown to bind cGMP. These conserved motifs occur in other signaling proteins and have also been noted as “GAF domains” (31a). The open block in PDE3 catalytic domain indicates a -44-amino acid insertion that characterizes this family.

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

5

ber of species has not been systematically examined. Because properties of the catalytic activities of PDEs commonly overlap, associating specific physiological functions with an apparent selective tissue distribution of PDEs should be approached with caution. Only three PDEs have been classified as class II PDEs; these include a PDE from S. cerevisiae (product of the PDEl gene) (32), a periplasmic PDE from Vibrio fischeri (33), and a PDE from Dictyostelium discoideum (34) that occurs as an extracellular enzyme as well as being associated with the cell membrane. At this time, no mammalian PDE has been included in class II. Class II PDEs share -32% sequence homology. Due to the absence of sequence similarities between class I and class II PDEs, the evolutionary relationship between these classes cannot be ascertained at this time; similar function in the absence of structural homology could have arisen either by a convergent or a divergent evolutionary process. However, it is likely that additional members of both classes of PDEs and perhaps even additional classes of PDEs will be identified. The focus of this article is on mammalian class I PDEs.

IV. Structural Features of PDEs PDEs from mammalian tissues have been subdivided into 11 families that are derived from separate gene families (Fig. 1) (15, 35-39). PDEs within a given family may differ significantly, but the members of each family are functionally related to each other through similarities in amino acid sequences, specificities and affinities for cGMP and CAMP, inhibitor specificities, and regulatory mechanisms. Comparison of amino acid sequences of PDEs suggests that all are chimeric multidomain proteins possessing distinct domains that provide for catalysis and a number of regulatory functions (40). A phylogenetic tree of the class I PDE superfamily has been constructed based on the amino acid sequences of the mammalian PDE families 1-7, the Saccharomyces PDEQ, the two nematode PDEs (PDEl and PDE4), and the four freshwater sponge PDEs (PDEs l-4) (Fig. 2) (28). From this analysis, vertebrate PDEs are proposed to have diverged from a common ancestral gene by gene duplication and domain shuffling early in the evolution of animals. This comparison reveals that the catalytic domain, the calmodulinbinding domain, the upstream conserved regions (UCRs), and the allosteric cGMP-binding domain are ancient in origin. Furthermore, both the amino acid sequences and the arrangement of the functional domains within the respective PDE structures that served to generate the different families of PDEs had been achieved prior to the evolutionary divergence of sponges and eumetazoans (28).

-

L

TWD3.3

R153.1

human PDE4A

human PDE48

human PDMD

human PDE4C

Drosophila dunce

nematode

human PDEIB

human PDESA

human PDElB

human PDElC

human PDElA

nematode

human PDE7A

PDE3

PDEl

PDE4

1

3

11

PDE7

PDEP

1

1

PDE6

m

-

hydrophobic membrane association domain

cGMP binding domaln

PDE catalytic domain

CAAX motif

@

conservec

CaM-interaction domain

upstream region 2 0

ea

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

A. Catalytic Domain The 11 PDE families exhibit the greatest sequence similarities (-2O45% identity) in the carboxyl-terminal segments, and among the members of a respective family, the homology is much higher (70-80%). This region was predicted to contain the PDE catalytic domain (31). This proposal was proved correct by experiments demonstrating that products derived from partial proteolysis of PDEs 1,2, and 3 and expressed truncation mutants containing the respective catalytic domains of PDEs 3,4, and 5 exhibited PDE catalytic activity (41-49) (49u). Deletion of a 99-amino acid region at the carboxyl terminus of the conserved catalytic domain of a PDE4 isoform altered the kinetic properties, but catalytic function was retained. Internal deletion of a 96-amino acid segment from this PDE4 produced an enzyme with greater activity, thus suggesting the presence of an autoinhibitory domain, but expression of the conserved catalytic core of PDE4 produced inactive protein (46). Studies with PDE5 demonstrate that the catalytic properties (Km for cGMP, and IC,, values for inhibitors) of a monomeric PDE5 catalytic domain closely resemble those of native or wild-type PDE5 (49u). These results indicate that the components required for catalyzing hydrolysis of the phosphodiester bond are contained within a single catalytic domain and that interactions between two catalytic domains within a dimer or between the catalytic domain and the regulatory domain are not required for this process. These results are particularly important because all known mammalian PDEs are either dimeric or oligomeric, and the functional importance of this quatemary structure is not known. Although regions outside the catalytic domain can alter catalytic efficiency for many of the PDEs, the catalytic domain alone is sufficient for catalysis. Notably, the catalytic rates of different PDEs vary markedly and are likely to result from subtle differences in the microarchitecture of the respective catalytic sites. If the metals associated with the respective PDEs vary, this might also account for or contribute to the wide variation in catalytic rates observed with different PDEs. Attempts to specifically photoaffinity label the catalytic sites of a number of PDEs using compounds related to cyclic nucleotides have been modestly successful (42, 50- 53). Using a CAMP analog, S-[(4-bromo-2,3-dioxobutyl) 4 FIG. 2. Phylogenetic tree of PDEs. The tree includes seven vertebrate PDE families, four sponge PDE families, two nematode PDE families, one Drosophila PDE, and one PDE derived from Sacchuromyces. This evolutionary tree was inferred from the amino acid sequences of the catalytic domains; the phylogeny was derived using the NJ method with a fungal PDE as the outgroup. 0, Parazoan-eumetazoan split; 0, human-Drosophila (or nematode) split; + , gene duplications that gave rise to different subtypes; 0, gene duplications whose divergence time is unknown; 0, gene duplications in the same subtype. The branch length is proportional to the number of accumulated amino acid substitutions. Domains that were integrated during evolution by domain shuffling are shown. Reproduced from Ref. 28 with permission.

8

SHARRON

H. FRANCIS

ET AL.

thioladenosine 3’,5’-cyclic monophosphate, Colman and co-workers (51) covalently labeled a peptide in the carboxyl-terminal portion of PDE4A; the labeled peptide was isolated and the sequence was determined to be 6g7GPGHPPLPDK706, but this peptide is located carboxyl terminal to the core of the conserved catalytic domain. The catalytic domain of the rod outer segment PDE6 has been affinity labeled using an inhibitory y-subunit derivatized at the carboxyl terminus; this construct covalently linked the labeled y-subunit specifically into the conserved sequence near the carboxyl terminus of the rod PDE6 catalytic domain (54). This suggests that the inhibitory action of the y-subunit results from its direct interaction with this region of the PDE6 catalytic domain.

B. Regulatory Domains The more amino-terminal and extreme carboxyl-terminal sequences in the various PDEs are highly divergent and vary in length. Numerous studies have established that the amino-terminal sequences in PDEs contain distinct domains that provide for quite varied modes of regulation of PDE function, e.g., allosteric cyclic nucleotide-binding sites, phosphorylation sites, Ca2+/ calmodulin binding sites, inhibitory protein binding site(s), and autoinhibitory domains. In some instances, sequences have been identified that provide for specific subcellular localization of a given PDE when overexpressed in a heterologous cell line. The functional features of the various regulatory mechanisms for each of the PDE families will be discussed in the following respective sections.

V. Features of Catalysis The molecular mechanisms that provide for specific binding of cyclic nucleotides and for hydrolysis of the phosphodiester bond are poorly understood. Although development of selective PDE inhibitors for therapeutic uses has been a major interest for many years, information about catalytic domains of these enzymes is surprisingly modest. The novel six-member phosphodiester ring of CAMP or cGMP is critical for interaction with PDEs because other nucleotides, including ATP, ADP, GTP, GDP, and the corresponding 5’-nucleoside monophosphates, do not interact appreciably with PDEs (Fig. 3). Cyclic AMP and cGMP are resistant to other phosphoesterases, such as intestinal phosphatases and ribonucleases. Enthalpies of hydrolysis of phosphodiester bonds of CAMP and cGMP have been estimated at - 14.1 and - 10.5 kcabmol,

respectively,

which approximates

the ener-

of ATP or GTP (55). Cleavage of the cyclic phosphodiester bond of either CAMP or cGMP by PDEs requires a negative charge,

gy in the y-phosphate

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

cGMP

CAMP

FE. 3. Molecular structures of cGMP and CAMP.

an equatorial oxygen atom in the ring, and the presence of divalent cations (56).

A. Catalytic

Mechanism

The phosphohydrolase action of PDEs involves the nucleophilic substitution of a solvent hydroxyl at the phosphate, resulting in the disruption of the P-O bond at the 3’-oxygen of the ribose. When catalysis is conducted in [r80]water, a single atom of IsO is introduced as one of the three equivalent terminal oxygens of the 5’-nucleotide product (5’~AMP or 5’-GMP); this stoichiometry of incorporation has been interpreted to indicate that the reaction proceeds without exchange of oxygen between phosphate and water (57). Other studies that examined the stereochemical course of catalysis using a CAMP analog with a chiral phosphorous [CAMP(S)] demonstrated that, on lysis of the phosphodiester bond, there is an inversion of configuration at the phosphorous group (58,59). Although other mechanisms are possible, the results are consistent with an in-line mechanism of nucleophilic substitution of the hydroxyl of a water molecule at the phosphate.

B. Determinants

of Nucleotide

Specificity

Two structural components of CAMP and cGMP, i.e., the cyclic phosphodiester ring and the purine moiety, provide for the specificity with which these compounds interact with PDEs. Several PDE families are highly selective for either adenine (PDE4, PDE7, and PDES) or guanine (PDE5, PDEG, and PDES), whereas others (PDEl, PDEB, PDE3, PDElO, and PDEll) accommodate both. In solution, CAMP and cGMP are in equilibrium between two conformations, syn and anti, based on the orientation between the purine and the ribose, whereas the spatial arrangement of the cyclic phosphate ring

10

SHARRON H. FRANCIS ET AL.

and ribose are fixed. Cyclic nucleotide analog studies have suggested that the preferred configuration, either syn or anti, varies among the PDE families (60- 62). The selectivity of the respective catalytic domains for either CAMP or cGMP is undoubtedly determined by interaction of select residues within the cyclic nucleotide-binding pocket with particular chemical properties of the purine, such as the relative hydrophobicity or direction and magnitude of the dipole moments of the adenine and guanine bases (Fig. 3) (56, 63, 64). However, for some PDEs, sequences outside this region have also been implicated in producing effects on the relative cAMP/cGMP specificity of the enzyme (65) or on the potency with which PDE inhibitors (compounds that are analogs of cyclic nucleotides) interact with the enzyme (11, 66- 70). Guanine has a higher dipole moment than does adenine and could therefore form stronger stacking interactions with hydrophobic amino acids. A conserved hydrophobic residue (Tyr/Phe) has been shown to be important for substrate interaction in PDE5 (71). Cyclic GMP and CAMP have similar hydrogen bonding potential in the imidazole ring, but quite different hydrogen bonding potential in the N-l, N-6, and C-2 positions. The purine selectivities of the substrate-binding sites of PDEs most likely reflect constraints in the spatial structure within the substrate-binding site as well as positive and negative chemical interactions between specific catalytic site residues and the adenine or guanine. Hydropathy analysis of the catalytic domains of CAMP- and cGMP-specific families of PDEs suggests that PDE substrate selectivity could result from the pattern of hydrophobic/hydrophilic residues in a short segment of sequence surrounding an invariant Glu; this Glu (Glu-775) has been shown to be critical for cGMP binding in the catalytic domain of PDE5 (64). Sitedirected mutagenesis of PDE5 was used to replace within this segment the residues that are conserved in cGMP-specific PDEs with the conserved residues in the corresponding positions of CAMP-specific PDEs (Table I). The results indicate that the substrate-binding site of PDE5 may contain positive elements for accommodating cGMP, as well as negative elements that disTABLE I CHANCES IN SELECTED RESIDUES ALTER SUBSTRATESELECTIVITY OF THE cCMP-BINDING

cCMP-SPECIFIC

Km(44 PDE5 Wild type A769T/L771R W762L/Q765Y W762L/Q765Y/ A769T/L771R

cGMP

CAMP

2 8 36

330 84 77

43

67

PDE (PDE5)

Relative affinity for substrate (Km cGMP& CAMP) 165 11 2 1.6

CYCLIC

NUCLEOTIDE

11

PHOSPHODIESTERASES

criminate against binding of CAMP Furthermore, the cGMP/cAMP selectivity of PDE5 can be shifted -SO-fold by substituting only two residues (Trp762 and Gln-765) of PDE5 (Table I) with the residues in the corresponding positions (Leu and Tyr, respectively) of PDE4, a CAMP-specific PDE. This produces a 4-fold gain in the affinity of PDE5 for CAMP and an l&fold decrease in the affinity of PDE5 for cGMP, so that the affinity of the PDE5 double-mutant for CAMP is only twice that for cGMP These studies provide further evidence that a significant portion of the interaction of PDEs with cyclic nucleotide is provided by this region.

C. Structural

Determinants

for PDE Catalytic

Activity

Catalytic domains of all mammalian PDEs contain three highly conserved regions of sequence (Fig. 4); the first, a histidine-rich region of 80120 amino acids, is located in the amino-terminal region of the catalytic domain and contains two Zn2+-binding motifs (HX,HX24_2,E), each of which mimics the single motif for coordination of a catalytic Zn2+ in metalloendoproteinases such as thermolysin (Fig. 4) (72- 75). The five histidines that are invariant in all class I PDEs are designated as H-l through H-5 for ease of comparison among PDEs. H-l and H-2 denote the two histidines in Zn2+-binding motif A; H-3 and H-4 denote the 2 histidines in Zn”+ -binding motif B. H-5 indicates the invariant histidine represented by His-675 in PDE5 (Fig. 4). Th e second conserved sequence in the catalytic domain of PDEs includes a conserved dyad of residues, threonine-aspartic acid, and then another cluster nearer the carboxyl terminus. It is now clear that each of these conserved regions in the catalytic domain participates in generating a functionally efficient catalytic site (71).

ZnZ*-Binding Motif A

Znz+-Binding Motif B

/

754DLSAITWWPIQQRIAELVATEFFDQGDRE783 FIG. 4. Features of the conserved catalytic domain in PDEs. General arrangement of the catalytic domain and partial amino acid sequence of catalytic domain of bovine PDES. Bold letters indicate residues that are highly conserved among class I PDEs. Zn2+-binding motifs A and B indicate the two Zn2+-binding motifs (HXaHX,,E/D) that include invariant histidines (H-l through H-4) and are crucial to catalysis. Components of motifs A and B provide for interaction with metal(s) required to support catalysis.

12

SHARRON

HI. FRANCIS

ET AL.

Point mutations of a number of invariant residues in PDEs have been made and assessed for their effects on catalytic function. From these studies, it has been concluded that several of the conserved histidine residues as well as a conserved threonine are critical for efficient catalytic function (46, 68, 71, 76). I n dr‘vid ua 1 point mutations of all invariant residues within the catalytic domain of PDE5 have been made in order to construct a comprehensive structure-function map of the conserved elements within the catalytic domains of PDEs (71). Because these residues are conserved in all mammalian PDEs, the conclusions regarding the function of these amino acids in PDE5 are likely to apply generally to other mammalian PDEs, although minor variations will almost certainly occur. Each of two mutations in PDE5, Y602A and E775A, caused a dramatic increase (-35-fold) in the Km of the enzyme for cGMP (Fig. 5), whereas the kcat for each was reduced only 3- to 4-fold (Fig. 6). Substitution of a phenylalanine for Tyr-602 (Y602F) caused minimal perturbation of the kcat of the enzyme, and the Km was actually slightly improved (4-fold) (no t sh own). These results are consistent with the interpretation that an aromatic residue that occurs at this position in all class I PDEs is probably involved in stacking interactions, which improves catalytic function. Significant deterioration (lo- to 15-fold) in the Km for cGMP

FIG. 5. Comparison of K_ for cGMP and IC,, for zaprinast of catalytic domain mutants of PDE5. The values for the Km for cGMP and IC,, for zaprinast of wild-type PDE5 were taken as 1.0, and the corresponding values for each mutant are expressed as a fold increase with respect to the wild-type PDE5. Reproduced from Ref. 71 with permission.

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

13

also occurred with substitutions at two other conserved residues (Glu-672, and Thr-713), thereby establishing a potentially important role for these residues in cyclic nucleotide binding as well. Other studies using site-directed mutagenesis of the catalytic domain of PDE5 have shown that the cGMP/ CAMP selectivity of PDE5, an enzyme highly specific for cGMP, can be shifted 106-fold by substituting four residues of PDE5 with those in the corresponding positions of the CAMP-specific PDE family, PDE4 (64). Several mutations of PDE5 [His-603 (H-l), His-607 (H-2), His-643 (H-3), His-647 (H-4), Asp-714, and Asp-7541 profoundly disrupt catalysis by reducing kcat of PDE5 with only modest changes in Km for cGMP (Figs. 5 and 6). The decrease in free energy of binding for several of these mutants is within the range predicted for loss of a hydrogen bond or an electrostatic bond involving a charged residue. It must be emphasized that these conclusions depend on specific assay conditions, because subsequent studies reveal that when the divalent cation supporting catalysis is changed, much of the catalytic activity can be restored in some mutants (76~). Several of these residues, His-607 (H-2) and His-643 (H-3) have now been shown to be important for the affinity with which a divalent cation binds to the enzyme as well as important for the relative specificity for different divalent cations (76~). Deciphering the precise roles of each of these various residues in PDE catalytic function will require much additional investigation,

FIG 6. Changes in the kot associated with site-directed mutagenesis of the conserved residues in the catalytic domain of PDE5A. Reproduced from Ref. 71 with permission.

14

SHARRON

H. FRANCIS

ET AL.

D. Metal Requirements Divalent cation is required to support PDE catalytic activity, and the more amino-terminal conserved region in the catalytic domain of PDEs has been shown to provide a major portion of the metal binding by these enzymes (Fig. 4) (72, 76~). Traditionally, Mg2+ has been used to support catalysis, but Mn2+ and Co’+ are also quite effective. A potentially prominent role for Zn2+ has emerged because this cation potently promotes catalysis in a number of PDEs (69, 72, 77). However, a clear picture of the metal requirements for each of the PDEs has yet to be definitively established. Based on the results of a number of studies, it is likely that metal requirements of PDEs are complex and may involve more than one cation. Kinetic studies using bovine rod outer segment PDE (PDEGoB) suggest that Mg2+ and cGMP interact with the PDE in a rapid equilibrium random binding order to form a ternary complex between the PDE, Mg2+, and cyclic nucleotide (78). These same studies discount the likelihood of the formation of a free Mg 2+/cGMP complex due to the high K, of this complex. Independent association of cGMP and Mg2+ with the catalytic site is also supported by studies in PDE5 utilizing cyclic nucleotide analogs (3). Other workers have proposed that divalent metal ions may facilitate catalysis by stabilizing the charged transitional intermediate (59). Work by our own laboratory demonstrated that for PDE5 and PDEGc$, Zn2+ is the most potent cation for supporting catalysis. For PDE5, catalytic activity was supported by submicromolar concentrations of Zn2+ (72), and for PDEGaB, Zn2+ was required even in the presence of 10 mM Mg2” (Fig. 7A) (S. Francis, unpublished results). For these two PDEs, order of potency for these cations is Zn2+ >>Mg 2+. Subsequent reports demonstrate that catalytic activity of recombinant PDE4A is supported by submicromolar concentrations of Zn2+ compared to significantly higher concentrations of other divalent cations (69, 77). However, Zn2+ apparently does not activate certain PDEs (51, 79, 80). Using atomic absorption spectroscopy and 65Zn binding, PDE5 has been shown to bind -3 Zn2+/PDE5 monomer (Fig. 7B) (72). PDE4A is reported to bind specifically Zn 2+ (77, 80), and both class I and class II PDEs in yeast bind Zn2+ specifically (81). Th ese combined results strongly support an important role for Zn 2+ in PDE catalytic function. The strict conservation of a histidine-rich region in catalytic domains of class I PDEs compares well with other catalytic Zn 2+-binding sites, for which X-ray crystal and the importance of two histidines (H-2 structures are known (82-84), and H-3) in this region for metal coordination in PDE5 has now been established (76~). Thus, it seems highly likely that this segment of conserved sequence in PDEs provides the salient features for coordinating required metals and participates in producing a nucleophilic hydroxyl ion from water, as suggested by earlier studies using isO-labeled water and cyclic nucleotide analogs (57- 59).

CYCLIC

NUCLEOTIDE

15

PHOSPHODIESTERASES

A 2.1 Metals Added After Preincubation with EDTA 4

Control

Treated Control

190 gM

1 mM

zn2+

Mn2+

>

B 1.6 -

0

0

I 0.6

I

I 1.2

I 1.6

Zn2+ (PM) FIG. 7. Importance of Zn2+ in PDE function. (A) Requirement of the rod PDE for Zn2+ to support catalysis. Following pretreatment of the rod PDE6 with EDTA, PDE catalytic activity was measured in the presence of 10 mM Mg 2+ either in the absence of other metals or in the presence of the indicated metals. PDE6 that was not pretreated with the chelator was used as control. (B) Saturation curve for 65Zn2+ binding to bovine PDE5A. Reproduced from Ref. 72 with permission.

The presence of two catalytic Zn 2+-binding motifs separated by 10 residues in the PDEs is novel, because other enzymes that utilize this motif to bind Zn2+ contain a single copy of this sequence. Studies in PDE5 have now shown that metal binding by PDEs has unique features compared to the proteases (76~). Stoichiometries of Zn 2+ binding to PDE4, PDE5, and PDE6

16

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suggest that PDEs may contain a multinuclear metal-binding site. Site-directed mutagenesis of select residues that comprise the two Zn2+-binding motifs causes a marked loss in PDE catalytic activity of each PDE that has been studied (Fig. 6) (46, 53, 68, 71, 76, 76u, 80). However, the relative contribution of these residues in maintaining catalytically active enzymes has varied. In PDE5, each of the residues in the two Zn2+-binding motifs have been individually mutated; five of the six residues proved to be critical to normal catalytic function (Fig. 6) (71). Th e exception was the glutamic acid in Zn2+-binding motif A. In studies of PDE5, other functions, such as allosteric cGMP-binding, were intact, suggesting that the deleterious effects of these mutations on catalysis are specific. These combined results indicate that residues in both Zn2+-binding motifs in PDEs are important for efficient catalysis. The particular importance of a single his&line in each Zn-binding motif (H-2 and H-3, respectively) for interaction with catalytic metal establishes that metal binding in this region of PDEs will not mimic that in metalloendoproteinases such as thermolysin, which contain only a single motif. In this region, the importance of another conserved residue (Tyr-602 in PDE5), which is not part of the metal-binding motif, is also established by these studies (71). Based on metal requirements, metal binding, and results of site-directed mutagenesis of several PDEs, it seems likely that class I PDEs use a multinuclear metal binding site to effect catalysis. At least a portion of the metalbinding sites is provided by the conserved histidine-rich region in these PDEs, including H-2 and H-3 (76~). Metal-binding site in PDEs could resemble complex metal-binding sites found in other phosphohydrolases, such as alkaline phosphatase or phospholipase C; in each of these sites, one metal is considered the “catalytic” metal whereas the other facilitates the catalytic process (75,84,85). Current evidence strongly suggests that Zn2+ plays a key role in catalysis effected by several PDEs. However, whether a particular metal is absolutely required for rupture of the phosphodiester bond by PDEs and whether one or more metals act directly or indirectly as “cocatalytic metals” to facilitate this process are still open questions. Given significant differences in the Vmax of PDE families, it is also possible that the complement of metals that optimally provides for support of catalysis may vary among PDE families. Last, if multiple metals are involved in catalysis, it is possible that cyclic nucleotide substrate binding may be independent of occupation of one of these metal-binding sites, but dependent on occupation of another.

E. Inhibitors The ognized phylline for use

prospect for utilizing PDE inhibitors therapeutically was quickly recwhen the inhibitory effects of agents such as caffeine and theowere discovered. The quest for specific and potent PDE inhibitors in physiological studies and therapeutic settings has continued un-

CYCLIC

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PHOSPHODIESTERASES

abated, and compounds that exhibit high selectivity for the various PDEs are continually reported (Table II) (13, 86-98). In some instances, these inhibitors are highly selective for a particular PDE family, as in the case of the inhibition of PDE4 by rolipram; however, most PDE inhibitors exhibit some inhibitory effect on multiple PDE families. For this reason, it is risky to invoke physiological roles for specific PDEs based exclusively on the effects of “specific” inhibitors. For example, zaprinast, a compound that for years has been referred to in the literature as a PDE5-specific inhibitor, also inhibits the photoreceptor PDEs (PDEG) and the PDElC isozyme quite well. Likewise, sildenafil (marketed as Viagra) is quite selective for PDE5, but it also inhibits the PDE6 family to some extent (93). Kinetic analysis of PDE inhibition by many of these agents commonly shows competitive kinetics with cyclic nucleotide substrate, thus suggesting that the compounds interact within the same binding pocket on the enzyme, but in other instances, a more complex pattern of interaction has been documented. For example, the kinetics of interaction of rolipram with some members of the PDE4 family suggests multiple sites of interaction (99). Therefore, careful molecular characterization of the interactions of these compounds with PDEs is crucial in order to more effectively design pharmacologically active inhibitors that exploit selective active site features of various PDEs. For PDE5, zaprinast and sildenafil (Viagra) are competitive inhibitors with respect to cGMP, which suggests that the two compounds interact with the same site. However, when catalytic domain mutants are

TABLE II COMMONINHIBITORSOF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES Family PDEl PDEZ PDE3 PDE4 PDE5 PDE6 PDE7 PDES PDE9 PDElO PDEll

Inhibitor” Vinpocetine, zaprinast, SCH51866 EHNA Milrinone, cilostamide, amrinone, enoximone Rolipram, Ro 20-1724, denbufylline, zadarverine, RP73401 CDP840, SB207499, RS25344, LAS31025 Sildenafil, zaprinast, DMPPO, dipyridamole, E4021, SCH51866, GF248 Sildenafd, zaprinast, DMPPO, E402 1, GF248 Benzotbieno- and benzotbiadiazine dioxides Dipyridamole Zaprinast ? Dipyridamole, zaprinast

~‘3.Isobutyl-1-methylwanthineis commonly used as a general inhibitor for most known PDEs, but it is ineffective for inhibition of PDE8 and PDES.

18

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compared for changes in Km for cGMP and IC,, for zaprinast, the two parameters do not correlate exactly (Fig. 5). For PDE5, the most dramatic changes in IC,, for zaprinast occur in D754A and G780A mutants, whereas the greatest change in Km for cGMP is observed with the Y602A, T713A, and E775A mutants. Interestingly, effects of the mutations on the potency of sildenafil more closely mimic the pattern for cGMP, with the greatest change (25-fold) occurring in the Y602A mutant (100). These results suggest that the site occupied by zaprinast and sildenafil overlaps the substrate cGMP-binding site, but the residues for binding these ligands differ in some instances. These results emphasize the importance of profiling PDE mutants with respect to these parameters because these types of studies may more fully define important interactions that provide for selectivity and potency of interactions between the respective PDEs and inhibitors. The interaction of the various PDE4 isoforms with rolipram is complex; in some binding studies two types of rolipram binding can be observed, i.e., a high-affinity component and a low-affinity component (99). Amino-terminal deletion of the regulatory region of the PDE4 eliminates the higher affinity binding site, whereas the lower affinity interaction and inhibition of PDE activity is retained (49). Studies using the human recombinant PDE4B suggest that the enzyme contains a single binding site for (R)-rolipram and that different conformational states of the enzyme determine the affinity with which rolipram is bound (101). These studies also support the involvement of both the amino terminus and the catalytic domain in high-affinity rolipram binding by this enzyme.

VI. Mechanisms Utilized for Regulation of PDEs The PDEs are highly regulated enzymes. The general schemes by which the rate of cyclic nucleotide breakdown can be regulated include (1) the availability of substrate, (2) regulation by extracellular signals, (3) short-term feedback regulation, and (4) long-term changes in PDE protein levels. Because the Km values for many PDEs are relatively high compared to cellular cyclic nucleotide levels, it is likely that many of these enzymes are functioning at less than maximum rates. Consequently, PDE activity would increase due to mass action on elevation of cyclic nucleotide levels. Furthermore, when one of the cyclic nucleotides is selectively increased, the rate of hydrolysis of both nucleotides may be changed due to competition by the respective cyclic nucleotides for the catalytic sites in the dual-specificity PDEs (such as PDEs 1, 2, 3, 10, and 11). Extracellular signals modify intracellular PDE activities through a panoply of signaling pathways, e.g., phosphorylation events, vtiations in the levels of small molecules such as Ca2+, phosphatidic acid, in-

CYCLIC

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PHOSPHODIESTERASES

ositol phosphates, and protein-protein interactions. Examples of this type of regulation include the stimulation of PDE3 activity by insulin, leptin, or insulin-like growth factor (16, 102-106), stimulation of PDE5 by atrial natriuretic factor (107), stimulation of PDE6 activity by photons through the transducin system, which alters PDE6 interaction with its inhibitory y-subunit (6, 18, IO??), and stimulation of PDEl activity by its interaction with Ca2+/ calmodulin (10). Short-term feedback regulation of PDEs is an important and immediate physiological response that can involve many of the schemes described above; examples include (1) phosphorylation of PDE3 or PDE4 catalyzed by PKA after CAMP elevation and resulting in an increased Vmax for each of these enzymes (109-111) and (2) allosteric cGMP binding to PDEB, which lowers the Km of PDE2 for substrate, thereby promoting breakdown

A MEETTGKV INO..... KDG..... KDG..... KDG.....

f

N-

-C cGMP-blndlng

snea

cGMP-blndlng

sitek

cattdytk

domain

B

FK. 8. (A) Conserved sequences in the allosteric cGMP-binding sites in PDEs 5,2, and 6. (B) Possible interactions of cGMP with the putative NKX,,D motif based on comparison with interactions of GTP in GTF-binding proteins. Reproduced from Ref. 261 with permission.

20

SHARRON H. FRANCIS ET AL.

of CAMP or cGMP after cGMP elevation (Fig. 8) (112-114). Long-term changes in the level of PDE protein are caused by a persistent change in the cellular environment. Examples of this type of regulation include (1) the densensitization that occurs by increased levels of PDE3 or PDE4 following chronic exposure of cells to CAMP-elevating agents (46, U-122) (2) the developmentally related changes in PDE5 protein (123) or enterotoxin-induced increases in PDE5 level (124, 125), and (3) the selective induction of particular PDE families or isoforms such as the induction of PDE7 that occurs in T cell activation (126) and specific expression of PDE4 isoforms (127, 128). Additional considerations that could influence regulation include selective cellular compartmentalization of PDEs effected by covalent modifications such as prenylation or by specific targeting sequences in the PDE primary structure, and perhaps translocation of PDEs between compartments within a cell (129-133).

VII. PDE Families At least 11 gene families are included in the mammalian PDE gene family. Most of these families have been shown to contain two or more genes; in certain instances, alternative mRNA splicing or utilization of different translational initiation sites produces multiple protein products that exhibit distinct kinetic and regulatory properties. Variations produced by alternative splicing will be discussed for each family. Regulation of gene expression of mammalian PDEs has previously been the topic of discussion in this series (134) and will not be discussed further here.

A. PDEl Family CALCIUMKALMODULIN-BINDING

PDEs

Ca2+/calmodulin (CaM) binds to and activates members of the PDEl family, also known as CaM-PDEs. The Ca2+/calmodulin PDEs are derived from three CaM-PDE genes; alternative mRNA splicing of these gene products produces a number of amino-terminal and carboxyl-terminal PDEl splice variants (135-141). Comparison of nucleotide and amino acid sequences has been used to categorize the PDEl as follows: PDElAl (-59 kDa) and PDElA2 (-61 kDa) are products of the same gene, PDElBl is the product of a separate gene, and PDEl Cl - 5 are products of a separate gene, with variants resulting from alternative splicing at both the amino and carboxy1 termini. The predicted molecular masses of the PDElC variants approximate the determined size of a 75-kDa CaM-PDE that has been described (142).

CYCLIC

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PHOSPHODIESTERASES

Each of the CaM-PDEs hydrolyzes both CAMP and cGMP, but PDElAl, PDElA2, and PDElBl exhibit higher affinity for cGMP (Table III); the five splice variants of PDElC h ave high affinity for both cGMP and CAMP and hydrolyze the two nucleotides with equal efficiency, but the potency with which Ca2+/calmodulin stimulates catalysis varies somewhat (139, 143). Both PDElAl and PDElA2 are dimeric (subunit molecular masses of -59 and 61 kDa, respectively), and their amino acid sequences are identical except for a segment near the amino terminus. Catalytic activity in the PDEl family can be regulated by both Ca2+/ calmodulin binding and phosphorylationdephosphorylation. Ca2+/calmodulin allosterically activates these PDEs through interactions with sequences near the amino termini, although the affinities with which the respective PDEl isoforms interact with calmodulin differ (10, 244-146). For example, half-maximal stimulation of catalysis in PDElAl occurs at a lo-fold lower concentration of Ca2+/calmodulin (Scam - 0.3 nM) than is required for activation of PDElA2 (-4 nM). A sequence near the amino terminus of PDElAl was predicted to form an amphipathic helix suitable for binding Ca2+/calmodulin (43). Synthetic peptides based on this sequence block activation of PDElA2 by Ca2+/calmodulin, thus supporting this interpretation. However, only a portion of this sequence is conserved in PDElA2, which is still dependent on Ca 2+/calmodulin; following deletion of this sequence in PDElAl, the PDE activity can still be activated -3-fold by calmodulin -3 nM). Using several experimental approaches, a region of sequence (%&Xl has been identified in PDElAl and PDElA2 that is likely to account for at least one site of Ca2+/calmodulin binding to these enzymes; the putative Ca2+/calmodulin binding sequence includes residues 115-126 in PDElA2 or residues 97-110 in PDElAl (147). PDElA and PDElB can be phosphorylated in this region by PKA and calmodulin-dependent protein kinase

TABLE III KINETIC PROPERTIES OF CALMODULIN-DEPENDENT

CYCLIC

NUCLEOTIDE PDEs (PDEl)”

Km(FM) PDElA2 PDElBl PDElC2

V miLXratio

CAMP

cGMP

112.7 24.3 1.2

5.0 2.7 1.1

(cAMP/cGMP) 2.9 0.9 1.2

aFrom Ref. 143,A. Z. Zhao, C. Yan, \V K. Sonnenburg,and J. A. Beavo; “Advancesin SecondMessengerPhosphoproteinResearch” (Corbin et al., Eds.), Vol. 3 1, pp. 237-251. Lippincoti-RavenPress, 1997.

22

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II, resulting in decreased sensitivity to activation by Ca2+/calmodulin (148 152). In PDElA2, phosphorylation of a single serine (Ser-120) accounts for much of the decreased affinity for calmodulin; whether PKA phosphorylates PDEl in viva has not been established. The two calmodulin-binding regions in PDElAl have been dubbed domains A and B, and although the role of the B domain in activating PDEl is assured, the role of the A domain is unclear and may be to somehow enhance the affinity for binding calmodulin. A number of studies have determined a stoichiometry of one calmodulin bound per monomer of PDElA (149, 153, 154); thus, it is possible (1) that one PDElA2 monomer has two sites of interaction with a single calmodulin molecule, (2) that calmodulin interacts with binding sites on two separate monomers, (3) that only one of these domains in PDElA2 is crucial to the activation by calmodulin whereas the other may enhance the binding affinity for Ca2+/calmodulin, or (4) that the stoichiometry of calmodulin binding has been underestimated. Modulation of the catalytic activity of PDEl family members by an autoinhibitory process has been well documented. Limited proteolysis of PDElA2 fully activates the enzyme, but the Ca2+/calmodulin binding function is retained (41). Likewise, using amino-terminal truncation of PDElA2, a region of sequence [residues 89-98 (-VPSEVBDWLA-)] has been identified that fulfills the criteria of an autoinhibitory domain, i.e., deletion of this sequence activates the enzyme, but Ca2+/calmodulin binding is preserved. Furthermore, other parameters (Vm,, oligomeric structure) in this truncated, activated PDElA are apparently unperturbed. Whether other PDEs contain a well-defined autoinhibitory domain or whether in some instances autoinhibition is effected through more general steric constraints on the enzyme structure remains to be determined. Selective tissue distribution of the various CaM-PDEs has been interpreted to implicate the respective enzymes in specific physiological functions, and the distinctive kinetic and regulatory features of the CaM-PDEs may contribute to their roles in these tissues. The PDEl family has a broad tissue distribution, but it is particularly well represented in testis, heart, and neural tissues, e.g., in cerebellar granule cells as well as in olfactory epithelial cells (138, 139, 155). PDEl is selectively expressed in different areas of the brain, and the particular abundance of PDEl in neurons of the central nervous system suggests that the PDEl family may participate importantly in a variety of neural functions. PDElC is particularly abundant in olfactory cilia and has been suggested to play an important role in the Ca2+ modulation of odorant signaling, perhaps in conjunction with a Ca2+-regulated olfactory adenylyl cyclase (138, 143, 155-157). Proliferating vascular smooth muscle cells contain high levels of PDEl, in contrast to the paucity of this PDE family in nonproliferating cells (158).

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

23

Given the broad tissue distribution of the PDEl family members and their diverse kinetic properties, these enzymes must always be considered as potential participants in signaling processes involving cGMP/cAMP and/or Ca’+-mediated events.

B. PDE2 Family The PDE2 family (also known as the cGMP-stimulated PDE, even though CAMP also stimulates the enzyme) occurs in both cytosolic and membranebound forms: it has a broad tissue distribution, but it is particularly abundant in the central nervous system and adrenal cortex (159-162). These enzymes are typically homodimers with a molecular mass of -240 kDa, with two subunits of -105 kDa each. Sequences of cDNAs derived from several species and tissues suggest that there are at least three isoforms of PDE2 that are thought to be derived from one gene. The respective PDE2 isoforms differ in their amino termini; alternative splicing of the PDESA mRNA is likely to account for the differences (138,162-164). Forms of PDE2 that are localized to the particulate fraction of the cell contain more hydrophobic amino-terminal domains that may specify their membrane association. Members of the PDE2 family are multidomain proteins with a catalytic domain and an amino-terminal cGMP-binding domain (Fig. 8). Interactions contributed by structures within the latter domain are thought to provide for dimerization (45, 165). Cyclic AMP and cGMP are hydrolyzed with similar Vmax values (-150 ~mol/min/mg); the affinity for cGMP is -twofold greater. Although FM the reported Km values vary, they generally fall in the range of -15-30 for cGMP and 30-50 pM for CAMP. Catalysis exhibits positive cooperativity (Hill coefficient ranging from 1.2 to 1.6 for cGMP and 1.6 to 1.9 for CAMP); hydrolysis of either CAMP or cGMP can be markedly stimulated (as much as 50-fold) by the other nucleotide, although cGMP is preferred both as substrate and effector (113, 166-169). The stimulatory effects of CAMP or cGMP are due to an increased affinity for substrate, i.e., a Km effect, with no change in the V_, in the enzyme; activation is mediated through interactions of CAMP or cGMP with the allosteric cyclic nucleotide-binding domains in the amino-terminal regulatory portion of PDE2 (Fig. 8). Occupation of the cyclic nucleotide-binding allosteric sites induces a conformational change in PDE2, converting it to a more active form that is also more sensitive to proteolysis. Studies of the molecular requirements for cyclic nucleotide interaction with either the allosteric cyclic nucleotide-binding sites or the catalytic site of PDEs have been performed using PDEB. Low levels of 3-isobutyl-1-methylxanthine (IBMX), a classical PDE inhibitor, can also stimulate catalytic activity by interacting with the allosteric cyclic nucleotide-binding sites on PDE2 (61, 62, 113,166,170-174). Based on predictions from the amino acid sequence, there are two putative allosteric

24

SHARRON

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cyclic nucleotide-binding sites per PDE2 monomer, but the stoichiometry of cGMP binding is -2 cGMP bound per dimer (45,175). The apparent activation constant for cGMP stimulation is substantially lower (-0.2-0.4 FM) than that for CAMP, and cGMP at concentrations as low as lo-7 M can stimulate PDE2 hydrolysis of micromolar levels of CAMP. Therefore, at concentrations of CAMP and cGMP that are estimated to occur in most cells (O.l- 1 t.&), it is predicted that PDE2 would preferentially hydrolyze CAMP. Elevations of cGMP in such conditions would lower CAMP by the action of PDEB, which could provide for cross-talk between these two signaling pathways. The PDE2 associated with the particulate fraction of the bovine brain can be phosphorylated in vitro by PKA, but no functional changes in the enzyme’s properties were detected (160); the PDE2 isolated from bovine heart is not phosphorylated by PKA in vitro (86). Among the group of PDE families that bind cGMP at allosteric sites in their regulatory domains, the role of these cyclic nucleotide-binding sites is best understood in PDES. There is significant experimental evidence to suggest that hydrolysis of CAMP by PDE2 is important in a number of physiological processes, including regulation of Ca2+ channels (176, 177), olfactory signaling pathways (155), platelet aggregation (178), and aldosterone secretion (179, 180). PDE2 is particularly abundant in the adrenals. Adrenocorticotropic hormones such as ACTH induce increases in CAMP, thereby promoting increased synthesis and secretion of aldosterone, which promotes an expansion in the blood volume. Atria1 natriuretic peptide, the release of which is sensitive to blood volume, also binds to these cells and activates a guanylyl cyclase that increases cGMP synthesis; subsequent cGMP binding to cGMP-binding sites in PDE2 is thought to stimulate hydrolysis of CAMP (179). As CAMP is lowered, aldosterone production diminishes, which reduces blood volume and blood pressure. This effect on steroidogenesis is one example in which a cGMP-binding PDE may regulate the cellular response to changes in cyclic nucleotide. Given the broad tissue distribution of the PDE2 family and the capacity of this family to hydrolyze both CAMP and cGMP, the PDE2 family has the potential to play vital regulatory roles in many tissues, including cross-talk between the CAMP and cGMP signaling pathways. In addition, because PDE2 degrades both cGMP and CAMP in vitro, it could also mediate negative feedback regulation of cGMP signaling in certain tissues.

C. PDE3 Family The PDE3 family hydrolyzes both CAMP and cGMP (reported Km values for CAMP and cGMP range from 0.1 to 0.8 FM), albeit with a 2- to lo-fold higher Vmax for CAMP (3-9 p,mol/min/mg); the purified enzyme exhibits normal Michaelis-Menten kinetics (181, 182). Because the two nucleotides

CYCLIC

NUCLEOTIDE

PHOSPHODIESTERASES

25

compete for hydrolysis at the same catalytic site, this family has been dubbed with the misleading name of cGMP-inhibited PDE (cGI-PDE). Cyclic GMP can compete with CAMP hydrolysis with a Ki of -0.2 FM; such a competition could play an important role in certain biological effects because elevation of either cGMP or CAMP might result in increased hydrolysis of that nucleotide while sparing the other (114, 183-186). This would effectively increase the intracellular levels of the latter nucleotide and enhance signaling through that pathway. Members of the PDE3 family are widely distributed dimeric enzymes, are highly susceptible to partial proteolysis during purification (187) and occur as both cytosolic and “peripheral” membranebound species [in both the endoplasmic reticulum and the plasma membrane (188, ISS)]. PDE3 s are particularly abundant in adipose tissue, cardiac muscle, vascular smooth muscle, liver, and platelets (17, 35, 190). The particulate PDE3s from adipocytes, hepatocytes, and platelets are regulated by a number of hormones, including insulin, glucagon, catecholamines, and prostaglandins; the activation induced by these agents ranges from 1.5- to 4-fold and is the result of a change in the Vmax (16, 102, 103, 191-193). Leptin has also been reported to activate the PDE3B in pancreatic beta cells (104) and in hepatocytes (104~). PDE3 plays a central role in a short-term negative feedback loop that modulates intracellular levels of CAMP (191, 1944196). Th is action of PDE3 contributes substantially to the hormonal regulation of glycogenolysis, to the antilipolytic effect of insulin, and to the leptin-induced decrease in insulin secretion from the pancreas (16, 104,191,195,197-201). PDE3 s are targets for the action of many drugs [such #as cilostamide, amrinone, fenoximone, and mihinone (Table I)] that exhibit cardiotonic, vasodilatory, thrombolytic, and antiplatelet aggregation properties (17, 86, 90, 95, 190,202). A mong these inhibitors, cilostamide (IC,5,, -40 nM) is widely used as a specific inhibitor of the PDE3 family. Two PDE3 cDNAs (PDE3A and PDE3B) that are predicted to encode proteins of -122,000-125,000 kDa have been cloned from a number of species (47,203-206). The PDE3A gene product encodes the PDE3 that is abundant in the cardiovascular system, including the myocardium and the vascular smooth muscles. It is also expressed in the smooth muscle of the airways and of the genitourinary and gastrointestinal tracts (207). The PDESB gene product encodes the PDE3 that is abundant in adipose tissue, hepatocytes, spermatocytes, and the renal collecting duct epithelium; the human genes for both PDE3s are located in chromosomal region 11~15 (205, 206). These predicted proteins have very similar domain structures and are clearly distinguished from other PDE families in that each contains a novel insertion of 44 amino acids in the amino-terminal conserved sequence in the catalytic domain (Fig. 1) (203). Th is insertion appears to be unique to PDE3s and is positioned within the Zn 2+-binding motif A (Figs. 1 and 4). Deletion

26

SHARRON

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ET AL.

of this insertion produces an inactive PDE (65, 80, 208). The more aminoterminal regulatory region in the PDE3s contains hydrophobic regions that may contribute to membrane association, and a number of consensus phosphorylation sites (-RRXSX-) for PKA are located in this region. PDE3s in adipocytes and hepatocytes are activated by phosphorylation of their regulatory domains by PKA (193, 201, 209, 210). In rat adipocytes treated with either isoproterenol and/or insulin, PDE3 is phosphorylated at a single serine (Ser-302) in a PKA consensus sequence (-RRPSLPC-) (211). However, treatment of adipocytes with both insulin and catecholamines causes phosphorylation and activation of the enzyme that are greater than additive (212). Cross-talk between the two signaling pathways involving upstream regulatory components may account for this apparent disparity. An insulin-stimulated PDE3 kinase that is distinct from PKA has been partially purified from platelets and has been shown to phosphorylate and activate PDES. Some evidence suggests that this kinase may be protein kinase B (213-215). The PDE3 family of enzymes clearly plays a major role in modulating cAMP/cGMP levels in many tissues. This family has a broad tissue distribution, occurs in both the soluble and particulate compartments of the cell, is hormonally regulated, and perhaps most importantly, the enzymes have Km values for CAMP and cGMP that approximate the levels of cyclic nucleotides in cells. Thus, even slight changes in either nucleotide would have the potential to markedly increase the catalytic rate in PDE3s. Last, although the PDE3 family has been convincingly demonstrated to participate in a rapid negative feedback control of CAMP levels that is mediated through PKA, a similar pathway has not been demonstrated for the cGMP pathway. However, the possibility that this type of regulation occurs is entirely feasible.

D. PDE4 Family The PDE4 family is characterized by being highly specific for CAMP as substrate, having a low Km for CAMP (l-3 FM), being insensitive to cGMP and Ca2+/calmodulin, and being potently and specifically inhibited by rolipram and RO 20-1724. It is commonly described as the “rolipram-inhibited CAMP-specific PDE.” The PDE4 family has been the focus of vast research efforts over the years because this family is considered to be a prime target for therapeutic intervention in a number of maladies. A comprehensive overview of the PDE4 family is beyond the scope of this article, but several recent reviews cover this topic in depth (11,14,21,23). The PDE4 family has a broad tissue distribution, is typically expressed in small amounts, and contains a highly diverse number of variants, many of which occur as both soluble and membrane-bound species. In human, rat, and mouse cells, the PDE4 family is encoded by four sep-

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27

arate genes (PDE4A-4D) located on three chromosomes, and there is high sequence conservation among species. Genomic organization of human and rat PDE4 genes is very similar, suggesting a unique ancestor (23, 134, 216, 217). Use of different promoters, multiple transcription start sites, and alternate splicing in these genes generates a multiplicity of mRNA and protein products for this large PDE family, and regulation of members of the PDE4 family is remarkably complex. PDE4A, PDE4B, PDE4C, and PDE4D genes are differentially spliced to generate more than 15 isoenzymes with aminoterminal heterogeneity that can be generally classified into long (85-110 kDa) and short (68-75 kDa) forms. Products of PDE4 genes show greater than 80% sequence identity over their catalytic regions, although there are segments of sequence in these regions that are unique to each PDE4 gene family. Despite differences in these various PDE4s, they share many of the same features, and genomic sequences of rat PDE4A, PDE4B, and PDE4D show high conservation of intronexon boundaries (218 -225). Mice lacking PDE4D exhibit growth and fertility abnormalities (22%). 1. ROLEOFTHEAMINO-TERMINALSEQUENCESINPDE~S The PDE4 gene family in rat has multiple members that utilize altemative splicing to form proteins with distinct amino-terminal regions. These distinct amino termini affect a number of properties of the PDE4s, including catalytic activity, inhibition of catalysis by rolipram, temperature stability, response to ligands such as phosphatidic acid, and localization to specific subcellular compartments (129,132,133,226-231). The membrane localization of rat PDE4A and PDE4B activity expressed in COS cells depends on the presence of a sequence of some 25 amino acids in the amino-terminal region of the protein (232). One of these (RNPDE4Al) is a membrane-bound PDE that can be solubilized by mild detergent treatment; two other PDE4A varants (RNPDE4A and RNPDE4A6) are present in both the cytosolic and particulate fractions of cells. Houslay and colleagues have shown that the aminoterminal 25 amino acids of RNPDE4Al account for its membrane localization; creation of a chimera between this sequence and the classically cytosolic protein, chloramphenicol acetyltransferase, localized the transferase to the particulate component of the cell (228, 233, 234). lH nuclear magnetic resonance (NMR) analysis of a synthetic peptide representing the amino-terminal 25 amino acids of RNPDE4A reveals an amino-terminal amphipathic a-helix connected by a hinge region to a compact, tryptophan-rich helix (235). The latter helix, which is formed by a hydrophobic dodecapeptide, contains a novel heptapeptide (-PWLVGWW-) that is apparently responsible for the membrane-targeting properties. This targeting apparently involves specific hydrophobic interactions because substitution of these residues by alanine disrupts PDE4 association with the membrane. Howev-

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er, the membrane components that interact with this peptide to anchor the PDE4A to specific regions have not been identified. The functional properties of the different particulate and cytosolic forms of human PDE4B and PDE4D have been described in much detail as well (132, 236). The aminoterminal domains of certain PDE4s may also provide for specific interactions with components of other signaling pathways. The long form of rat PDE4A (RNPDE4A5) in t eracts with the SH3 domain of v-Src by its amino-terminal splice region (237,238). A shorter amino-terminally truncated PDE4A splice variant fails to associate with v-!&c SH3. 2. VARIATIONSINTHEREGULATORYDOMAINFUNCTIONSIN PDE4s In addition to the differences in subcellular localization, the long and short forms of PDE4 differ in the presence or absence of two upstream conserved regions (UCRl and UCRB), which are unique to PDE4s and are located in the amino-terminal portion of these enzymes (Fig. 1). UCRl (approximately 55 amino acids) and UCR2 (approximately 75 amino acids) show no homology to any other known protein sequence; however, these sequences are preserved in PDE4s from evolutionarily distant species, which implies their importance in PDE4 function (216). UCRl and UCR2 differ markedly in sequence and are separated by a short linker region (LRl) of -33 amino acids that shows no homology between PDE4s (217); UCR2 is separated from the catalytic domain by a second linker region (LR2) of lo-28 residues. LR2 is hydrophobic and shows some homology between members of the PDE4 family. Full importance of the UCRl/UCRB region for PDE4 functions has not been established, but several lines of evidence support the likelihood that important regulatory features of PDE4s are contributed by this region. It has been proposed that the linker sequences could influence or provide for specific regulation of UCRl/UCR2 interactions in various PDE4 isoforms (238). Long and short forms of PDE4 can be divided into two groups: (1) those that contain UCRl and UCR2 and (2) th ose in which UCRl and/or portions of UCR2 are absent. To investigate functional significance of these domains, deletion mutagenesis has been peformed for PDE4A (239). Results indicate that catalytic activity of a long form of PDE4A is not significantly affected by amino-terminal truncation to remove either or both UCRs from the sequence. The truncated PDE4A s were less sensitive to rolipram inhibition, were not stereoselective for the enantiomers of rolipram, and no longer contained the high-affinity rolipram binding site. These combined results suggest that alternative splicing of PDE4 mRNA, particularly in the region involving the UCRs and the amino terminus, produces PDE4 isoforms with different potentials for regulation. Evidence has been presented that rolipram

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interacts with two different affinities at a single site on PDE4 and this may relate to different conformational states of the enzyme (67, 101, 240). A number of agents produce a rapid activation of PDE4 (-twofold increase in the VmJ (234, 241, 242). Th is activation of PDE4D provides for short-term feedback regulation of CAMP levels because this enzyme is activated by a CAMP-induced phosphorylation by PKA. The amino-terminal regulatory portion of PDE4D contains two PKA phosphorylation consensus sequences (Ser-13 and Ser-54 in PDE4D3), and Ser-54 is located in the UCRl (109, 111). Site-directed mutagenesis studies also support a crucial role for phosphorylation of Ser-54 for the activation of the enzyme by PKA-catalyzed phosphorylation, and the activated enzyme has a lower IC,, for rolipram (243). Replacement of Glu-53 with alanine blocked the activation of the enzyme despite rapid phosphorylation at Ser-54. These results suggest that disruption of hydrogen bonding in this region of the protein is involved in the effects manifested following phosphorylation (243). Persistent long-term treatment with hormones that elevate CAMP promotes an increase in the level of transcription of PDE4Dl and PDE4D2 variants and an increase in PDE4D protein (116, 120, 244). There has been a sustained and widespread interest in PDE4 for many years. Although low in abundance, the PDE4 family is homologous to the products of the dunce gene that accounts for learning defects in Drosophila (245) and is implicated as a major participant in signaling processes in immunocompetent and proinflammatory cells. The relatively low Km of this enzyme for CAMP, its high specificity for CAMP, its molecular complexity, and its abundance in neural and endocrine tissues implicate it as a major determinant in the modulation of CAMP levels in these tissues.

E. PDES Family The PDE5 family belongs to the group of PDEs that contain allosteric cGMP-binding sites in their regulatory domains (Figs. 1 and 4); it is known as the cGMP-binding cGMP-specific PDE. In this family both the allosteric sites and the catalytic site are highly specific for cGMP PDE5 is a homodimer of -99-kDa subunits and is abundant primarily as a cytosolic enzyme in lung, platelets, vascular smooth muscle, and kidney (246, 247, 247a). PDE5AI gene product was first cloned from bovine tissues (248). Subsequently cDNAs (PDE5A1, PDESA2, and PDE5A3) representing splice variants of PDE5A gene have been found. Although the tissue distribution of these differs (249, 250, 25Oa), the catalytic properties and inhibitor profiles do not. Bovine and human PDE5Als are 96% identical after excluding an insertion of a 10 amino-acid glutamine-rich segment near the amino terminus in the human enzyme. PDESAI, PDE5A2, and PDESA3 cDNAs differ only at the 5’ end and encode proteins that are 100 kDa (875 amino acids), 95 kDa (833 amino

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acids), and 95 kDa (823 amino acids), respectively. Human PDE5A maps to chromosome 4q25-27 (250-252).

gene

1. CATALYTICDOMAIN The catalytic site of PDE5 has significantly higher affinity for cGMP (Km - l-5 pv, although CAMP (Km 2 300 FM) is hydrolyzed with a higher V may.PDE5 was the first mammalian PDE for which Zn2+ was shown to potently support catalysis and that specifically binds Zn2+ with a stoichiometry of -3 mol Zn2+ per PDE5 monomer (72). Although Mn2+ and Mg2+ also support catalysis, Zn2+ is significantly more potent. Site-directed mutagenesis has been used to introduce alanine substitutions at 23 conserved residues in the catalytic domain of PDE5 (Fig. 4B). Under the conditions used for assaying these mutant PDE5s, substitution of His-603 (H-l), His-607 (H-2), His-643 (H-3), His-647 (H-4), Glu-672, Asp-714, and Asp-754 produced marked changes in kcat (Fig. 6); therefore, all of these residues appear to be highly important for efficient catalysis. The most dramatic decrease in kcat occurred with substitutions at either His-643 (H-3) or Asp-754, which decreased free energy of binding -4.5 kcal/mol for each; this is within the range predicted for loss of a hydrogen bond involving a charged residue. These PDE5 catalytic domain mutants were initially studied using standard assay conditions that include Mg2+ as supporting cation (71). More recent studies that in many instances, substitution of Mn2+ for Mg2+ dramatically increases the catalytic rate in certain mutants. These studies reveal that the mutations at His-607 (H-2), His-643 (H-3), and Glu-672 impaired metal binding to the PDE5, which then manifested as dramatic changes in kcat (76~). The Km for cGMP was most profoundly altered by alanine substitutions at Tyr-602 and Glu-775. The Km of the Y602F mutant was actually fourfold improved compared to that of the wild-type enzyme. These results suggest that Tyr-602 forms important stacking interactions in the substrate-binding site of PDE5 and is likely to serve a similar role in other PDEs. An acidic residue is important at the Glu-775 position and is suggested to interact with the N-l nitrogen of the guanine ring (71). The two putative Zn 2+-binding motifs that are conserved in mammalian PDEs include His-603, His-607, Glu-632, His-643, His-647, and Glu-6 72 in PDE5 (Fig. 4). Five of these residues were implicated as critical for efficient catalysis (Fig. 6). The only residue in this group that seems to be nonessential for catalysis is Glu-632; this residue is not conserved in the class I yeast PDE, so this finding is not surprising (27). However, the results support an important role for His-603 (H-l), His-607 (H-2), His-643 (H-3), His-647 (H-4), Glu-672, Asp-714, and Asp-754 in catalysis, and one possible function of this combination of residues is to provide metal-binding sites. The results establish that mutations in either Zn 2+-binding motif are profoundly delete-

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rious to catalysis and that a single Zn 2+-binding motif is insufficient to support normal catalysis in PDEs. With sufficiently high levels of the appropriate metal, significant activity can be largely restored in certain mutants [His607 (H-2), His-643 (H-3), Glu-6721, but not in others (76~). Results of these mutagenesis studies demonstrate that residues located in each of the three conserved subdomains of the catalytic domains of PDEs provide for efficient catalysis and for establishing the Km of PDE5 for cGMP. This distribution suggests that the active site may be formed at the interface between these blocks of conserved residues. Thus, catalytic and substratebinding components are overlapping and utilize several subdomains. 2. INHIBITOR SPECIFICITY AND INTERACTION PDE5 is potently inhibited by sildenafil (IC,, - l-4 nM), DMPPO (I% -3 nA4), UK-122764 (IC,s, - 5 nM), WIN 65579 (IC,, - 2-3 nM), zaprinast (IC,, - 300 nM), and dipyridamole (IC,, - 1 k,M) (3, 93, 96, 253). Inhibition by sildenafil, zaprinast, and IBMX shows competitive kinetics, and the inhibitors interact with the catalytic site of PDE5 in a mutually exclusive manner (100). The IC,, values of sildenafil and IBMX for isolated catalytic domain of PDE5 are essentially the same as for the wild-type enzyme (49u). Among the inhibitors that have been examined using a range of PDE5 catalytic domain mutants, sildenafil exhibits a pattern of changes in the IC,, most similar to that found for the substrate, cGMP. This implies similar interactions of cGMP and the inhibitors with the catalytic domain. However, the potencies of a number of inhibitors of PDE5 (sildenafil, DMPPO, UK122764, and zaprinast) are significantly higher than the affinity for cGMP. Thus, these inhibitors must form additional novel contacts with PDE5 to provide for the tighter binding (100). 3. ALLOSTERIC cGMP-BINDING SITES The two homologous allosteric cGMP-binding sites (a and b) are kinetically distinct, and cGMP occupation of both sites is required for phosphorylation and activation of bovine PDE5Al at Ser.92 by either PKG or PKA (Fig. 1) (247u, 254-255~). Whether these sites have other effects on PDE5 function is unknown. The demonstration that both cGMP-binding sites are required for phosphorylation of Ser-92 provided the first evidence that both cGMP-binding sites in PDE5 are important for a structural change in this enzyme. Furthermore, occupation of the catalytic site by IBMX, cyclic nucleotide analogs, or substrate promotes cGMP-binding to the allosteric sites (3, 60, 247). Th us, the allosteric cGMP-binding sites clearly influence interdomain communication in PDE5. Phosphorylation of Ser-92 in PDE5A by PKG proceeds with at least a lofold higher rate than that by PKA. The primary sequence surrounding Ser-

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92 provides a phosphorylation site that is relatively selective for PKG over PKA (256). Phosphorylation of the PDE5 by PKG has been shown to cause an increase in the activity of the enzyme using low substrate concentrations with no discernible change in IC,, for the inhibitors sildenafil and zaprinast (25%). Using a partially purified PDE5, Bums and Pyne reported that phosphorylation of PDE5 by addition of exogenous PKA increased the V,,, of the enzyme and weakened the potency of inhibition of the enzyme by zaprinast (257). However, the site phosphorylated in these studies was not identified, and the phosphorylation was not dependent on cGMP binding to the allosteric sites on the enzyme, indicating that the differing results of the respective phosphorylation studies may result from different processes. Wyatt et cd. reported a correlation between phosphorylation of PDE5 and increased catalytic activity in cultured vascular smooth muscle cells treated with atria1 natriuretic peptide (107). Last, Lochhead and co-workers reported that activation of partially purified PDE5 by PKA is modulated by interaction with low-molecular-weight proteins (- 14 and 18 kDa) that resemble the inhibitory y-subunits present in the photoreceptor PDEs (258). However, other workers have been unable to demonstrate an interaction of purified -y-subunit with PDE5 or with a chimeric PDEGo/PDE5 (54). The allosteric cGMP-binding sites (a and b) of PDE5 are highly specific for cGMP, and at least a portion of the cGMP/cAMP selectivity may result from the ionization state of a specific aspartic acid residue in these sites (Fig. 8) (259); the more amino-terminal site a exhibits higher binding affinity compared to site b (t,,, - 0.26 and 1.0 hr-r, respectively) (260). Half-maximal saturation of the allosteric cGMP-binding sites is achieved at -0.2-2 pM cGMP, but the stoichiometry of cGMP binding approaches one cGMP per PDE5 monomer. The reason for this low stoichiometry of binding is unclear given the presence of two homologous sequences that provide for cGMP binding to allosteric sites in each PDE5A monomer and the kinetically distinct binding sites. However, a similarly low stoichiometry of binding has been observed for other families of cGMP-binding PDEs, i.e., PDE2 and PDEG, so it is possible that the actual binding stoichiometry for these cGMPbinding PDEs is one cGMP per PDE5 monomer. The allosteric cGMP-binding sites in cGMP-binding PDEs represent a newly recognized class of cyclic nucleotide-binding site that is distinct from those sites in the catabolite activator protein (CAP) family, i.e., PKA, PKG, and cyclic nucleotide-gated channels (40). The cGMP-binding sites in PDEs have no sequence homology with CAP-related proteins, and the analog specificities of the PDE sites differ markedly from those sites in the CAP-related proteins. Site-directed mutagenesis of residues in the cGMP-binding sites of PDE5 has established that a conserved -N(K/R)XnD- motif is critical for cGMP binding (260,261). Th is motif (Fig. 8) resembles the canonical NKXD

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motif that participates in binding guanine in GTP-binding proteins (262). Because the NKXnD motif is conserved in the allosteric cGMP-binding sites of other cGMP-binding PDEs, it is likely that these residues will prove to be important in cGMP binding in the PDE2 and PDE6 families. Based on changes in free energy of binding of cGMP on mutation of these conserved residues, on studies using cGMP analogs, and on the known functions of the respective residues in the NKXD motif in GTP-binding proteins, a functional role for each residue in the NKXnD motif in PDEs has been proposed (261). In PDE5A, disruption of cGMP binding in either site by site-directed mutagenesis had no apparent effect on the function of the other cGMP-binding site or on catalytic function. This result is further supported by studies of the properties of a truncation mutant containing the PDE5 catalytic domain (49~). This catalytically active PDE5 fragment was shown to be monomeric and to exhibit catalytic properties that are comparable to those of native PDE5. However, a subtle, but physiologically relevant, difference in catalytic activity of this mutant could not be ruled out. The remarkable therapeutic success of sildenafil (Viagra), a highly specific inhibitor of PDE5, in treating male erectile dysfunction has generated much interest in this enzyme, and the potential for other drugs that might target PDE5 is now well appreciated (93, 263). The tissue distribution of the PDE5 family is relatively restricted compared to some other PDEs; this makes the use of agents that selectively target PDE5 and particular physiological processes somewhat more practical. Given the abundance of PDE5 along with other components of the cGMP signaling pathway in smooth muscle, it is likely that regulation of PDE5 activity is a central feature in modulating contractile tone in the vasculature, airways, and gastrointestinal smooth muscle. Although significant progress has been made in defining the molecular features that provide for the properties of PDE5, definitive data regarding the catalytic mechanism and the manner in which substrates and inhibitors interact with the enzyme are still very limited.

F.PDE6 Family Members of the PDE6 family are highly specific for cGMP as substrate and are found in the rod and cone cells of the vertebrate retina, where they function as key participants in the visual response to light. The population of PDEs in the photoreceptor cells is almost exclusively restricted to the PDE6 family. The PDE6 family is characterized by (1) its high catalytic specificity for cGMP, (2) th e p resence of allosteric cGMP-binding sites that are located in the amino-terminal half of the enzyme, (3) a high kCat,and (4) the fact that this is the only PDE family known to be regulated by G proteins. Numerous visual defects have been traced to mutations affecting the various protein subunits of the rod and cone PDEs (1, 9,18,19,108,264,265).

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1. STRUCTURALFEATURES The PDE6 family is a member of the cGMP-binding subgroup of PDEs because enzymes of this family bind cGMP with high specificity at allosteric sites in their amino-terminal regulatory domains. PDE6 family is closely related to the PDE5 family, and the allosteric cGMP-binding sites are homologous with those in PDEs 2, 5, and 10. Photoreceptor PDEs occur as heterotetramers that contain two dimerized homologous catalytic subunits [either 01and B (rod PDEG) or two (Y’(cone PDEG)]; each of these three catalytic subunits is the product of separate genes. The predominant form of rod PDE holoenzyme contains one PDE-ol and one PDE-B subunit (-99 and 98 kDa, respectively), although minor components involving PDE-ecu. and PDE-BB have been detected; cone PDE6 is a homodimer of two catalytic PDE-a’ subunits (-94 kDa) (266-268). In each instance, two inhibitory y-subunits of -9.7 kDa that are specific for either rod PDE or cone PDE are complexed with the respective PDE dimers, and the affinities of these y-subunit interactions with either rod or cone catalytic subunits differ (269, 270). Th e majority of PDE6 activity is associated with the membrane fraction of the cell. This may result from posttranslational modification of the PDE6 subunits in a CAAX sequence at their respective carboxy1 termini; PDE6o. is farnesylated and PDEGB is geranylgeranylated (271,272). The soluble portion of PDE6 contains an additional subunit, i.e., the S-subunit (-15,000 Da) (273-275). 2. ~LOSTERIC

CAMP-BINDING

SITES

PDE6 family members bind cGMP with high selectivity and affinity to the allosteric cGMP-binding sites (276-279). Both allosteric cGMP-binding sites contribute to cGMP binding by the regulatory portion of PDEG, and the two binding sites appear to be coupled (54, 280). Like other cGMP-binding PDEs, the best estimate of cGMP-binding stoichiometry is one cGMP bound per PDE6 monomer. Affinity for cGMP binding varies substantially (Kd 60 nM-1 (IM)depending on the activity state of PDE6 (278, 281), and the dynamics of the protein-protein interactions that determine overall activity of PDE6 appear to be modulated by state of occupation of the cGMP-binding allosteric sites (278, 281, 282). Cyclic GMP binding to the allosteric sites is increased by binding of Py to the enzyme, and the polycationic region of Py (residues 24-45) accounts for this increased affinity (283, 284). Activation of PDE6 by transducin causes an initial loss of cGMP-binding affinity of -lO-fold, and subsequent removal of Py an&or delta subunits from the catalytic subunits decreases affinity further (278, 284~). Although cGMP binding to allosteric cGMP-binding sites in PDE6 does not appear to affect catalytic function directly, occupation of these sites alters binding affinity of PDE-CYB for Py and thereby modulates catalytic function.

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3. PDE6 INVISUALTRANSDUCTION In dark-adapted rods and cones, cellular cGMP (-60 FM) is quite high, but a significant portion is apparently bound to allosteric cGMP-binding sites of the photoreceptor PDEs. Concentration of unbound cGMP (-5 t&I) is still sufficient to provide for cGMP occupation of the cGMP-gated cation channels; under these conditions, the channel exists in an open state, thereby facilitating inward conductance of Naf/Ca2+. The membrane potential is thus relatively depolarized. Absorption of light converts rhodopsin to a photoactivated rhodopsin, which, in turn, activates a heterotrimeric GTP-binding protein, transducin (265). The GTP-bound o-subunit of transducin (CIJ interacts with the inhibitory y-subunits of PDEG, thereby relieving inhibition and causing a rapid increase in PDE catalytic activity. GDP-bound ot can bind to the P-y, but with markedly lower affinity (284). The increased PDE activity rapidly lowers intracellular cGMP, such that cGMP dissociating from the channel is not replaced. Consequently, channel pores then close, thereby abolishing the depolarizing influx of cations into the photoreceptor. However, activity of a Na+ exchanger persists, so that the membrane potential of the cell becomes increasingly hyperpolarized. This provides the signal that is perceived as light in higher integrative centers. Specifics of this pathway continue to be studied intensely. Amplification of a light signal occurs at the levels of the G protein, the PDE, and the cGMP-gated channel. A single photon converts rhodopsin into photoactivated rhodopsin, which promotes GDP/GTP exchange on as many as 500 transducins in 1 sec. Hundreds of o-GTPs dissociate from transducin free ot-GTP concomplexes, reaching a local concentration as high as 80 p,M; centration is estimated to approximate available binding sites for (w,-GTP on the PDE-Py complex. The resulting au,-GTP forms a tightly membranebound complex with PDE, thereby activating the enzyme. Two c-w,-GTPscan be bound per PDE, but whether one or two o,GTPs are required to activate each PDE molecule fully is not clear (285). However, the physiologically activated PDE is most likely to be complexed with two CY,-GTPs. Estimates of affinity with which o,GTP binds to PDE vary widely, and binding is suggested to be positively cooperative; intrinsic fluorescence studies of the interaction between ot and PDEy determined the binding constant to be co.1 nM (286). The o,GTP activation occurs in a membrane-bound PDE complex, and a number of studies have shown that presence of membranes also enhances the process (287, 288). Each activated PDE molecule then hydrolyzes perhaps 1000 cGMP molecules to 5’-GMP. The maximal activity of rod PDE is estimated at -3000-4000 cGMP/sec/PDE molecule, and ‘Y+ GTP-activated PDE is likely to reach -90% of this rate due to the positive cooperativity of CY~-GTPbinding. Lowered cGMP causes closure of the cGMP-gated channel, and the influx of thousands of cations is blocked.

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4. CHARACTERISTICSOFTHEINTERACTIONSTHATCONTRIBUTE TOTHEREGULATIONOFPDEGBYPDE-?/SUBUNIT ANDTHETRANSDUCINIX~-GTP/GDP Interactions that occur between PDE-v and PDE6 catalytic subunits, and transducin o-subunit, are central to regulation of photoreceptor PDE activity. Myriad advances have been made regarding the complex mechanisms that contribute to modulation of the interactions of these and other components both in the dark, in the acute response to light, and in events associated with light adaptation. Furthermore, the number of proteins participating in modulating this pathway has progressively increased (280, 285, 289-293). Extensive experimental evidence suggests that the whole process involves direct contacts between Py and PDE6 catalytic subunits, between transducin o-subunit, ot, and Py, and between ot and PDE catalytic subunits (195, 291, 294,295). More recently, proteins from the RGS family have been shown to interact with o,-GTP to stimulate GTP hydrolysis (293, 296-298). The inhibitory y-subunits interact with PDE-c+ with a very high affinity (Kd < 10 100 PM) to maintain the enzyme in an inactive state in absence of light. The high affinity of interaction between Py and PDE-c@ is provided by at least two subdomains on Py (54,283,299-302). One set of contacts involves the amino-terminal portion of a centrally located polycationic region between Py residues 24-45, which interacts with a region in PDE-o subunit (residues 46 l- 553) that is located between the catalytic domain and the cGMP-binding domain. A synthetic peptide corresponding to residues 517-541 of the PDE-a subunit blocks inhibition of PDE activity by Py and also competes with interaction between Py and qGTP. A second subdomain in Py, the carboxyl-terminal5 -7 amino acids, also interacts with the PDE-of3, and this contact is critical to effect the inhibition of catalysis. Carboxyl-terminal deletion of these residues renders Py ineffective, and peptides corresponding to the carboxy-terminal region of Py inhibit PDE catalysis (299). Studies have shown that the carboxy-terminal 5-7 amino acids of Py interact directly with a site (residues 751-763) in the catalytic domain of one of the catalytic subunits of rod PDE, i.e., PCL Using a fluorescent probe attached to the carboxyl terminus of Py, it was shown that K, for this interaction was -4 nM and a competitive inhibitor, zaprinast, displaced the probe from the interaction on PoyP. Furthermore the carboxyl terminus of Py could be directly cross-linked with a specific region in the catalytic site of PDE6 near an NKXD motif, thought to provide for specific interaction with the guanine of cGMP (303). These studies suggest that the carboxyl terminus of Py inhibits photoreceptor PDE by physically blocking access of cGMP to the catalytic site. Alternatively, Py could cause a conformational change near the catalytic site that would block catalysis.

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Interaction between the Py and transducin or,GTP complex occurs through two major regions of contact. On Py this involves the carboxyl-terminal portion of the Py polycationic region and a segment surrounding Cys-68 in the carboxyl-terminal region (Py 63-76) (54, 284, 290, 299). Studies involving modification of several residues, including two an&nines (Arg-33 and -36) and a threonine (Thr-35) located in the polycationic region of Py, support the importance of this region in forming contacts with o+, Both arginines can be covalently modified in the absence of ot, but when complexed with ot (GTPor GDP-bound forms), they appear to be masked (304). Phosphorylation of Thr-35 in Py by PKA functionally alters Py so that its potency of inhibition of PDE is increased, and its potency of interaction with the ot is also altered (305). Most recently a highly important role has emerged for participation of RGS proteins in photoreceptor signaling. The RGS proteins interact with otGTP to increase the GTPase activity and thereby increase responsiveness of this whole pathway (280, 293, 296-298, 306-308). Evidence has been presented that supports a direct interaction between ot and catalytic subunits of PDE6 as well as with a specific RGS protein (291, 309, 310). The structure of ot contains a GTP-binding domain and an o-helical domain (HD); th ere is now evidence that a 22-amino acid sequence in the HD region of (3~~ interacts with catalytic subunits of PDEG, a site that is distinct from the site on PDE-cxB that interacts with Py (291), and that this interaction facilitates interaction of the at-GTF-binding domain with Py, thereby enhancing activation of PDE6 (291, 309). It has been proposed that this additional interaction of o+ with catalytic subunits of PDE6 may contribute to the high efficiency of PDE activation/inactivation. The investigation of the proteins involved in determining the visual phototransduction pathway has been unrelenting, and despite the monumental amount of effort we still have only a general picture of the molecular events that provide for this highly complex process. However, the major advances that have been made in studying this pathway have yielded important insights into the central role of PDE6 in this system and into the basic properties of the PDE superfamily, as well as the complexity of the controls that can overlay a relatively simple catalytic event.

G. PDE7 Family The PDE7 family is characterized by having a high specificity for CAMP (K,,, = 0.2-l ~_LLM) an d resistance to inhibition by a variety of common PDE inhibitors, including rolipram, zaprinast, Q-(2-hydroxy-3-nonyl)adenine (EHNA), Ro 20-1724, and enoximone. There are no known PDE7-selective inhibitors, but PDE7B is inhibited by IBMX (IC,, -2 t_J4), dipyridamole (IC,, = 1.5 PM) (313~2,b). PDE7 catalytic (I%, -2 FM), and SCH51866 activity is unaffected by either Ca’+/calmodulin of cGMP By comparison of

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amino acid sequences, PDE7 family is most closely related to PDE4 family, but the similarity is modest. Two cDNA clones (PDE7Al and PDE7A2) representing variants of the PDE7A gene and one cDNA clone representing PDE7B have been isolated (311-313b). Amino acid sequence of PDE7B is - 70% identical to PDE7A, and PDE7A variants differ in their amino termini. These variants arise through alternative mRNA splicing with the divergence occurring just amino terminal to the second conserved -RRGAIS- sequence: PDE7A2 has one of these sequences beginning at Arg-21, whereas PDE7Al has two copies, one beginning at Arg-2 1 and the second at Arg-45. Protein products of PDE’IAl, PDE7A2, and PDE7B are 481-, 456. and 446amino acid proteins (-57, 50, and 52 kDa), respectively. PDE7Al was detected in both cytosolic and particulate fractions, whereas PDE7A2 is primarily associated with the particulate fraction in skeletal and cardiac muscle. Proteins of the predicted sizes are detected in Sf9 cells overexpressing the respective cDNAs, and a PDE activity consistent with that of PDE7Al has been detected in adult skeletal muscle. PDE7Al is particularly abundant in human lymphoid tissue (126, 311); in several cell lines of T lymphocytes the PDE7Al represents -40% of total CAMP PDE activity. PDE7A mRNA is broadly distributed in fetal tissues, but in some instances PDE7 protein levels do not correlate well with PDE7 mRNA content. Although PDE7 mRNA is abundant in adult skeletal muscle, T lymphocytes, and B lymphocytes, PDE7 protein and activity are readily measurable only in the T lymphocytes (311). The basis for this disparity and the physiological relevance of such a difference is unclear, although it has been suggested that PDE7A2 predominates in skeletal muscle and is largely particulate. Increased level of PDE7 in T cell lymphocytes is implicated as a major determinant in T cell activation and proliferation (126). PDE7B transcripts are abundant in the putamen, caudate nucleus, heart, skeletal muscle, and pancreas (311-313b).

H. PDE8 Family The PDE8 family was discovered only recently and has been descriptively dubbed as the high-affinity CAMP-specific and IBMX-insensitive PDE (36, 38). PDE8 has a very low Km for CAMP (0.06-0.15 pJ4 compared to a Km of 124 ~.LLM for cGMP). Cyclic GMP at concentrations as high as 100 p~J4 do not significantly inhibit CAMP catalytic activity. When compared to PDE4A, PDE8 has a 40-fold higher affinity for CAMP, but PDE4A has a lo-fold higher V,,. PDE8 is a soluble PDE with a predicted sequence of 823 amino acids. The catalytic domain of PDE8 contains 22 of the 24 invariant amino acids conserved among the seven earlier recognized PDE families, including the two histidine-rich metal-binding motifs. Although PDES is inhibited potently by dipyridamole (IC,, - 5 k&f), it is notably insensitive

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to a number of other well-known PDE inhibitors, including IBMX, zaprinast, rolipram, vinpocetine, and SKF-94 120. In the catalytic domain, the primary sequence is most similar to those of the PDE4 and PDE7 families (39 and 34% identity, respectively) and least similar to those of PDE5 and PDE6 (23 and 200/o,respectively). The PDE8 mRNA is a single species of 4.5 kb, is expressed widely, and appears to be most abundant in testis, ovary, small intestine, and colon. However, the amount of PDES protein in these tissues is relatively low. Maximum catalytic activity of PDE8 requires 1 mM Mn2+ or Mg”+, whereas Ca2+ (100 kh’l) is significantly less effective (20% maximum).

I. PDE9 Family The PDE9 family is also a recently recognized family and is described as a very high-affinity cGMP-specific PDE. This PDE family has been cloned from both murine and human tissues, and four different PDE9 mRNA transcripts have been identified (PDESAl, PDE9A2, PDE9A3, and PDE9A4); these are produced as a result of alternative splicing of 5’ exons. The predicted protein products from these transcripts would encode proteins of 593, 533, 466, and 465 amino acids (37, 39, 314). The primary structure of the catalytic domains of these PDE9s is most similar to that of PDE8 (34%) and least similar to that of PDE5A (28%). Of the 22 residues considered to be invariant in other mammalian PDE families, 21 residues are present in the PDE9 catalytic domain. The PDESA mRNA (-2 kb) is widely expressed and is particularly abundant in spleen, intestine, kidney, heart, and brain. However, the abundance of PDE9 protein and activity in tissues has not been assessed, so the possible involvement of the PDE9 family in physiological processes is still unclear. In humans the PDESA gene contains 20 exons that extend over I22 kb; it maps to chromosome 21q22.3, a region of the genome that is associated with a number of medical disorders (314). The PDE9 family is highly selective for cGMP and has an affinity for cGMP that is 40-170 times higher than that of the two other families of cGMP-selective PDEs, PDE5 and PDEG. PDE9 has been expressed in insect cells and in COS-7 cells and has a Km of 70-170 nA4 for cGMP versus 230 $!4 for CAMP; the Vmax for cGMP is -e-fold greater than PDE4 for CAMP. Due to the high affinity of PDE9 for cGMP and the broad tissue distribution of the mRNA, this PDE family has been proposed to participate in maintaining basal cGMP levels in the cell (3 7). In the concentration range of l10 mM Mn2+ or Mg 2+, the catalytic rate in the presence of Mn”+ is twice that of comparable concentrations of Mg 2+. PDE9 is inhibited by zaprinast (IC,5, - 35 ~~,n/l) and by PDEs 1 and 5 inhibitor SCH51866 (IC,, - 1.6 $t4), but is insensitive to a wide range of other PDE inhibitors, including sildenafil, dipyridamole, IBMX, rolipram, vinpocetine, and SKF-94 120.

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J. PDElO Family PDElO is dual substrate specificity PDE family. There are at least two variants (PDElOAl and PDElOA2) that have 766 residues in common (315318). Although Km for CAMP is much lower than that for cGMP (0.05 and 3 @4, respectively), Vm, for cGMP is -5-fold greater than that for CAMP. Both human, mouse, and rat PDElO clones have been isolated. Predicted sequences include a region that is homologous to the conserved catalytic domain found in other PDEs, but there is less than 50% sequence identity with catalytic domains of other PDE families. Predicted sequence of PDElOA also contains a segment that is homologous to the cGMP-binding domain in cGMP-binding PDEs (PDES, PDE5, and PDEG), but cGMP binding to PDElO has not yet been demonstrated. Mouse and rat PDElOA encodes proteins of 779 amino acids and 794, respectively (88,516 Da) (315, 316), and a novel form in rat has an amino terminus that is distinct from the human enzymes (317). The mRNA for mouse PDElOA is most abundant in brain and testis. Human PDElO maps to chromosome 6q26-27 (316). Based on Northem blot analysis, the human enzyme shows a broad tissue distribution.

K. PDEll Family A PDEll family has recently been recognized (Fig. l), and human PDEllAl has been cloned, expressed and characterized (318). The cDNA for PDEllAl encodes an open reading frame for a 56-kD protein (490 amino acids), and the sequence of its catalytic domain is most like that of PDE5 (-50%). The regulatory domain of PDEllAl contains a single sequence that is homologous to the two cGMP-binding motifs located in the regulatory domains of PDEs 2, 5, 6, and 10, although cGMP-binding to PDEll has not been demonstrated. This is the first recognized PDE to have a single such site in the regulatory region, and no effect of either cGMP or CAMP to stimulate hydrolysis of the other nucleotide has been observed. Investigators conjecture that this domain may bind either cGMP or perhaps another unknown ligand to regulate function. PDEll Al hydrolyzes both CAMP and cGMP with similar V,= values and similar affinities, with Km values of 0.5 and 1 k.M, respectively. Based on these kinetic values, PDEll may contribute significantly to modulating both CAMP and cGMP in the cell. PDEll activity is inhibited by IBMX (IC,, - 50 FM), and among other PDE inhibitors tested, dipyridamole is the most potent compound (IC,, - 0.4 I.LM) is not inhibited by either followed by zaprinast (IC,, - 12 yM). PDEllAl milrinone or rolipram, selective inhibitors for PDE3 and PDE4, respectively. There are at least three mRNA transcripts (10.5,8.5, and 6 kb, respectively) for PDEllA, suggesting that there may be additional isoforms in this family, and based on RNA expression patterns, PDEll may have a broad tissue

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distribution. Immunoblots of human tissues detect three protein bands (78, 65, and 56 kD); the smallest form, which co-migrates with recombinant PDEllAl, is abundant in prostate gland, while this form, as well as two additional protein bands, are found in skeletal muscle.

VIII. Concluding Remarks Appreciation of the complexity of the PDE superfamily and its importance in regulating physiological processes continues to increase. The diverse features of the various families of PDEs emphasize the potential for selective regulation and function, and it is clear that PDEs provide a critical component of cyclic nucleotide signaling in all tissues. Exploiting the novel and shared features of these enzymes for developing improved therapies for a number of maladies continues to be a major challenge in this field. An improved understanding of the molecular basis for catalytic function and regulation of these enzymes will undoubtedly provide the framework for success in systematically approaching this goal.

ACKNOWLEDGMENTS Supported by NIH Grants GM41269 (JDC) and DK40029 (JDC), and by the American Heart Association (SHF).

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Thyroid

Hormone Regulation

of Apoptotic Tissue Remodeling: implications from Molecular Analysis Amphibian

of

Metamorphosis YUN-BO SHI* AND ATSUKO ISHIZUYA-OKAY

1

*Laboratory of Molecular Embryology National lnstitute of Child Health und Human Development National Institutes of Health Bethesda, Maryland 20892 fDepartment of Histology and Neurobiology Dokkyo Unioersity School of Medicine Mibu, Tochigi 321-02, Japan

I. Introduction .................................................. II. Thyroid Hormone and Vertebrate Development .................... A. Thyroid Hormone in Mammals ............................... 8. Thyroid Hormone in Anurans ................................ III. Mechanism of Thyroid Hormone Action .......................... A. Thyroid Hormone Receptors ................................. B. Transcriptional Regulation by TRs ............................ IV Thyroid Hormone-Dependent Morphological and Cellular Changes during Intestinal Remodeling ................................... ............................................ A. Larval Intestine ............................... B. Metamorphic Transformations C. Organ Autonomous Response to TH .......................... V TR Expression and Function during Intestinal Remodeling .......... A. Correlation of Receptor mRNA Levels with Intestinal Remodeling . B. TH Activation of TRR Genes ................................. C. Cell Type-Dependent Temporal Regulation of TRB Genes ........ VI. Thyroid Hormone Response Genes in the Intestine ................. A. Early TH Response Genes ................................... B. Late TH Response Genes .................................... VII. Functions of TH Response Genes: Implication from Studies on Matrix ............................................ Metalloproteinases ............. A. ECM Remodeling during Intestinal Metamorphosis .............. B. Regulation of MMP Genes during Metamorphosis ............... C. Extracellular Matrix and Cell Fate Determination VIII. Conclusions and Prospects ..................................... References ...................................................

Progress in Zlucleic Acid Research and hlolecular Biology, Vol. 63

53

54 ,5s 55 56 58 !59 61 70 70 73 74 77 77 77 79 80 80 83 84 85 85 91 91 94

54

YUN-BO

SHI AND ATSUKO

ISHIZUYA-OKA

Organogenesis and tissue remodeling are critical processes during postembryonic animal development. Anuran metamorphosis has for nearly a century served as an excellent model to study these processes in vertebrates. Frogs not only have essentially the same organs with the same functions as higher vertebrates such as humans, but also employ similar organogenic processes involving highly conserved genes. Development of frog organs takes place during metamorphosis, which is free of any maternal influences but absolutely dependent on the presence of thyroid hormone. Furthermore, this process can be easily manipulated both in intact tadpoles and in organ cultures by controlling the availability of thyroid hormone. These interesting properties have led to extensive morphological, cellular, and biochemical studies on amphibian metamorphosis. More recently, the cloning of thyroid hormone receptors and the demonstration that they are transcription factors have encouraged enormous interest in the molecular pathways controlling tissue remodeling induced by thyroid hormone during metamorphosis. This article summarizes some of the recent studies on the mechanisms of gene regulation by thyroid hormone receptors and isolation and functional characterization of genes induced by thyroid hormone during Xenopus metamorphosis. Particular focus is placed on the remodeling of the animal intestine, which involves both apoptosis (programmed cell death) of larval cells and de no00 development of adult tissues, and the roles of thyroid hormone-induced genes that encode matrix metalloproteinases during this process. 8 2000Academic PIZSS.

I. Introduction For nearly a century, biologists have used amphibian metamorphosis, particularly anuran metamorphosis, as a model to study postembryonic development in vertebrates. This process is independent of maternal influences but absolutely requires the presence of thyroid hormone (TH) (see below). It is easy to manipulate this process in vitro and access the developing organs and animals for various analyses. Such properties have made anuran metamorphosis a long-lasting model to study the biochemical and morphological changes associated with organogenesis and tissue remodeling in postembryonic animals. All three classes of amphibians-caecilians, urodeles, and anurans-undergo varying degrees of metamorphosis during their development. Anurans have the most dramatic and complete larva/adult transformation. Their metamorphosis involves changes in essentially all organs and tissues of the tadpole. Three major types of changes occur during this process (1,2). The first is the complete resorption of larva-specific organs such as the tail and gills. Tail resorption is one of the best studied processes during anuran metamorphosis. It involves systematic resorption of different tissues, such as the epidermis, connective tissue, and muscles. At the other extreme, limbs develop de nova from undifferentiated blastema cells to form structures that consist

THYROID

HORMONE-REGULATED

TISSUE

REMODELING

55

of tissues similar to those of the resorbed tail. The vast majority of the organs, such as the intestine and liver, are present in both tadpoles and frogs. They undergo a partial but dramatic remodeling to form organs suitable for the frog environmental habitat. In this article, we will focus on this kind of transformation. In particular, we will review our current understanding of the molecular pathways involved in the remodeling of the intestine. We will focus primarily on our studies in Xenopus laevis. Where appropriate, we will describe related studies in other organs and in Rana catesbeiana, another anuran that has been studied at molecular level in considerable detail, to illustrate the potential generality of the basic conclusions. We will begin by reviewing the roles of TH in vertebrate development and the current understanding of the molecular mechanisms of gene regulation by thyroid hormone receptors.

II. Thyroid Hormone and Vertebrate Development A. Thyroid

Hormone

in Mammals

Thyroid hormone is known to affect diverse biological processes in vertebrates. The earliest observed effect associated with TH is human cretinism due to the lack of TH caused by iodine deficiency (3). Human cretins were observed as early as 2600 BC; cretinism is characterized by short stature, mental retardation, and the presence of a goiter, due to overgrowth of the thyroid gland (3). Such developmental defects can be prevented by supplementing TH during fetal and postnatal development, indicating key roles of TH in human development. Consistently, TH levels in human fetal plasma rise sharply a few months prior to birth and remain high during the first several months postbirth (Fig. 1) (4). On birth, the human neonate has a higher rate of metabolism and an increased demand for oxygen (3). TH is known to stimulate metabolic rate both in vitro and in vivo in humans and animals (5 - 7). In addition to increased metabolism after birth, the period of perinatal development is also associated with increased proliferation of glial and neuronal cells and acquisition of several brain functions, as exemplified by the sensory processes (4). Again, the high levels of TH at this period are critical for these and other neural development processes, although the exact processes affected by TH in the developing human brain are not clear. Animal studies have shown that TH influences many aspects of brain development (8). At anatomical and histological levels, both the forebrain and the cerebellum require TH for normal maturation. For example, hypothyroidism delays the appearance of the external germinal layer and decreases the number and density of synaptic contacts

56

YUN-BO SHI AND ATSUKO ISHIZUYA-OKA

with the already defective Purkinje cells, leading to a permanent impairment of neuronal connectivity. The anatomical alterations are accompanied by extensive biochemical changes in the brain, including changes in oxygen consumption and the metabolism of glucose and polyamines. TH continues to play important roles during human postnatal development well past birth (3). Thyroid h ormone is essential for normal growth and development, and deficiency is associated with severe retardation of growth and maturation processes of almost all organ systems. Body weight does not increase and bone growth is also retarded if TH is deficient. The most dramatic effects are seen in tissues that are rapidly proliferating. The severe consequence of insufficient TH or lack of TH during fetal and postnatal development is cretinism, which affects many aspects of the patient (3, 9-13). In addition to the developmental effects, TH deficiency is known to lead to reduced metabolic rate (5 - 7). Similarly, abnormal TH levels are associated with a number of cardiovascular symptoms (14, 15). Thus, proper levels of TH are critical for mammalian development and organ function. 6.

Thyroid

Hormone

in Anurans

Nearly nine decades ago, Gudematsch (16) found that a substance(s) in the thyroid gland could induce metamorphosis. Shortly after, Kendall (17,18) showed that the active ingredient is thyroid hormone. These studies led to the isolation and structural characterization of two natural thyroid hormones, 3, 5,3’, 5’-tetraiodothyronine (TJ, commonly known as thyroxine, and 3, 5, 3’triiodothyronine (T,) (Fig. 1A). Subsequent investigations revealed three independent lines of evidence that firmly establish the causative role of TH in anuran metamorphosis. First, elevations in circulating plasma concentrations of thyroid hormones T, and T, correlate with metamorphosis (Fig. lB, for X. laeuis) (19, 20). In X. Zuevis, there is little TH before stage 54, when tadpoles grow rapidly but exhibit few morphological changes (21). During prometamorphosis (stages 54-58), synthesis of endogenous TH allows for the accumulation of increasing levels of T, and T, in the plasma (Fig. 1B). Accompanying this, tadpoles undergo both growth and morphological transformation, most noticeably the development of the hind limbs. Finally, at the climax of metamorphosis (stages 58 -66), TH is at peak levels and the tadpoles stop feeding and undergo a rapid metamorphic transition. On the completion of metamorphosis at stage 66, plasma TH levels are also reduced. The second line of evidence supporting the role of thyroid hormone in metamorphosis comes from the ability of TH to induce precocious metamorphosis. The first such experiment was the landmark study by Gudernatsch (16), which has since been reproduced for many different anuran species with pure T, and T, at concentrations comparable to endogenous

THYROID

HORMONE-REGULATED

TISSUE

57

REMODELING

T3: 3,.5,3’-triicdothyronine

T4: 3, 5, 3’, 5’-tetraiodothyronine=thyroxine

B

Human Age I,,I

I w

-6

-4

-2

I

I

I

2 0 (Birth)

I

I

(month)

4

6

6

I

I

I

10

12

14

III

100 -

.z LJ 76 F ;

50-

3 2

25-

1

I,,,

I 35/36

I

I

45

51

1

61

63

66

Xenopus Stage

FIG. 1. (A). Structures of two natural thyroid hormones. (B). Plasma TH levels during Xenopus Zaevis and human development. The Xenopus stages are based on Nieuwkoop and Faber (21). The TH levels are based on Leloup and Buscaglia (19) for X. la&s and Tata (4) for humans.

plasma TH levels (1, 20,22).For example, addition of 5 nM of T, to the rearing water of X. laevis tadpoles at stages 55156 leads to drastic changes within 5 days (Fig. 2). Most notable are the external changes, including hind limb morphogenesis and cranial restructuring. Finally, amphibian metamorphosis can be prevented by blocking the synthesis of endogenous TH. In fact, shortly after the landmark experiment of Gudernatsch (16), Allen (23) sh owed that thyroid gland removal resulted in formation of larger than normal tadpoles that were not capable of metamorphosis. However, such giant tadpoles can resume metamorphosis when exogenous TH is added to their rearing water. In addition to thyroidectomy, chemical inhibitors (goitrogens) can be used to inhibit the synthesis of endogenous TH and block metamorphosis (1).

58

YUN-BO

Control

SHI AND ATSUKO

ISHIZUYA-OKA

TH-treated

FIG. 2. TH induces precocious metamorphosis. Stages 55156 Xenopus tadpoles untreated (left) or treated (right) with 10 nM T3 for 5 days showed TH-dependent limb developmentand morphological changes in the head and body.

Their action can again be reversed

by exogenous

evidence

unambiguously

collectively

demonstrates

TH. Thus, all of the above that TH is the causative

agent of anuran metamorphosis.

III. Mechanism of Thyroid Hormone Action Both of the naturally

occurring

thyroid hormones,

T, and T,, are synthe-

sized in the thyroid gland (1, 24). T, can be either secreted ing plasma

or converted The T,

to T, through and T,

deiodination

thyroid

gland.

quently

carried by the plasma to different

ert their biological effects. T, can also be converted (25, 26). In addition,

secreted

into the circulat-

by 5’-deiodinase

.from the thyroid

in the

gland are subse-

organs and tissues, where they ex-

to T3 in various target tissues by 5’-deiodinase

both T3 and T, can be inactivated

through

the action

of 5-deiodinases, producing T, and reverse T,, respectively. Two different 5’deiodinases have been identified in various animal species (25,26). A 5’-deiodinase and a 5-deiodinase

from

R. catesbeiana (27-29) and a 5-deiodinase

THYROID

HORMONE-REGULATED

59

TISSUE REMODELING

from X. lamis (30) have been cloned. They have distinct developmental regulation patterns in different organs, suggesting that T, and T, levels can vary in different organs and tissues. It is unclear whether T, and T, have different biological functions. Some evidence suggests that, at least in certain cases, T, may be first converted to T, to exert its biological effects (29). The biological effects of TH are mostly, if not entirely, mediated by thyroid hormone receptors (TRs). TRs are high-affinity TH-binding proteins with dissociation constants of less than 1 nM. They are localized in the nucleus both in the presence and the absence of TH (31, 32). Using photoaffinity labeling, Samuels and colleagues (33) showed that TRs are chromatin associated. These and other studies show that TRs can regulate gene expression even in the absence of TH and the binding of TH to TRs alters this regulation.

A. Thyroid

Hormone Receptors

There are four TR genes in Xenopus-two TRo genes and two TRB genes (34, 35). Alternative splicing of the TRB transcriptions gives rise to two different isoforms for each TRB gene (Fig. 3). In higher vertebrates, there is only one TRa and one TRB gene, with the latter producing two TRB isoforms due to alternative splicing (36). TRs belong to the superfamily of nuclear hormone receptors, including receptors for glucocorticoid and retinoic acid (37- 41). Members of this family share many structural features. In general, each can be divided roughly into five domains, A/B, C, D, E, and F, respectively, from the amino terminus to the carboxyl terminus (Fig. 3) (42). The DNA-binding domain (domain C) is located in the amino half of the protein and is the most highly conserved domain among different receptors (Fig. 3). The large ligand-binding domain (domain E) is in the carboxyl half of the protein and is conserved among TRs

DNA

Hormone Binding Domain

Binding Domain AIB

C

E

D

TRaAIB 1 tY?&SY

V/////////////////A

TRPAl

f\\\\\‘y

mu

TRW2

p

TRPB 1 TRPB2

F

0

mu m

m

I

FIG. 3. Domain structures of Xenopus TRs. The A/B domain has been shown to have transcriptional activation properties for some TRs, although its role in frog TRs is uncertain. Domain C encodes the DNA-binding domain and E encodes the hormone-binding domain. A transactivation domain (AF2) is located at the carboxyl terminus encompassing the F and part of the E domain. Different boxes for A/B and F domains indicate divergent sequences among different receptors. The C,D, and E domains and part of the F domain are highly conserved.

60

YUN-BOSHI AND ATSUKOISHIZUYA-OKA

in different species. The rest of domains vary in size and sequences among different nuclear receptors. The N-terminal A/B domain is highly variable in sequence and length, the shortest being the TRB in X. la&s (Fig. 3) (35). At least in some TRs, this domain contains a transactivation function (Al?), although its role in amphibian TRs is unclear. Another transactivation function domain is the AF-2 domain, which is located at the very end of the C terminus (F domain and part of the E domain). Between the DNA- and ligand-binding domains is the D domain, or variable hinge region. This region often contains a nuclear localization signal and influences both DNA binding and transactivation (38,43,44), although the underlying mechanisms are unclear. 1. THYROID HORMONE BINDING BY TRs Specific binding of thyroid hormone by TRs requires the minimum thyroid hormone binding domain, which spans the C-terminal domain of about 250 amino acids (36, 45). Th is d omain shares only a low level of homology among different nuclear receptors. However, TRs from species as diverse as frogs and humans are over 90% identical in this region (35). Such conservation accounts for the essentially identical high affinities of TRs for T, (with K, in the subnanomolarity range) (29, 44, 46, 47). The crystal structure of the domain bound with a ligand reveals that the holo-ligand-binding domain consists of 12 o-helices with the ligand buried inside (48). The last helix (helix 12) encompasses the carboxyl terminus where the AF-2 domain (F domain) is located. Although the sequence of the ligandbinding domain for different nuclear receptors diverges extensively, the domain seems to maintain a similar overall structure. The crystal structure of the holo-TH-binding domain is remarkably similar to that of the holo-retinoic acid @.A)-binding domain of the retinoic acid receptor y (RARy) (49). It also resembles that of steroid hormone receptors (50, 51). Interestingly, the apoRA-binding domain of RXRa (52) h as a slightly different structure. Although it has the 12 a-helices in similar locations as seen in RARy and TRa, its last helix (helix 12) stretches away from the rest of the protein, in contrast to the ligand-bound RARy and TRo, where helix 12 wraps tightly against the rest of the ligand-binding domain, with the ligand buried inside. This difference suggests that on ligand binding, there is a conformational change in the ligand-binding domain that leads to the inward folding of the helix 12. Indeed, biochemical studies have provided direct evidence for conformational changes in TRs induced by TH binding (38,39, 53 - 55). 2. DNA BINDING BY TRs The DNA-binding domain of TRs mediates specific recognition of the thyroid hormone response elements (TREs) present in TH response genes

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(38, 39). The DNA-binding domain consists of two adjacent Zns+ fingers, each of which contains two histidine and two cysteine residues that coordinate a Zn2+ ion in a tetrahedral configuration (56). This coordination of the Zn2+ ions by the two Zn 2+ fingers determines the overall structure of the DNA-binding domain. TRs can bind to DNA as monomers, homodimers, and heterodimers formed with other members of the thyroid-retinoid receptor subfamily (36, 38, 40,45). The most stable complexes are those formed by heterodimers of TRs and RXRs (9-cis-retinoic acid receptors). A number of in vitro and in viva studies support the view that TIQRXR heterodimers are the true mediators of the biological effects of TH (38, 44, 57- 61). Many of the natural occurring TREs consist of two direct repeats with a 4-bp spacing and of sequences highly similar to AGGTCA (62-64). The binding of such a TRE by a TR/RXR heterodimer involves the recognition of the 5’ repeat by the RXR and the 3’ repeat by the TR of the heterodimer (65, 66). Such a complex has been directly visualized in an X-ray structure of a cocrystal of a TRE made of two direct repeats separated by 4 bp and a heterodimer consisting of the DNA-binding domain of a TR and an RXR (56). 6.

Transcriptional

Regulation by TRs

TH can both up- and down-regulate gene expression in target tissues or cells. Thus, depending on the target promoters and/or cell types, TH-bound TRs can either activate or repress transcription. The vast majority of the known TH response genes are up-regulated by the hormone and most studies of receptor function have been on these up-regulated genes. Relatively little is known about how liganded TRs repress transcription. The discussions here focus only on the mechanisms involved in regulating the transcription of TH up-regulated genes. 1. ACTIVATIONVS.REPRESSION Transcriptional activation by TH requires the binding of TRs, most likely as heterodimers with RXRs, to TREs present in the regulatory regions of the TH response genes. The binding of TREs by TR/RXR heterodimers is, however, independent of TH both in solution and in chromatin (33, 60). TH appears to bind to TRs and trigger conformational changes in TRs that activate the receptor function (53 - 55). Various experiments have revealed that in the absence of TH, TRs repress the transcription of TRE-containing promoters. In the presence of TH, TRs enhance the transcription from these same promoters (38, 57, 60, 67). Thus, unliganded TRs seem to function as repressors. The binding of TH leads to not only the relief of the repression by unliganded TRs but often, if not always, additional activation of the target gene. Thus, TR/RXR functions as a

62 dual transcriptional regulator-a activator when bound by TH.

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repressor in the absence of the TH and an

2. COACTIVATORSANDCOREPRESSORS Both transcriptional repression by unliganded TRs and activation by Tabound TRs involve TR-interacting cofactors. Many such factors have been isolated based on their ability to interact with TRs in the presence or absence of T, or under both conditions (68 - 75). Th e corepressors bind preferentially or exclusively to unliganded TR while the coactivators have the opposite preference. a. Corepressors. Several corepressors have been cloned, including SMRT, N-CoR, SunCoR, and Alien (73, 75- 78). All are capable of interacting with unliganded TRs and have transcriptional silencing activity. Among them, SMRT and N-CoR have been studied extensively. These are two large proteins of about 2400 amino acids (SMRT also has a smaller form of 1500 amino acids, which was the original form identified). SMRT and N-CoR appear to be members of a related family, sharing considerable structural and sequence similarity. Both interact with the D domain of TRs. Interestingly, a mutation in the D domain of TR that abolishes the ability of the TR to suppress transcription also fails to interact with SMRT, again pointing out the potential involvement of SMRT in TR-mediated repression (76). In addition to the TR, these corepressors also interact with other members of the nuclear hormone superfamily. Both SMRT and N-CoR appear to form multimeric complexes through their interactions with corepressor Sin3. Sin3 in turn binds to histone deacetylases such as RPD3 (79, 80). Th us, the corepressor complexes are expected to be able to modulate the acetylation levels of histones and/or other proteins such as transcription factors. 6. Coactivators. The transcription coactivator proteins interact with nuclear receptors in the presence of the ligand. Often they can interact with many different nuclear hormone receptors and enhance the activities of these receptors when cotransfected with the receptors into mammalian tissue culture cells (68, 69, 71, 72). Further evidence for a role of coactivators in nuclear hormone receptor function has come from studies with gene knockout in mice. For example, inactivation of the coactivator SRC-1 in mice leads to partial resistance to steroid and thyroid hormones (81, 82). Like the corepressors, coactivators also form multimeric complexes containing other proteins and at least in the case of SRC-1 complex, an RNA (71, 74, 84). There are at least two types of coactivator complexes, those with histone acetyltransferase activity and those without. Many of the TR-interacting

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coactivators, such as CBP/p300, SRC-1, and P/CAF, are histone acetyltransferases. Interestingly, at least some coactivator complexes contain different TR-interacting coactivators with histone acetyltransferase activities (71, 83), although the functional significance of the inclusion of multiple histone acetyltransferases in a single complex is unclear. A TR-interaction coactivator complex has been characterized that apparently lacks any histone acetyltransferase activity (84-86). On the other hand, it contains many subunits homologous to proteins of the yeast mediator complex, which associates with RNA polymerase II through the carboxylterminal repeat domain of the polymerase large subunit. Thus, it is very likely that the recruitment of this complex by TH-bound TR/RXR will activate the target promoter directly through the RNA polymerase complex. 3. ROLE OF CHROMATIN Most of the functional studies of hormone receptors were carried out in vitro or by transient transfection experiments in tissue culture cells. However, genomic DNA in eukaryotic cells is associated with histones and other nuclear proteins and is assembled into chromatin. Growing evidence indicates that chromatin structure plays important roles in regulating gene transcription (87- 89). It is known that transcriptionally active chromosome domains have distinct structure and protein compositions compared to repressed chromatin. In addition, transcriptional activation is often accompanied by chromatin reorganization. Thus, to understand the mechanism of TR action, it is important to use properly chromatinized templates. The Xenopus oocyte offers a unique system to study transcription due to its large storage of basal transcription factors as well as many other proteins important for early embryogenesis, including histones for chromatin assembly. Furthermore, it is easy to introduce exogenous genes into oocytes through microinjection. DNA injected into the oocyte nucleus is assembled into chromatin (90). Interestingly, the types of chromatin formed differ depending on the forms of the injected DNA. When double-stranded promoter-containing plasmid DNAs are used, they are chromatinized in 5-6 hr with less welldefined nucleosome arrays such that the transcription from the promoters is at high levels (Fig. 4A). In contrast, when single-stranded plasmid DNAs are used, they are quickly replicated (l-2 h r) an d assembled into chromatin in a replication-coupled chromatin assembly pathway, mimicking the genomic chromatin assembly process in somatic cells (90). The resulting templates produce much lower levels of transcriptional activity (Fig. 4A). Thus, by using different forms of promoter-containing DNA, it is possible to study the transcriptional regulation under different chromatin conditions. Xenopus oocytes have only a very low level of endogenous TR, insufficient to affect the transcription of a TRE-containing promoter (59, 61, 91).

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FIG. 4. (A) Schematic diagram showing the difference in chromatin assembled from double-stranded (ds) and single-stranded (ss) plasmid DNA injected into a frog oocyte nucleus (NU) and the resulting transcriptional activity from the plasmid. (B) The histone deacetylase inhibitor TSA releases the transcriptional repression instigated by both chromatin and unliganded TR/ RXR. Groups of oocytes were first injected with (+) or without (-) TSA (5 ng/ml) or T, (50 nM) overnight. RNA was then prepared from the injected oocytes and the transcription from TH-dependent Xenopus TRpA promoter (pTRPA) was analyzed by primer extension (Expt.). The internal control represents the primer extension product derived from the endogenous storage pool of histone H4 mRNAs, serving as an RNA isolation and primer extension control. Levels of transcription from pTRPA were quantitated by phosphorimaging and were normalized against the internal control. The level of transcription from control ds pTRPA was designated as 1 (lane 1) and the other lanes were compared with it. (C) Liganded TR/RXR can relieve the repression

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However, when exogenous Xenopus TRs and RXRs are cointroduced into the oocytes, they can repress the transcription from the double-stranded template containing a TRE (Fig. 4B; compare lanes 1 and 3) (TRs function much less efficiently in the absence of injected RXRs) (59, 60). Transcriptional repression of the promoter by the TR occurs even when single-stranded DNA is injected to allow the promoter DNA to be assembled into chromatin in the replication-coupled chromatin assembly pathway (Fig. 4B; compare lanes 6 and 8). Independent of whether double- or single-stranded promoter DNA is used, addition of TH leads to transcriptional activation (59, 60). The final transcriptional activity is essentially identical when either single- or doublestranded promoter DNA is used (Fig. 4B; lanes 5 and lo), indicating that THbound TRs can overcome any repression incurred by chromatin. Therefore, the central question for transcriptional activation is how the TR/RXR heterodimer does so. Recent studies have suggested that two levels of chromatin remodeling contribute to this gene regulation-histone acetylation and chromatin disruption.

a. Regulation of Hi&one Acetylation Levels through H&one AcetyltransHistone acetylation has long been implicated in ferases and Deacetylases. influencing gene expression (92- 96). Histone acetylation occurs at the lysine residues on the amino-terminal tails of the histones, leading to the neutralization of the highly positively charged histone tails and reduced affinity toward DNA (97). These changes alter the nucleosomal conformation and chromatin accessibility, allowing easier excess of transcription factors to chromatin templates (98-100). C onsequently, histone acetylation can increase gene transcription. Many TR-interacting coactivators are histone acetyltransferases (68, 69, 71, 72). Thus, liganded TR/RXR heterodimers may activate gene transcription in part through the recruitment of such coactivator complexes to alter histone acetylation levels. The opposite appears to be true for transcriptional repression by unliganded TR/RXR. This is because the corepressors NCoR and SMRT, which bind to unliganded but not Ta-bound TR/RXR, have been shown to form a complex containing histone deacetylases (79 - 80). This deacetylase complex formation involves the binding of N-CoR or SMRT to

established in the presence of histone deacetylase xRPD3. Groups of oocytes were injected with dsDNA of pTRPA, and with or without increasing amounts of xRPD3 mRNA as indicated (0.5 ng, lanes 2 and 4; 1 ng, lanes 3 and 5). After 14 hr some oocytes were injected with (+) TR/RXR rnRNAs and treated with (+) Ta for 14 hr, before the levels of transcription were analyzed by primer extension. From Ref. (101); J. Wong, D. Patterton, A. Imhof, D. Guschin, Y.-B. Shi, and A. P. Wolfe (1998). EMBOJ. 17,520-534, by permission of Oxford University Press.

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the transcriptional repressor Sin3A, which in turn interacts with histone deacetylases such as RPD3. Studies on the regulation of Xenopus TRRA gene by TR/RXR have provided direct evidence for a role of histone acetylation in promoter activation (101). As described earlier, a double-stranded plasmid containing the TRRA promoter is highly transcribed when injected into the oocyte nucleus, and this transcription can be repressed by unliganded TR/RXR (Fig. 4B; lane 3). The addition of a specific inhibitor of histone deacetylase, trichostatin A (TSA), can reverse this repression, mimicking the addition of TH, while having no effect on the transcription in the absence of TR/RXR (Fig. 4B; lane 2), indicating the involvement of histone deacetylase in the repression by TR/ RXR. In addition, replication-coupled chromatin assembly of the singlestranded promoter injected into the oocyte nucleus also repressed gene transcription to a very low basal level (Fig. 4B; lane 6), which can be further repressed by unliganded TR/RXR (Fig. 4B; lane 8). TSA treatment relieves both types of repression (lanes 7 and 9), again just like the addition of T, (lane 10). In contrast to the deacetylase-blocking experiments, overexpression of the catalytic subunit RPD3 of a frog histone deacetylase complex leads to transcriptional repression of the promoter, and this repression can be reversed by the expression of TR/RXR in the presence of T, or the addition of TSA (Fig. 4C) (101). Th us, these two sets of complementary experiments strongly support a role of histone acetyltransferases/deacetylases in transcriptional regulation by TR/RXR. b. Chromatin Disruption. The frog oocyte system has also allowed us to investigate the structural changes of the plasmid minichromosome in response to gene regulation by TR/RXR. This has been carried out by using two assays (60, 61). The first is the plasmid DNA supercoiling assay. This assay is based on the fact that the wrapping of DNA around a nucleosome generates one negative supercoil on deproteinization. Any loss of nucleosomes or changes in the nucleosome-DNA wrapping will lead to alterations in superhelical density of the circular plasmid, which can be detected on an agarose gel (Fig. 5B). The second assay is based on the ability of micrococcal nuclease to digest preferentially the intemucleosomal spacer region. Thus, a partial digestion of the minichromosome with ordered nucleosome will provide a nucleosomal DNA ladder (Fig. 5C). Using these assays, we have found that T, binding to TRs bound to chromatinized templates causes the disruption of the ordered chromatin formed during replication-coupled chromatin assembly (Fig. 5B and 5C). Furthermore, this chromatin disruption occurs even when transcription is blocked by cx-amanitin (Fig. 5A and 5B). Thus, Ta-bound TR/RXR heterodimers can disrupt chromatin structure through an active process. These and other stud-

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FE. 5. The transcriptional activation by liganded TR/RXR is accompanied by extensive chromatin disruption. (A) The transcriptional activation by liganded TR/RXR can be inhibited by a-amanitin. Groups of 20 oocytes were injected with ss pTRRA (Fig. 4) (100 ng/ml, 23 nhoocyte) and TR/RXR mRNAs (100 ng/ml, 27 nboocyte) and treated with (+) TH as indicated; o-amanitin was coinjected with ssDNA at a concentration of 10 ng/ml. The transcription was analyzed by primer extension, and the internal control is the primer extension product from an unknown endogenous mRNA (60). (B) The DNA topology assay indicates that liganded TR/RXR also induces extensive chromatin disruption and that this chromatin disruption is not the by-product of processive transcription. The injections were the same as in A. The DNA was purified from each group and the topological status of the DNA was analyzed using chloroquine agarose gel. The top band in each lane represents the nicked form of the plasmid. (C) Liganded TR/RXR instigates extensive chromatin disruption. The oocytes were injected with ssDNA (100 ng/ml, 23 nhoocyte) and TR/RXR mRNAs (100 ng/ml each, 27 nhoocyte) and treated with (+) hormone and processed for micrococcal n&ease (MNase) assay. The amounts of MNase used are 0, 10, 5, and 2.5 U, respectively. Note the disruption of the orderly nucleosomal ladder in lanes 14-16 when both TH and TR/RXR are present. Adapted from Ref. (61); J. Wong, Y.-B. Shi, and A. P. by permission of Oxford University Press. Wolffe (1997). EMBOJ. 16,3158-3171,

+ -

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ies suggest that chromatin disruption and transcriptional activation are separable but both require the binding of transcriptionally active TR/RXR heterodimers (61). It is unclear how liganded TR/RXR disrupts chromatin. It does not appear to be due to changes in histone acetylation levels, because TSA treatment or overexpression of histone deacetylase RPD3 in frog oocytes has no detectable effects on chromatin structure as measured by these assays (101). On the other hand, studies ranging from yeast to mammals have suggested the involvement of the SNF/SWI family of proteins in chromatin remodeling (88, 89). Similar protein complexes may be involved in chromatin disruption by liganded TR/RXR. 4. PUTATIVE MODEL

OF TR ACTION

The cumulative information of transcriptional regulation by nuclear receptors has clearly indicated a complex, multistep, multicomponent nature of the underlying mechanism. A potential model for TR/RXR function is outlined in Fig. 6. In the absence of TH, TR/RXR recruits a corepressor and its associated deacetylase complex to the promoter, leading to histone deacetylation and transcriptional repression. On TH binding, the corepressor complex is dissociated and a coactivator complex is recruited to the promoter. This recruitment may lead to histone acetylation (102), chromatin disruption, and transcriptional activation. Although the studies so far are supportive of an important role of histone acetylation in transcriptional activation, other pathways are likely involved. First of all, histone acetyltransferases can also acetylate other proteins, such as general transcription factors (103), and other transcription factors, such as

, FIG. 6. A proposed mechanism for transcriptional regulation by TRs. TR functions as a heterodimer with RXR. In the absence of TH, the heterodimer represses gene transcription, likely through the recruitment of a corepressor complex containing the corepressor N-CoR or SMRT. The corepressor interacts with Sin3A, which in turn recruits a histone deacetylase such as RPD3 to deacetylate histones, thus affecting transcription. On binding by TH, a conformational change takes place in the heterodimer, which may be responsible for the release of the corepressor complex. Liganded TR also recruits a coactivator complex containing coactivators such as SRC-1, CBP/pSOO, and PCAF, and/or the DRIP/TRAP coactivator complex. The DRIP/TRAP complex may contact RNA polymerase directly to activate gene transcription. On the other hand, the SRC-1, CBP/pSOO, and PCAF complexes may function through chromatin modification because they possess histone acetylase activity. In addition, transcriptional activation is associated with chromatin disruption, which may be due to the recruitment of chromatin remodeling machinery by TR/RXR. This chromatin disruption may be necessary for transcriptional activation by TR/RXR. The corepressor and coactivator complexes as well as the chromatin remodeling machinery are multicomponent complexes and only a limited number of subunits are shown for simplicity.

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p53 (104). Thus they may also affect transcription independent of histone acetylation. Furthermore, there is evidence that corepressors such as N-CoR can interact with basal transcription factors and inhibit transcription independent of their ability to recruit deacetylases. Likewise, the DRIP/TRAP coactivator complex can interact with the transcriptional machinery directly. In addition, chromatin disruption as detected by micrococcal nuclease digestion or plasmid DNA supercoiling assay appears to be necessary but not sufficient for transcriptional activation by liganded TR/RXR (61). On the other hand, overexpression of RPD3 or TSA treatment has little effect on chromatin structure based on two assays, despite their strong influence on transcription (101). Finally, there are many other TR-interacting proteins of yet unknown function, and they are likely to affect transcription through distinct mechanisms. Thus, further studies on the different cofactors and characterization of the nature of chromatin disruption and histone modification are needed to clarify the exact mechanism governing transcriptional regulation by TR/RXR.

IV. Thyroid Hormone-Dependent Morphological and Cellular Changes during Intestinal Remodeling The presence of a free-living larval form necessitates the remodeling of many existing organs for adult use as an animal changes often from being aquatic and herbivorous to being terrestrial and carnivorous. The intestine is one such organ that has been under extensive investigation (2,105, 106).

A. Larval Intestine Intestinal tissues are derived from the endoderm (the epithelium), the mesoderm (the connective tissue, muscles, and serosa), and the neural crest (the nerve). The epithelium is the tissue responsible for the principal physiological function of the organ, i.e., food digestion and absorption. It initially appears as a solid cell mass containing large amounts of yolk granules, which rapidly decrease after hatching (21, 107). This multilayered endoderm then gradually proliferates and differentiates into a simple columnar cell layer, the larval or primary epithelium (Fig. 7) (108). Unlike higher vertebrates, there are no villi and crypts in anuran tadpoles. There is a single fold, the typhlosole, in the anterior one-third of the small intestine in X. Zuevis (Fig. 8A), which is absent in other larval anurans. In addition, the differentiated larval epithelial cells are capable of dividing independently of their location within the typhlosole (109), in contrast to adult frogs or higher vertebrates, where the proliferating cells are less or not differentiated and are localized in the trough of an epithelial fold (frog) or crypt (higher vertebrates). Finally, there has been

\ Multi-layered endodermal cells

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FIG. 7. Comparison of intestinal development in Xenopus la&s and higher vertebrates. The primordial endodermal cells first form a multilayered cell mass. The endodennal cells are then converted into a monolayer of columnar epithelial (EP) cells tightly associated with the connective tissue (CT), which is derived from the mesoderm, through a basement membrane (basal lamina). Further development in amphibians diverges from that in higher vertebrates. In the latter, the columnar cells develop into multiply folded epithelium surrounded by elaborate connective tissue (stippled area) and muscles (derived from the mesoderm) (hatched area; MU). In Xenqnu.s, the epithelium remains as a simple tubular structure with only a single fold, the typhlosole. The differentiated epithelial cells in both cases have numerous microvilli in the brush border (bb) on the luminal surface for efficient nutrient processing and absorption. The Xenqnx intestine then undergoes a second phase of development that results in the replacement of larval epithelium with adult epithelium as well as extensive development of connective tissues and muscles.

Mesoderm

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FIG. 8. Metamorphic transformations of the anterior intestine of Xenopus tadpoles, stained with hematoxylin (A, C-F) or methyl green-pyronin Y (B), or labeled by bromodeoxyuridine immunohistochemistry (G). (A) Cross-section of the larval intestine at stage 54. The larval epithelium (le) is a monolayer of columnar cells. The layer of connective tissue (ct) is thin except for the typhlosole (Ty). The muscle (m) is also thin. (B) Appearance of small islets of the adult epithelium between the larval epithelium and the connective tissue (arrows) at stage 60. (C, D) Development of adult epithelial( ae ) 1s1e t s and degeneration of the larval epithelium at stage 61. Mitotic cells are numerous in the islets (arrows in D). (E) F ormation of intestinal fold (F) at stage 62. (F) Many intestinal folds developed at stage 64. The adult epithelium differentiates into a simple columnar epithelium. (G) Adult intestine at the completion of metamorphosis at stage 66. Labeled nuclei of the adult epithelium (arrows) are localized in troughs of the intestinal folds. Bars, 50 km.

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no evidence for the existence of undifferentiated stem cells in the primary tadpole epithelium similar to those in the mammalian small intestine (110).

B. Metamorphic

Transformations

Around the onset of metamorphic climax the long larval small intestine begins to shorten, and this process continues until the end of metamorphosis (108, 111, 112). Following the beginning of this shortening, the morphogenesis of the intestinal folds occurs. These appear as several circular folds that run longitudinally and are straight along the gut axis, gradually increasing in number and height, and finally being modified into longitudinally zigzagged folds. Accompanying those anatomical changes are the transformations of all major tissues within the intestine. 1. EPITHELIAL

TRANSFORMATION

The epithelial transition from larval to adult form of the amphibian intestine can be divided into two processes, degeneration of the larval (primary) epithelium and development of the adult (secondary) epithelium (Fig. 8). Degenerative cellular changes occur around the onset of metamorphic climax. For example, the microvilli composing the brush border decrease in number and height, whereas lysosomes increase in number and in hydrolytic activity (113,114). At the cellular level, the larval epithelial cells die through apoptosis (programmed cell death). The resulting membrane-bound cellular and nuclear fragments, i.e., apoptotic bodies, are at least partially phagocytosed by macrophages (115). The macrophages are eventually extruded into the lumen while still retaining the apoptotic bodies. Concurrent with larval cell death, the adult epithelial cells are detected at the epithelial-connective tissue interface as small islets consisting of undifferentiated epithelial cells (Fig. SB). It is still unclear whether these adult epithelium cells are derived from a pool of undifferentiated cells in the larval epithelium (113) or transformed from differentiated larval cells (109). In any case, the primordia rapidly grow into the connective tissue through active cell proliferation and differentiate to form the secondary epithelium, replacing the degenerating primary epithelium (112,116), (Fig. 8D). With the progression of fold formation (Fig. SE and 8F), proliferative cells of the adult epithelium become localized in the trough of the folds (Fig. 8G) like those in the mammalian crypts (117), in contrast to the primary epithelium (see above). Thus, during metamorphosis, the larval intestine transforms into a structure with a cell renewal system analogous to that in mammals (Fig. 7) (110). 2. CONNECTIVE

TISSUE REMODELING

Growing evidence suggests a close relationship between the epithelium and the connective tissue during intestinal remodeling. When the epithelial

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transition from the larval to adult form begins, the connective tissue suddenly increases in mitotic activity, cell number, and thickness (Fig. 8C). The connective tissue at this time consists of various types of cells, such as immature mesenchymal cells, fibroblasts, macrophages, and mast cells (106, 115, 118). Furthermore, remarkable changes in the connective tissue occur close to the epithelium. When the larval epithelium begins to degenerate, the basal lamina, which is thin through out the larval period, becomes thick in the entire region beneath the epithelium and remains thick until the larval epithelium disappears. In addition, throughout the thick basal lamina, fibroblasts, that possess well-developed rough endoplasmic reticulum often contact the epithelial cells. These cell contacts are most frequently observed around the primordia of the adult epithelium when the epithelial cells most actively proliferate. These observations suggest that the thickening of the basal lamina and the cell contacts are related to the larval epithelial cell death and the adult epithelial cell proliferation, respectively. At later stages, the basal lamina becomes thin beneath the adult epitheliurn. In addition, the cell contacts and all cell types of the connective tissue, except fibroblasts, decrease in number. By the end of metamorphosis, almost all of the connective tissue cells are ordinary fibroblasts. In the trough of the epithelial folds, these fibroblasts are close to the epithelium and aligned parallel to the curvature of the epithelial basal surface. This structure is similar to the subepithelial fibroblastic sheath reported to be present in the crypt of the mammalian small intestine. This sheath has been thought to play important roles in epithelial cell proliferation and/or differentiation (119). 3. OTHER TISSUES Very little information exists on other intestinal tissues. One such tissue, the muscle, becomes considerably thicker (Fig. 7) during metamorphosis, primarily due to the increase of the inner circular muscle layer. In contrast, after metamorphosis, the thickening is mainly due to that of the outer longitudinal muscle layer. Consequently, in adult Xenopus, the thickness of each layer is almost the same (220). Replacement of the neurons from larval to adult types in the myenteric plexus of the bullfrog intestine has been also observed during metamorphosis (121).

C. Organ Autonomous

Response to TH

Like other organs, the intestine can also be induced to undergo precocious remodeling by treating premetamorphic tadpoles with TH. In addition, this regulation is organ autonomous, for intestinal fragments cultured in vitro can be induced to metamorphose by including TH in the culturing medium. The changes induced by TH treatment mimics those in viva, that is, intestinal length reduction, degeneration of the larval epithelium, and devel-

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opment of the adult epithelium (Fig. 9) (122,123). Interestingly, in the organ cultures, the development of the adult epithelium requires the addition of glucocorticoid and insulin in addition to TH, whereas the larval epithelial degeneration can occur in the presence of TH alone (122). Thus, even though TH acts directly on the intestine, other nonintestinal factors also influence intestinal remodeling in viva. Organ culture studies have also revealed an important role of the connective tissue in adult epithelial development. When X. Zaevis anterior small intestine, which contains the connective tissue-rich typhlosole, is treated with TH in u&-o, it undergoes both larval epithelial cell death and adult epithelial development (Fig. 9A). The adult epithelium then differentiates into a simple columnar epithelium expressing intestinal fatty acid-binding protein (IFABP) (Fig. 9B), wh ic h marks the differentiation of absorptive epithelial cells (124). However, when posterior small intestine, which has little connective tissue, is cultured with TH, only cell death is reproduced (Fig. 9C). On the other hand, coculturing either posterior or anterior epithelium with anterior connective tissue in the presence of TH leads to both larval cell death and adult tissue development (125). Culturing epithelium alone only produces TH-dependent epithelial apoptosis. Thus, cell death appears to be tis-

FIG. 9. Larval small intestine of the Xenopus tadpole undergoes TH-dependent changes in organ cultures; stained with hematoxylin (A, C), or intestinal fatty acid-binding protein immunohistochemistry (B). (A, B) Explants of the anterior intestine cultured with T, insulin, and cortisol. On day 5 of cultivation (A), a typical islet (is) grows by rapid cell proliferation (arrows). The connective tissue (ct) develops. On day 7 (B), th e ep’ rth eliurn (e) differentiates into a simple columnar epithelium possessing intestinal fatty acid-binding protein. (C) Explant of the posterior intestine on day 5 in the presence of TH. No islets are formed. The number of epithelial cells is small. Connective tissue cells are few. Bars, 20 urn.

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sue autonomous whereas adult tissue development requires cell-cell and/or cell-extracellular matrix (ECM) interactions. More recently, by culturing isolated larval intestinal epithelial or fibroblastic cells in vitro, we have shown that these cells respond cell autonomously to TH in vitro. TH stimulates the proliferation of both cell types but causes apoptosis specifically for epithelial cells, leading to an increase in fibroblast cell number and a precipitous drop in epithelial cell number

b

bP 4,361 2322~ 2027'

A Fit?, +T3

564-

1250 0

1

2

3

4

5

Culture Time (Day) FIG. 10. (A) Contrasting effects of thyroid hormone on tadpole intestinal epithelial (Ep) and fibroblastic (Fib) cells. The fibroblasts and epithelial cells were isolated from stages 57158 of tadpole small intestine and then cultured on plastic dishes in the presence of 10% TX-depleted fetal bovine serum in the presence or absence of 100 nM T,. The live cells were counted daily by trypan blue staining. (B) The TH-induced intestinal epithelial cell death produces a typical nucleosomal-sized DNA ladder. Tadpole epithelial cells were cultured as in A for 1 day and total DNA was isolated and analyzed on a 1% agarose gel.

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(Fig. 10A) (126, 127). This TH-induced cell death is typical of mammalian apoptosis both in the requirement of caspases and nucleases and in the generation of a nucleosome-size DNA ladder (Fig. 10B) (126, 127). The cell death in vitro has apoptotic morphology similar to that seen during natural development. Thus, at least for larval epithelial cells, TH functions by directly inducing a cell death program within the target cells. However, it is possible that extracellular events can modulate this process as well (see below).

V. TR Expression and Function during Intestinal Remodeling TRs are the presumed mediators of the causative effects of TH on anuran metamorphosis. Their function in vivo depends on the presence of RXRs. Thus, extensive studies have been carried out to analyze the expression of both TR and RXR genes in various organs in both X. Zaevis and R. catesbeiana. In general, strong correlations exist between TR and RXR expression and organ transformations (based on studies of the mRNA and to a lesser extent, protein levels) (91, 128-133).

A. Correlation

of Receptor mRNA Levels

with Intestinal Remodeling In the Xenopus intestine, the TRB and RXRy genes are up-regulated during intestinal remodeling (Fig. llA), paralleling the rise in plasma TH levels (19). Both TRa and RXRo mRNAs are expressed at similar or higher levels compared to TRB and RXRy, respectively, during intestinal development, although their expression does not change substantially (Fig. 11A) (59). Expression of the third RXR gene, RXRB, has not been studied in the intestine. These results indicate that all receptors are likely to be present during intestinal remodeling to mediate the organ autonomous effects of TH.

B. TH Activation of TRP Genes The Xenopus TRB genes are regulated by TH. This regulation appears to be ubiquitous. Furthermore, the up-regulation occurs within a few hours of TH treatment of premetamorphic tadpoles and is independent of new protein synthesis, arguing that the TRB genes are regulated by TRs (134-136). Analysis of the TRB genes shows that Xenopus TRB and TRBB genes span at least 70 kb of genomic DNA. Each gene has two alternative promoters. One of the promoters is expressed at low but constitutive levels whereas the other is highly expressed only in the presence of TH (134, 135). Analysis of

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54

62

58

66

Developmental Stages

B connective tissue and muscle cell differentiation

I

connective tissue and muscle cell proliferation

Connective tissue

L__

Stage

54

56

58

60

61

+

62

63 epithelial

64

66

molphogenesis

,y’

FIG. 11. Correlation of receptor expression with intestinal remodeling. (A) Coordinated regulation of TR and RXR genes in the intestine. The mRNA levels were quantified from Northern blots of total intestinal RNA. Note that both TRf3 and RXRy are up-regulated during metamorphosis, whereas TRcx and RXRa expression remain fairly constant. However, the absolute levels of TRa and RXRa mRNAs appear to be higher than those of TRS and RXRy mRNAs, respectively (59). (B) TRS mRNA levels correlate with tissue-specific transformations in the intestine [based on in situ data of Shi and Ishizuya-Oka (141)]. For clarity, the mRNA levels in different tissues are plotted on different scales.

the inducible promoter reveals that both genes contain at least a strong TFtE consisting of two nearly perfect direct repeats of AGGTCA separated by 4 bp (60, 64, 137,138), which is likely responsible for the up-regulation of the TFtp genes by TH during metamorphosis in different organs. Consistent with the above data, up-regulation of the TRP genes in the in-

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testine is not only independent of protein synthesis (139) but also is cell autonomous, because isolated larval epithelial cells are induced to expressed TRR genes when treated with TH in cell culture in vitro (T Amano and Y.-B. Shi, unpublished observation).

C. Cell Type-Dependent Temporal Regulation of TRP Genes Different tissues within the intestine undergo distinct changes at different developmental stages during metamorphosis. How such tissue specificity is controlled remains to be investigated. Several factors could contribute to this (140). First, TH levels within individual cells may be regulated through import and export controls. Second, cellular free TH levels may depend on the levels of cytosolic TH-binding proteins and intracellular TH metabolism. Finally, TR levels may vary in a tissue-dependent manner. By using in situ hybridization, we have carried out a detailed analysis of TRR gene expression during intestinal metamorphosis (141). Little or no TRP mRNA is present in any tissues within the premetamorphic intestine. Around stage 5 7, TRP mRNA becomes detectable in some larval epithelial cells, and by stage 59, just prior to the onset of larval epithelial apoptosis, all larval epithelial cells express high levels of TRR mRNA (Fig. 11B). Subsequently, as apoptosis takes place in the larval epithelium, the TRR mRNA levels are down-regulated (stage 61). Similarly, TRP expression is high in the proliferating adult epithelial cells as soon as they can be identified as cell islets, between the larval epithelium and the connective tissue. The TRR expression in the adult epithelial cells remains high until stage 62, when adult epithelial cell differentiation begins. Likewise, in the connective tissue and muscles, TRR mRNA levels are high when cells proliferate but are down-regulated as these cells differentiate to form the adult connective tissue and muscles, respectively (Fig. 11B). Thus, TRR appears to be involved in promoting both cell proliferation and apoptosis, depending on the cell types in which they are expressed. In particular, high levels of TRR mRNA appear to be incompatible with high degrees of differentiation. In the differentiated larval epithelial cells, high levels of TRR expression are associated with apoptosis. On the other hand, as the cells of the adult epithelium, connective tissue, and muscles begin to differentiate, they need to shut down their TRR expression to prevent likely deleterious (apoptotic) consequences associated with high levels of TRl3 mRNA. Although is it unknown whether TRo and RXR genes have similar cell typespecific regulations, the results suggest that TR levels may control the temporal regulation of metamorphosis and that cell type specificity of tissue transformations is determined by genes other than TRs.

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VI. Thyroid Hormone Response Genes in the Intestine Like other tissue remodeling processes, intestinal metamorphosis involves many genes at various stages of development. The binding of TH to TRs will likely lead to the activation of some genes and repression of others. For simplicity, those genes that are regulated within 24 hr of TH treatment of premetamorphic tadpoles are referred to as early TH response genes, whereas the later ones are the late TH response genes. The direct TH response genes are those that are regulated at the transcriptional level by TRs. They may be either early or late response genes; in the latter case, the synthesis of another protein(s) is required for TR to regulate the target genes, consequently requiring longer TH treatment. To understand the molecular pathways leading to intestinal metamorphosis, a key step is to isolate and functionally characterize these various TH response genes.

A.

Early TH Response Genes

A differential screen is a powerful method to isolate genes whose mRNA levels differ in two samples (142). To isolate low-abundance mRNA species, various polymerase chain reaction (PCR)-based methods have been developed to remove selectively genes that are expressed at similar levels in the two samples being compared, while enriching and amplifying the genes whose mRNA levels differ in the two samples. One such method has been applied to amphibian metamorphosis in several organs (136, 143). This method has been used to identify early TH response genes in the remodeling intestine. This was done with intestinal RNAs isolated from stage 54 premetamorphic tadpoles that had been treated for 18 hr with or without 5 n MT,, close to the endogenous plasma T, concentration at the metamorphic climax (stage 62) (19). A total of 22 up-regulated and one down-regulated genes was isolated (139). Most of the genes respond to T, treatment very quickly (within a few hours) and their regulation by T, appears to be independent of new protein synthesis (Table I), suggesting that they are direct TH response genes and represent the first wave of gene regulation induced by TH. The identities of many of the up-regulated genes have been revealed through sequence analysis (Table I). Several of the direct response genes encode transcription factors and thus are likely involved in directly activating or repressing transcription of intermediate and/or late TH response genes. In addition, several genes encoding proteins varying from a transmembrane amino acid transporter to extracellular enzymes are also found to be among the early response genes in the intestine. These results suggest that TH simultaneously induces many intra- and extracellular events, which in turn cooperate to effect intestinal remodeling.

Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated Down-regulated Down-regulated

TH/bZip Zn finger TF (BTEB)

Stromelysin-3 Sonic hedgehog Na+/PO, cotransporter IU12 Nonhepatic arginase

Collagenase3 Collagenase-4 Gelatinase-A

Pleiotrophic factor BMP-4

Ubiquitin-activating Collagens Calbindin Villin IFABP

direct direct direct direct

Late Late Late Late Late

Late Late

Late Late Late

Early, direct Early, direct Early, direct Early Early

Early, Early, Early, Early, Early

Kinetics”

signaling signaling Protein degradation ECM proteins Calcium metabolism Brush border structural protein Fatty acid metabolism

Cell-cell Cell-cell

144 144 217 145 123

144 144

139,177,212 139,177,212 139, 175,212

ECM remodeling ECM remodeling ECM remodeling

139, 211, 212 139, 212 139, 141, 212 139,210,212 139,212 139,175,212 204 139,212,214 139,212,215,216 139,212,213

regulation regulation regulation regulation regulation

Ref.

ECM remodeling Cell-cell signaling PO, transport Amino acid transport Proline biosynthesis

Transcriptional Transcriptional Transcriptional Transcriptional Transcriptional

Possible function

“A directresponseindicates that the regulation by TH is resistant to prokin synthesis inhibition; “early”or “late” refers to regulation by TH in the intestine requiring less or more than 1 day of treatment, respectively.

enzyme El

Response to TH

Protein encoded

Tw NFI bZip (Fra-2)

TABLE I THYROID HOHMONE RESPONSE GENES IN Xenopus INTESTINE

a2

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B Stage

64 56 66 60 62 64 66

54565860

626466

56 54 58 62 66

012357

0

1

2

3

5

FIG. 12. Development of expression of three TH response genes during natural (A) and THinduced (B) metamorphosis. Intestinal RNA was isolated from tadpoles at different stages, or from stages 52/54 tadpoles treated with 5 nMT, for the indicated days, and subjected to North ern blot hybridization. TH/bZip and NFI-B are early response genes (210, 211), and IFABP is a late response gene (123). Adapted from Refs. 123,210, and 211 (Temporal and spatial regulation of a putative transcriptional repressor implicates it as playing a role in thyroid hormone-dependent organ transformation; A. Ishizuya-Oka, S. Ueda, and Y.-B. Shi, Deu. Genet. Copyright 0 1997, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)

The first indication that these genes are likely to play important roles in tissue remodeling comes from their dramatic regulation in the intestine during both natural and TH-induced metamorphosis (e.g., Fig. 12 for TH/bZip and NFI-B). Developmentally, these early response genes fall into three general classes (136, 139). The genes in the first class [for example, TRP, a basic and leucine zipper (TH/bZip) motif-containing transcription factor (Fig. 12), the extracellular matrix-degrading metalloproteinase stromelysin-3 (Table I)] are expressed strongly only at the climax of intestinal remodeling (around

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stages 60 - 62). Much lower levels of expression of these genes are present in pre- or postmetamorphic intestine. The second class of genes [for example, the NFI family of transcription factors (Fig. 12) (Table I)] are activated during metamorphosis and their expression remains high in postmetamorphic frog intestine. Finally, two genes, including the Na+/PO; cotransporter (Table I), are in the third class, They are expressed at high levels immediately before or after the climax or metamorphosis but minimally at the actual climax. Interestingly, most of the direct response genes are also regulated by TH in many other organs, even though different organs undergo vastly different transformations. For example, TRR genes are up-regulated by TH in all of the organs analyzed so far, including tail and limb, even though the tail resorbs and the limb undergoes de novo development. This contrasts with the late TH response genes, which are often intestine specific (see below and Table I). Thus, the ubiquitous early TH response genes are likely to function together with preexisting, intestine-specific factors to activate the downstream intestine-specific metamorphic pathways. For an unknown reason, there appears to be relatively few early response genes that are down-regulated by TH within 24 hr in the intestine (only one was isolated) (139). Th is is similar to the findings from similar screens in other Xenopus organs, with the exception of the brain (136, 143). At present, much less is known about the down-regulated genes. It is difficult to assess their importance during metamorphosis.

B. Late TH Response Genes A PCR-based subtractive screen has also been carried out to isolate intestinal genes regulated by a 4-day TH treatment of stage 57 premetamorphic Xenopus Zaevis tadpoles (144). This led to the identification of over 20 genes that are induced by TH. Most of these genes are distinct from the early TH response genes described above. Although no detailed kinetic study of their TH induction has been carried out, they are likely to be late TH response genes. Like the early response genes, these TH-induced genes also belong to distinct classes (144). They encode transcription factors, collagens, components of the ubiquitin proteasome pathway, morphogenetic and growth factors, etc. (Table I). They can potentially participate in the regulation of larval epithelial cell death and/or adult cell proliferation and differentiation at a step(s) downstream of the direct, early TH response genes. Through various means, a number of other genes have been identified as late TH response genes in the intestine. Many of these genes are specific to intestinal epithelium, such as the genes encoding intestinal fatty acid-binding protein (Fig. 12) and villin (an epithelial brush border structural protein).

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Interestingly, both IFABP and villin are initially down-regulated by TH as the larval epithelial cells undergo apoptosis. Subsequently, they are up-regulated again as adult epithelium develops (123,124,145,146). Presumably, many other epithelium-specific genes behave similarly. The regulation of these genes is likely reflective of the terminal changes of the intestinal epithelium during metamorphosis.

VII. Functions of TH Response Genes: Implication from Studies on Matrix Metalloproteinases Among the early and late TH response genes are those encoding matrix metalloproteinases (MMPs; Table I). MMPs are extracellular enzymes that are capable of degrading various components of the ECM (147-150). This growing family of enzymes includes collagenases, gelatinases, and stromelysins, each of which has different but often overlapping substrate specificity (151, 152). They are secreted into the ECM as proenzymes with the exceptions of stromelysin-3 (ST3), which appears to be secreted in the active form (153), and membrane-type MMPs, which exist as membrane-bound active enzymes. The proenzymes are enzymatically inactive owing to the presence of a propeptide (149, 154-157). The proenzymes can be activated in the ECM or on the cell surface through the proteolytic removal of the propeptide (155-158). Th e mature enzyme has a catalytic domain at the N-terminal half of the protein, which contains a conserved Zn2+ binding site. Once activated, these MMPs can degrade components of ECM. Thus differential expression and activation of various MMPs can result in specific remodeling of the ECM. Many MMPs have been implicated to play a role in the metastasis of cancerous cells owing to the up-regulation of their expression in this process and the fact that metastasis requires extensive ECM degradation/modification (148, 159-161). In addition, the developmental expression profiles of MMP genes suggest that MMPs are also critical players in a number of developmental processes (162-165). The importance of MMPs in development has been substantiated by gene knockout studies in mice (166-168). The activation of MMP genes by TH during metamorphosis suggests a role for these extracellular enzymes in tissue remodeling. Such an idea was first proposed over 30 years ago, based on the drastic increase in collagen degradation activity in the resorbing tadpole tail (169), which led to the identification of the first MMP, the collagenase. Further studies on the expression profiles of MMP genes during X. laevis metamorphosis suggest that MMPs participate in ECM remodeling to influence tissue transformation.

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A. ECM Remodeling during Intestinal Metamorphosis The intestinal epithelium is separated from the connective tissue by a special ECM, the basal lamina, which is composed of laminin, entactin, collagens, and proteoglycans, etc. (170, 171). In premetamorphic X. Zaevis tadpoles, the intestinal basal lamina is a continuous but thin structure separating the connective tissue and the epithelium. As the larval epithelium undergoes degeneration it becomes much thicker and multiply folded, and remains thick until the larval epithelium finally disappears, i.e., along with the massive epithelial apoptosis (118, 172). Interestingly, the basal lamina appears to be much more permeable at the climax of metamorphosis (stage 60- 63), in spite of the increased thickness. This permeability is reflected by the frequently observed migration of macrophages across the basal lamina into the degenerating epithelium, where they participate in the removal of degenerated epithelial cells. In addition, extensive contacts are present between the proliferating adult epithelial cells and the fibroblasts on the other side of the basal lamina (118, 125). L arval epithelial cell removal is essentially complete around stages 62-63. After stage 63, with the progress of intestinal morphogenesis, i.e., intestinal fold formation, the adult epithelial cells differentiate. Concurrently, the basal lamina become thin and flat again, underlining the differentiating adult epithelium. The mechanism governing the ECM remodeling are unclear at present. Several factors may contribute to it. First, as the larval intestine reduces its length, the ECM may increase in thickness due to contraction. Second, synthesis of new ECM components will lead to changes in the composition and nature of the basal lamina. This is supported by the finding that at least some collagen-encoding genes are activated by TH during intestinal metamorphosis (Table I) (144). Finally, ECM degradation by MMPs is likely to be an important aspect.

B. Regulation of MMP Genes during Metamorphosis Several frog MMP genes have been found to be up-regulated by TH during metamorphosis. Among them, the collagenase-1 (Coil) gene of R. cutesbeiana and stromelysin-3 (ST3) of X. Zuevis are early, direct TH response genes (173 - 175). The Xenopus Co/3 was isolated as an early gene in the tail, but its regulation in the intestine by physiological concentrations of TH requires more than 1 day of TH treatment, similar to Xenopus Co/4 (176,177). Thus, Co/3 and Co/4 are likely late TH response genes. In addition, Northem blot analyses with human gelatinase A (GeZA) and ST1 cDNA probes suggest that Xenopus GeZA is a late TH response gene whereas the expression of Xenops ST1 changes little during intestinal remodeling (175). Spatial and temporal expression ofxenopus MMP genes implicates different function for different MMPs.

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1. ST3 EXPRESSIONISASSOCIATED WITHTH-INDUCEDCELLDEATH

Among the known amphibian MMPs, the Xenopus stromelysin-3 gene is of particular interest. This is in part because its human homolog is expressed in most human carcinomas (178, 179). Furthermore, both the human and mouse ST3 genes are expressed during development in tissues where cell death takes place (178,180, 181). These results suggest that ST3 is involved in both apoptosis and cell migration, the processes that also occur during frog intestinal remodeling. The developmental expression of ST3 mRNA correlates strongly with organ-specific metamorphosis (Fig. 13A) (175). Furthermore, ST3 expression is temporally correlated with the stages when cell death occurs in all organs analyzed, and the levels of its mRNA appear to correlate with the extents of cell death in these organs (175). More importantly, the activation of the ST3 gene occurs at the onset of or prior to cell death. Thus, in the intestine, high levels of ST3 mRNA are already present at stage 60, when larval apoptosis is first detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (Fig. 13A) (182, 183). These results suggest that ST3 is involved in regulation of larval cell death. However, because adult epithelial proliferation in the intestine is rapid around stages 60-62 and differentiation also starts around stage 62, it is possible that ST3 may also be involved in adult epithelial development. In situ hybridization analysis has revealed that ST3 expression is spatially correlated with apoptosis in different organs during Xenopus metamorphosis (182-185). In the intestine, ST3 mRNA is localized in the fibroblastic cells adjacent to the epithelium, but not actually in the apoptotic cells of the intestine (Fig. 13B) (175, 183). Thus, as an MMP, ST3 influences epithelial apoptosis by modifying the ECM, in particular the basal lamina separating the STS-expressing fibroblasts and the dying epithelial cells. Consistent with this, ST3 expression is not only temporally but also spatially correlated with the modification of the basal lamina (the ECM that separates the epithelium and connective tissue) (Fig. 14) (182). In both pre- and postmetamorphic intestine, fibroblasts just beneath the thin and flat basal lamina do not express ST3. However, during metamorphosis, as STS-expressing fibroblasts increase in number near the muscular layers (Fig. 14A), the intestinal basal lamina adjacent to them begins to fold (Fig. 14B and 14C). Then at the highest levels of ST3 mRNA just beneath the degenerating epithelium (Fig. 14D), the basal lamina attains its maximal thickness (Fig. 14E) but becomes more permeable, as described above. It is, therefore, possible that ST3 causes specific degradation/cleavage of certain ECM components, aiding in the folding of the ECM and resulting in increased permeability.

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Such modifications may facilitate larval epithelial apoptosis and adult epithelial development. Although such a function for ST3 remains to be proved, it is interesting to note that overexpression of the MMP stromelysin-1 in the mammary gland of transgenic mice leads to similar changes in the basal lamina, alters mammary gland morphogenesis, and induces apoptosis (185a187). Furthermore, homologous deletion of ST3 results in mice with reduced incidence of carcinogen-induced tumors whereas overexpression of ST3 in cultured cells leads to increased tumor formation when the ST3-expressing cells are injected into mice (188,189), a g ain supporting a role of ST3 in ECM remodeling to influence cell behavior. 2. DIFFERENTIALEXPRESSIONOFDIFFERENTMMPGENES IMPLICATESDIFFERENTFUNCTIONS

The multicomponent nature of the ECM suggests the participation of different MMPs in its remodeling and degradation during metamorphosis. Thus, it is not surprising to find multiple MMP genes up-regulated during metamorphosis. Northern blot and in situ hybridization analysis reveal unique but overlapping expression profiles for the five Xenopus MMP genes in the intestine (Fig. 13) (175,177). ST3 is the most up-regulated gene whereas ST1 has relatively constant mRNA levels throughout development (Fig. 13A). The two collagenase genes have only a few fold higher levels of mRNA in the metamorphosing intestine around stage 62 compared with those at other stages (Fig. 13A). The putative Xmopus GelA has an expression profile that is most similar to that of ST3 (Fig. 13A). As described above, ST3 is expressed throughout the connective tissue underlying the remodeling basal lamina and degenerating larval epithelium during intestinal metamorphosis (Figs. 13B and 14) (182, 183). In contrast, Co13 is expressed only in sporadic regions/cells within the connective tissue, and little Co14 mRNA can be detected by in situ hybridization (Fig. 13B). Thus, Co13 and Co14 may be involved in connective tissue remodeling to facilitate adult intestinal morphogenesis and play at most a minor role in larval epithelial degeneration. GelA expression in the intestine resembles temporally that of ST3 (Fig. 13A). However, high levels of GelA mRNA in the intestine are reached later (at stage 62) than th ose of ST3 (at stage 60; Fig. 13A). This difference is also observed when premetamorphic tadpoles are treated with T, to induce precocious intestinal remodeling (175). Although the Xenopus ST3 gene is upregulated very quickly (within a few hours) by the T, treatment, the GelA gene up-regulation is detectable only after a treatment of 3 days or longer. As described above, stage 62 is the time when intestinal epithelial cell death is mostly complete and adult epithelial differentiation is taking place. The activation of GelA, and to a lesser extent Co14 (Fig. 13A), at this stage in the in-

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co13

co14

GelA

ST3

FIG. 13.

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b

ST3

co14

TUNElL

FIG. 13. (continued) (a) Northern blot analyses demonstrate differential regulation of different MMP genes during Xenqnus intestinal metamorphosis. Each lane had 10 pg total intestinal RNA. The probes used were human stromelysin-1 (STI), Xenopus collagenase-3 (Co13), Xenopus collagenase-4 (Co14), human gelatinase A (GelA), and Xenopus ST3. (b) association of ST3 but not Co13 or Co14 expression with larval epithelial cell death during intestinal remodeiing. Panels A-C: In situ hybridization with ST3, Co13, and Co14 antisense RNA probes, respectively, or anterior typhlosole-containing sections of the small intestine at stage 60. Note that ST3 is highly expressed in essentially all regions of the connective tissue (ct) underlying the larval epithelium (e), which undergoes apoptosis (see D), whereas Co13 is only sporadically expressed in the connective tissue and Co14 has no detectable expression. None of the MMPs are expressed in the epithelium. (D) TUNEL assay for apoptotic cells or anterior small intestine at stage 60. Note that labeled apoptotic cells (arrows) are present throughout but are essentially limited to the epithelium; 1, intestinal lumen; m, muscle. Bar, 300 pm.

testine suggests that these MMPs are involved in the removal of the ECM associated with the degenerated larval epithelium and/or remodeling of the ECM for adult epithelium differentiation. The earlier activation of the ST3 gene in the intestine is likely to be important for the ECM remodeling that facilitates larval epithelial apoptosis. How these MMPs influence tissue remodeling is yet unknown. Interestingly, mammalian ST3 is secreted in its enzymatically active form when overexpressed in tissue culture cells (153). This has allowed the identification of one potential physiological substrate, the ol-proteinase inhibitor, a non-

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FIG. 14. Remodeling of the basal lamina correlates with ST3 expression in the intestine during metamorphosis. (A and D) In situ hybridization using an antisense ST3 probe on cross-sections of the anterior region of the small intestine. (A) At stage 59, hybridization signals (arrows) are observed in some cells of the connective tissue (ct) near the muscular layer (m), but are weaker in the upper region of the typhlosole (Ty). (D) Small intestine at stage 61. Most of the connective tissue cells just beneath the epithelium (e) are positive, i.e., ST3 expressing (arrows). Bars, 20 (J-m. (B, C and E) Electron micrographs of the epithelial-connective tissue interface of the small intestine. (B) Upper region of the typhlosole at stage 59, when ST3 expression is weak. The basal lamina (Bl) remains thin. (C) Bottom region of the typhlosole at stage 59, when ST3 expression is strong. The basal lamina begins to fold. (E) Th’rck ened basal lamina at stage 6 1, when ST3 is highly expressed. The basal lamina is vigorously folding into accordion-like pleats. Bars, 1 km.

ECM-derived serine proteinase inhibitor (190). Although no in viva ECM substrates of ST3 have been identified, such substrates may exist. One the other hand, the ability of ST3 to cleave a non-ECM substrate raises the possibility that ST3 may affect cell behavior through both ECM- and non-ECMmediated pathways. The other frog MMPs-STl, GelA, Co13, and Col4-are expected to digest specific ECM substrates just like their mammalian coun-

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terparts, thus leading to the remodeling and degradation of the ECM during metamorphosis.

C. Extracellular

Matrix

and Cell Fate Determination

The remodeling of theECM during metamorphosis together with the correlation of the expression of MMPs, especially ST3, with apoptosis suggests that ECM remodeling plays a role in TH-induced apoptosis. Direct support for a role of ECM on cell fate has come from a number of studies with mammalian systems. One of the best-studied systems is the involution of the mammary gland. As the epithelial cells undergo postlactation apoptosis, a number of MMP genes are activated (191, 192). More importantly, by culturing the epithelial cells on different ECM matrices, it has been shown that the ECM can directly influence cell differentiation and survival (193, 194). Similarly, the ECM has been shown to be essential for survival of several other types of cells, and blocking the function of ECM receptor integrins can induce cell death (195-199). As described above, tadpole intestinal epithelial cells can be cultured in vitro and induced to undergo apoptosis by TH just as in viva (126, 127). When the plastic culture dishes are coated with various ECM proteins, the cells become resistant to TH-induced cell death (Fig. 15) (127). Consistent with this apoptosis-inhibiting effect of the ECM coatings, when proliferating/ differentiating adult epithelial cells of the intestine at stage 64 are cultured in vitro on plastic dishes, they too undergo TH-induced apoptosis. In viva, these adult cells proliferate and differentiate instead of undergoing apoptosis in the presence of high levels of circulating plasma TH. Thus, dissociating the adult cells from the ECM alters their response to TH. The mechanism by which the ECM influences cellular function is still unknown. Clearly, one way to transduce the ECM signal into the cells is through cell surface ECM receptors, especially integrins (195,200-203). In the case of mammary gland development, it has been proposed that the interaction of the ECM with its integrin receptors leads to the activation of a focal adhesion (tyrosine) kinase (FAK), which in turn transduces the signal through the MAP kinase pathway to the nucleus (194, 205). This or similar mechanisms may be responsible for ECM-dependent gene transcription and cell fate determination.

VIII. Conclusions and Prospects Anuran metamorphosis bears many similarities to postembryonic organ development in higher vertebrates (4, 206), including maturation of a number of adult tissues/organs such the intestine and brain. Similarly, transition

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. 08

+

Plastic

+

Laminin

+

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+

Collagen IV

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T3 OW FIG. 15. Epithelial cells from stages 57/58 intestine culture on matrix-coated dishes are more resistant to Ta-induced apoptosis. The epithelial cells were cultured on va.rious dishes in the presence of different concentrations of T, for 3 days and DNA fragmentation was then determined by an enzyme-linked immunoassay method.

from fetal to adult hemoglobin and the activation of liver albumin genes, etc., occur during postembryonic development of both mammals and anurans. Finally, a number of key processes during this period are dependent on the presence of TH in mammals as well. Clearly, TH plays a more critical role in anuran metamorphosis than in mammalian postembryonic development. The absolute dependence of anuran metamorphosis on TH has made it one of the oldest systems to study vertebrate development. This has led to the accumulation of extensive morphological, cellular, and biochemical information on the process and the establishment of a number of in vioo and in vitro systems to study TH-induced cell death and proliferation/differentiation required for tissue remodeling. The current molecular studies have strengthened the idea that TH induces a gene regulation cascade within each metamorphosing tissue through TRs. Many of the direct TH response genes have been isolated. These genes belong to several diverse groups encoding intracellular, extracellular, and membrane-bound proteins, suggesting that TH simultaneously induces intra- and extracellular signal pathways to effect tissue transformation (Fig. 16). The direct response genes encoding transcription factors are expected to di-

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Various functions

FIG. 16. A gene regulation cascade model for TH regulation of tissue remodeling. The binding of TH to TR/RXR heterodimers leads to conformational changes that activate the receptor complex. The activated TR/RXR heterodimers then up-regulate several classes of TH response genes, which in turn affect intra- or extracellular events, resulting in ultimate changes in cell fates. Note that TH-bound TRiRXR can also repress gene expression. However, such down-regulated genes in the intestine, if any, have yet to be identified and are therefore not included in the figure.

rectly regulate downstream gene transcription while others, such as the MMPs, will indirectly regulated gene expression through intracellular signaling cascades and/or cell-cell and cell-ECM interactions (Fig. 16). One key question that needs to be addressed is whether the early, direct TH response genes are involved in the regulation of the known late TH response genes. It is likely that other genes may play a mediatory role in transducing the upstream signals to the late response genes. The identification of such genes will also be important to the understanding of the TH-induced gene regulation cascade. Perhaps the most urgent need is to investigate directly the roles of various T, response genes during metamorphosis. Existing organ and cell culture systems will be very useful for this purpose, especially for genes that encode extracellular proteins, because overexpressed proteins or functionblocking antibodies, inhibitors, and dominant negatives can be added to the culture systems to modulate the levels and functions of the endogenous proteins. The ability to manipulate Xenopus embryos through microinjection of DNA or RNA encoding proteins of interest into fertilized eggs serves as another means to study gene function. For example, by overexpression TRs and RXRs in developing embryos, we have shown that both TRs and RXRs are required to efficiently mediate the developmental effects of T, and for specific regulation of several genes that are known to be regulated by TH during metamorphosis in embryos (207). Finally, the more recently developed transgenic Xenopus technology (208) will allow study of gene function directly in metamorphosing tadpoles, as has been done for the TH response gene encoding a type III deiodinase (209).

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ACKNOWLEDGMENT We thank Ms. Kieu Pham for preparing the manuscript.

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Role of S6 Phosphorylation and S6 Kinase in Cell Growth SINISA VOLAREVIC GEORGE

AND

THOMASI

Fried&h Miescher Institute CH-4058 Basel, Switzerland I. 40s Ribosomal Protein S6 ........................ A. S6 Phosphorylation ........................... B. Location .................................... C. Function .................................... D. Drosophila S6 and Extraribosomal Functions of S6 II. S6Kinase ...................................... A. IdentificationofSGKl......................... B. Structure of S6Kl ............................ C. Mechanisms of S6Kl Activation ................ D. Upstream Effecters ........................... III. Downstream Effecters of S6K ..................... A. S6 Phosphorylation and 5’TOP mRNA Translation B. S6Kl and Cell Cycle Progression ............... C. InsulinProduction ............................ IV. Physiological Importance ......................... A. S6Kl Deletion in Mice and Discovery of S6K2 .... B. Drosophila S6K Mutant ....................... . Future Perspectives .............................. References .....................................

This article reviews our current knowledge S6 phosphorylation

of cell growth and proliferation.

...

.

of the role of ribosomal

protein

Although 40s ribosomal protein S6 phosphory-

25 years ago, it only recently has been implicated in the

up-regulation

thetic apparatus.

103 103 10s 105 107 108 108 108 111 113 117 117 118 119 119 119 120 122 123

and the S6 kinase (S6K) signaling pathway in the regulation

lation was first described translational

. .

of mRNAs coding for the components

These mRNAs contain an oligopyrimidine

scriptional start site, termed

of protein syn-

tract at their 5’ tran-

a 5’TOP, which has been shown to be essential for

their regulation at the translational level. In parallel, a great deal of information has accumulated regulatory

concerning

phosphorylation

this knowledge

the identification

of the signaling pathway and the

sites involved in controlling

we are only beginning

involved in growth factor-induced

S6K activation. Despite

to identify the direct upstream

elements

kinase activation. Use of the immunosupres-

1 To whom correspondence should be addressed. Progress in Nucleic Acid Research and Molecular Biology, Vol. 65

101

Copyright 8 2001 by Academic P~PII All rights of reproduction in any form reserved. 0079-6603/01 $35.00

102

SINISA

sant rapamycin,

a bacterial

macrolide,

VOLAREVIC

in conjunction

AND GEORGE

THOMAS

with dominant interfering

and activated forms of S6Kl has helped to establish the role of this signaling cascade in the regulation employing

of growth and proliferation.

the mouse as well as Drosophila

sights into physiological

paramount

S6K2, whereas importance

have provided

function of S6K in the animal. Deletion

in mouse cells led to an animal of reduced homolog,

In addition, current studies

melanogaster

loss of dS6K

in development

size and the identification

function in Drosophila and growth control.

new in-

of the S6Kl gene of the S6KI

demonstrated

its

o 2000AcademicPWS.

Mitogens stimulate cells to exit the G, stage of the cell cycle, progress through the G, phase of the cell cycle, synthesize DNA during S phase, and ultimately pass through mitosis and cell division (1). Earlier studies in yeast indicated that passage through G, requires a coordinated increase in protein synthesis and cell mass. Hartwell and colleagues in their seminal studies demonstrated that conditions that prevented cell growth, such as nutrient deprivation, arrested cell proliferation (2). In contrast, mutations that blocked cell cycle progression did not affect cell growth (3). These findings demonstrated the dominance of cell growth over cell proliferation. However, the molecular mechanisms by which the processes controlling cell growth and proliferation are integrated to bring about the development of the organism are largely unknown. Although the signaling pathways that control cell cycle progression have been studied intensively (I), we know less concerning the signaling pathways that control cell growth, such as those associated with the regulation of protein synthesis. For some time, it has been evident that for the cell to grow it must synthesize new proteins and that this process relies on the activation and maintenance of high rates of protein synthesis to meet this demand (4). However, less recognized is the fact that the greatest demand is for those proteins that make up the translational apparatus, especially ribosomes (5- 7). The highly regulated relationship between ribosome biosynthesis and cell growth was first demonstrated in bacteria over 40 years ago (8). In this way, biosynthetic energy is conserved when cells are not growing, whereas the rate of protein synthesis increases as a function of physiological or pathological growth state, such as cancer (9). The control of ribosome biogenesis is closely monitored, as the energy investment in production of these large multiprotein-rRNA complexes has been calculated to be as much as 80% of the expended energy in a proliferating mammalian cell. The energy investment in ribosome biogenesis is further underscored by the approximate 5 million ribosomes present per mammalian cell, with each ribosome having an overall molecular mass of 4.2 X lo6 Da. These values mean that ribosomes make up greater than 80% of the cellular RNA and 5-100/o of cellular proteins (IO).

s6

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AND

s6

KINASE

IN CELL

GROWTH

103

Although, a number of phosphorylation events have been implicated in the regulation of global translation, mostly at the level of initiation of protein synthesis (11, 12), only recently was it demonstrated that the S6K is involved in the translational up-regulation of a specific family of mRNAs that contain an oligopyrimidine tract at their 5’ transcriptional start site (5’TOP mRNAs). This step is presumably regulated by the multiple phosphorylation of 40s ribosomal protein S6 (13). H ere, we will attempt to review the current status of the role of S6 phosphorylation in the regulation of 5’TOP mRNA translation, the molecular mechanisms of S6K activation, and the characterization of the upstream components in this signaling cascade. Recent insight into the physiological relevance of this pathway obtained from the manipulation of S6K in both mouse and Drosophila models also will be addressed.

I. 40s Ribosomal Protein S6 A. S6 Phosphorylation The S6 gene encodes for one of the 33 proteins that, in a complex with one molecule of 18s rRNA, comprise the mature 40s ribosomal subunit (14). Phosphorylation of the 40s ribosomal protein subunit was first observed in the livers of rats injected with 32P0, (15). Gressner and Wool (16) subsequently demonstrated that the only significantly phosphorylated 40s ribosomal protein, following partial hepatectomy in rats, was S6. Because partial hepatectomy induces the remaining hepatocytes to grow and proliferate, this finding led to a number of studies that demonstrated that growth factor stimulation of different cell types also induced hyperphosphorylation of S6 (1719). In addition to the induction of S6 phosphorylation following partial hepatectomy in rats, this response was also induced in vivo during refeeding after fasting, differentiation, fertilization, and infection with viruses (19-22). In general it was also observed that increased S6 phosphorylation correlated with increased rates of protein synthesis (23). Despite this correlation between protein synthesis and S6 phosphorylation, it was initially difficult to rationalize this response with the observation that protein synthesis inhibitors, such as cycloheximide and puromycin, also were found to elicit a potent S6 phosphorylation response (16,23,24). Nevertheless, two main observations prompted further research into the role of S6 phosphorylation in the regulation of the protein synthesis. First, the kinetics of S6 phosphorylation closely parallel changes in translational activity (23), and second, the observation that ribosomes with the highest proportion of phosphorylated S6 are selectively found in large polysomes (25, 26). To identify the sites of S6 phosphorylation, advantage was taken of the fact that

104

SINISA VOLAREVIC

AND GEORGE

Species:

Seauence:

Homo sapiens

RRRLSSLRASTSKSESSCIK’~~

Mus musculus

RRRLSSLRASTSKSESSQK’@

Ratus norvegicus

RRRLSSLRASTSKSESSQK’@

Xenopus laevis

RRRLSSLRASTSKSESSQK“@

Drosophila

RRRSASIRESKSSVSSDKK’”

melanogaster

Drosophila alternative isoform

RGRY’JTIRKPKSSVFSGKK’~~

Saccharomyces

KRRA.SSLKA’=

cerevisiae

THOMAS

FIG. 1. Comparison of the carboxy-terminal domain of ribosomal protein S6 from different species. The sequence alignment begins at the S6K RXFt recognition motif. Phosphorylation sites are shown in bold.

intraperitoneal injection of cycloheximide had been shown to induce S6 phosphorylation in all tissues examined (23). In this way it was found that cycloheximide induces the phosphorylation of S6 on five car-boxy-terminal serine residues, residing within a 32amino acid carboxyl-terminal fragment (24). The five phosphorylation sites were shown to be Ser-235, Ser 236, Ser240, Ser-244, and Ser-247 (Fig. 1) (24). In later studies it was shown that serum stimulation of mouse fibroblasts induced S6 phosphorylation on the same sites as had been shown in rats treated with cyclohexamide (27). Earlier studies had shown that phosphorylation of S6 appeared to proceed in an ordered fashion (28). Subsequent in vivo and in vitro studies showed that phosphorylation proceeds in an ordered manner: Ser-236 > Ser-235 > Ser240 > Ser-244 > Ser-247 (27, 29). With the identification of the S6 kinase (see below) it was possible to demonstrate that its recognition sequence resided immediately upstream of the principal site of phosphorylation, Ser236, in the sequence FtXRXXS, with arginines present in the -5 and -3 position (30). The mouse, rat, and human S6 protein sequences are identical to each other, whereas the identity of the Drosophila and yeast S6 protein with the mammalian sequence is 74 and 60%, respectively (31-34). The yeast homolog of mammalian S6, termed SlO, is present in two copies in the yeast genome, as are most ribosomal proteins. Compared to mammalian S6, SlO lacks the last 10 carboxy-terminal amino acids. Therefore SlO contains only two of the five potential mammalian phosphorylation sites, corresponding to S235 and S236 (Fig. 1). A yeast strain having a single copy of the S6 gene is viable, therefore Johnson and Warner (35) replaced this allele with a mutant S6 gene having the two putative phosphorylation sites changed to alanines.

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s6

KINASE

IN CELL

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105

Extensive analysis of this strain revealed no obvious alteration in the growth phenotype (35). Neverth e 1ess, it is important to emphasize that yeast do not regulate ribosomal protein expression at the translational level and that yeast ribosomal protein mRNAs do not contain 5’TOP sequences (36, 37). Furthermore, it appears to be the later sites of S6 phosphorylation that correlate with increased translation, whereas the early sites, S235 and S236, appear to regulate phosphorylation at the downstream sites (13).

B. Location Chemical cross-linking and protection studies with specific components of the translational apparatus have localized S6 to the small head region of the 40s subunit at the mRNA-tRNA binding site. This area of the 40s subunit resides at the interface with the larger 60s ribosomal subunit, where S6 apparently comes into direct contact with 28s rRNA (11,38). The location of S6 in the 40s subunit places it in a position where it could potentially have a functional impact on translation. This led to the hypothesis that S6 in the phosphorylated state, through steric effects or conformational changes (11, 19,38), may be involved in regulating specific steps in translation. Other studies have led to the hypothesis that differentially phosphorylated S6 could directly interact with a subset of mRNAs or associated proteins and lead to selective changes in the pattern of translation (see below).

C. Function Early studies correlated increased S6 phosphorylation with the up-regulation of global translation. However, in some instances the kinetics of these two events did not coincide. For example, in HeLa cells, S6 phosphorylation was shown to decline 6 hr after serum stimulation, whereas maximum levels of protein synthesis were achieved at later time points (23). An important observation that led to the suggestion that S6 phosphorylation may be involved in selective translational up-regulation of specific mRNAs was the finding that the levels of at least 16 proteins coincided with increased S6 phosphorylation in serum-stimulated Swiss mouse 3T3 cells (39). The increased synthesis of seven of these proteins was not blocked by actinomycin D, a potent inhibitor of transcription, suggesting that the up-regulation of the seven unaffected proteins was controlled at the posttranscriptional level. The protein with the most increased value was subsequently identified as eukaryotic elongation factor-lo, eEF-lo (40). Consistent with the actinomycin D experiments described above, the absolute levels of eEF-lo mRNA do not change within the first 3 hr of serum stimulation (41). In quiescent cells eEF-lo mRNA is translated inefficiently, with the bulk of the mRNA largely residing in mRNP particles, with a small amount of eEF-la transcript present on either disomes or monosomes (41).

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SINIL?A VOLAREVIC

AND

GEORGE

THOMAS

Following serum stimulation, both populations of eEF-la mRNA are selectively recruited to very large polysomes, of approximately 11- 12 ribosomes per transcript (41). Under these conditions the mean polysome size does not change and most mRNAs remain in the same position within the polysome profile. Thus, the selective changes in eEF-la mRNA usage correlated with the increased phosphorylation of S6. To obtain better insight into the relationship between selective translational up-regulation of eEF-lo and S6 phosphorylation, advantage was taken of rapamycin, a bacterial macrolide known to inhibit S6 phosphorylation due to its ability to inhibit S6K (42; and see below). Rapamycin selectively repressed recruitment of eEF-lo into large polysomes, without affecting global translation. Interestingly, eEF-lo belongs to a family of mRNAs that contain at their transcriptional start site S’TOP, an element known to act as a translational regulator (43). All mammalian ribosomal protein mRNAs in which the 5’ transcription start site has been mapped, as well as elongation factors, contain this motif (37). Th e motif starts with a cytidine and is followed by a stretch of 5 to 15 pyrimidine residues. However, for efficient translational up-regulation of S’TOP mRNAs, integrity of the region downstream of the S’TOP is also required (37,44). Although the S’TOP family of mRNAs contains only 100 to 200 genes, their transcripts can represent up to up 20% of total cellular RNAs (37, 44). More recent studies employing chimeric mRNAs, in which either the wild-type S’TOP tract or a disrupted S’TOP tract had been fused to a reporter transcript, demonstrated that an intact S’TOP tract is required for rapamycin to elicit an inhibitory effect on the translation of these mRNAs (45). Consistent with these findings, a dominant interfering S6Kl mutant repressed the mitogen-induced translational up-regulation of S’TOP mRNAs to the same extent as rapamycin, whereas expression of a rapamycin-resistant S6Kl mutant negates the inhibitory effects of rapamycin on S’TOP mRNA translation (45). These same constructs either block or promote S6 phosphorylation, respectively (46). Taken together, these results demonstrate that the rapamycin inhibitory block of S’TOP mRNA translation is mediated through S6Kl inactivation and strongly suggest that S6 phosphorylation mediates this response. Direct evidence for a role for S6 phosphorylation in the regulation of 5’TOP mRNAs is lacking. If phosphorylated S6 is functioning to regulate translation of S’TOP mRNAs, it will be of interest to determine the underlying molecular mechanism. It is possible that phosphorylated S6 directly interacts with the S’TOP, that it induces a conformational change in the 40s subunit that favors S’TOP mRNA translation, or that it mediates this interaction through other factors. In support of the latter possibility it has been found that the S’TOP region interacts in vitro with the La protein (47). In addition the cellular nucleic binding protein (CNBP) interacts with the region

s6

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s6

KINASE

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GROWTH

downstream of the polypyrimidine tract, a sequence required for efficient 5’TOP mRNA translation (48). The Ro60 autoantigen mediates the binding of the two proteins to their respective sequences, with the binding of either of the two proteins excluding the other (49). The fact that La and CNBP interact with two sequences required for translational up-regulation of 5’TOP mRNAs suggests that they may exert a modulating activity on the translational control of this family of mRNAs. Although it is unclear how these proteins may affect 5’TOP translation, it is tempting to speculate that they modulate interaction between S6 and the 5’TOP sequence.

D. Drosophilu

S6 and Extraribosomal

Functions

of $5

Drosophila S6 (DS6) is a single-copy gene. The genomic sequence of the DS6 gene has revealed that it is made up of three exons; however, for exon 3A, two alternative tandem repeats exist, 3B and 3C (50). If utilized, as has been claimed (32) exons 3B and 3C would produce a transcript that encodes a 189-amino acid protein that is considerably shorter than the 248-amino acid protein produced by exon 3A. In addition, the shorter protein would have a distinct carboxy terminus. Preliminary experiments indicate that only the longer form of DS6 is found in Drosophila 40s ribosomes and that it is also phosphorylated at five sites (51). This form of DS6 is almost equivalent in length to its mammalian ortholog, sharing 74% identity and 84% similarity with the mammalian protein (51). The correlation between increased DS6 phosphorylation and protein synthesis in Drosophila has not yet been examined carefully. It is clear that the translation of ribosomal proteins in Drosophila, as in mammals, is regulated at the translational level (52). However, the regulation of mRNAs coding for components of the translation apparatus may be different from that of mammals because not all ribosomal protein mRNAs appear to contain 5’TOPs (37). It should also be noted that P-element insertions in the 5’ untranslated (5’UTR) of the DS6 gene, which is X linked, leads to a decrease in DS6 gene expression, with hemizygous mutant males failing to emerge as adults after a prolonged third larval instar (31, 32). During larval development the mutants develop melanotic tumors in the hematopoietic system, with significantly overgrown lymph glands, implying an additional role for DS6 in tumor suppression of larval hemocytes, which mediate the immune response in insects (31, 32). Th e increased number of hemocytes and the presence of giant cells within the hematopoietic organs are not completely consistent with a role for DS6 in cell growth. However, the expression level of DS6 in these tumor cells has not been determined, nor whether DS6 phosphorylation is involved. Obviously the powerful genetics offered by the Drosophila system should prove advantageous in elucidating the role of S6 phosphorylation in translation. An additional indication that S6 may have an extraribosomal function

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comes from the finding of a free form of S6 in the nucleus of mammalian cells (53). This pool of S6 is also phosphorylated in response to growth factor stimulation, presumably by a nuclear form of S6Kl (54). However, there is no direct evidence for an extraribosomal function of S6 in mammals. It is interesting to note that the extraribosomal biochemical functions have been ascribed to a number of other ribosomal proteins (55). However, the biological significance of these biochemical functions has been difficult to demonstrate in vivo, although a patient with a mutation in S19 has been described. The mutation is genetically linked to Diamond-Blackfan anemia (56), a clinical feature that suggests an extraribosomal function.

II. S6 Kinase A.

Identification

of S6Kl

Although a number of kinases were initially implicated in S6 phosphorylation, based on their in vitro activities (57,58), it generally has been accepted that the S6K family of kinases mediates this response. However, studies in Xenopus oocytes suggest that in this system p90mk may function as an in vivo S6 kinase (59). S6Kl was first purified from extracts of mitogen-stimulated cells utilizing 40s ribosomes as substrate (60). Subsequent sequencing of the enzyme, followed by cloning and expression studies, led to the identification of two isoforms of S6Kl that are produced from the same transcript (61- 64) by alternative initiation translational start sites (Y. Chen, C. D. Hoemann, G. Thomas, and S. C. Kozma, unpublished data). The shorter form of S6K1, which is largely localized in the cytoplasm, was termed ~70~~~; a longer form, which exclusively localizes in nucleus, is termed ~85 S6K (Fig. 2). The larger isoform of S6Kl is distinct from the shorter form in that it contains an amino-terminal 23-amino acid extension, containing a nuclear localization signal (54). The functional significance of the differential subcellular localization of the two isoforms of S6Kl has not been established, although it is tempting to speculate that the nuclear form is involved in the phosphorylation of the nuclear pool of free S6 described above (53). It was found that deletion of the S6Kl gene in mice impaired neither S6 phosphorylation nor 5’TOP mRNA translation (65). This observation led to the identification of a second S6 kinase (S6K2) gene that is highly homologous to the first gene (65- 67) (Fig. 2).

B. Structure

of S6Kl

The mechanism by which kinase activation is presumed to take place has been acceded to by the dissection of the primary structure of S6Kl into do-

525

FIG. 2. Schematic representation of S6Kl and S6K2. Two forms of S6K1, p70 and ~85, are shown. The positions of S6K domains are indicated. The phosphorylation sites are numbered. Nuclear localization signals in ~85 and S6K2 are depicted. A proline-rich sequence, representing a putative SH3 recognition motif, is indicated in S6K2.

S6K2

356

110

SINISA VOLAREVIC

Kinase:

Seauence:

S6Kl

lVTH ~ TzaFCGT IEYMWE ~-

PKBa

ATM&“FCGTPEyL&&

PKCa

VTRRT@‘FCGTPDY_lE

CaMKlV

VLMKT’=VCGlPGYCAPE

AMPK

EFLRT’ =SCGSPNYAAPE _____

AND GEORGE

THOMAS

AGC family

CaMK family

FIG. 3. Comparison of the sequence surrounding the activation loop T229 site of S6Kl with those of related kinases. The S6Kl T229 phosphorylation site and equivalent sites in other kinases are shown in bold. The identical amino acids in S6Kl and other kinases are underlined.

mains and to the identification of specific regulatory phosphorylation sites (68). Such an understanding was instrumental in the identification of one of the upstream S6Kl kinases as the phosphoinositide-dependent protein kinase, PDKl (69, 70). P rior studies had revealed five distinct domains that cooperate to bring about S6 kinase activation (Fig. 2). The first is an acidic domain that extends from the amino-terminus to the beginning of the catalytic domain and confers rapamycin sensitivity on the kinase (71- 73). The second is the catalytic domain, which contains the mitogen-induced phosphorylation site T229, residing within the activation loop (74, 75). The catalytic domain as well as the sequence immediately amino terminal to the T229 phosphorylation site are highly conserved in the protein A, protein G, and protein C (AGC) family of protein kinases as well as the CaMK-dependent family (Fig. 3) (76). The third domain is the linker domain, which connects the catalytic domain to the autoinhibitory domain. The linker domain is also highly conserved in the AGC family of serine threonine kinases and contains two phosphorylation sites, S371 and T389, which are essential for S6Kl activation (77, 78). The motif surrounding T389 is flanked by hydrophobic amino acids in the + 1 and - 1 positions and is also conserved in a large number of kinases that belong to this family (Fig. 4) (77). In contrast to most members of the AGC family, which end with the linker domain, S6Kl has two additional carboxy-terminal domains. The first contains a motif that is homologous to the amino acid motif surrounding phosphorylation sites in S6, and was initially suggested to serve as an autoinhibitory domain (61, 79). I n support of this model synthetic peptides covering this sequence inhibit S6 kinase activity in vitro (30) and removal of the carboxy terminus raises basal kinase activity (73). This domain contains five

s6

PHOSPHORYLATION

AND

s6

KINASE IN CELL

GROWTH

Kinase:

Sequence:

S6Kl

VELGFT=YVAP

PKCa

DEEGFS=‘YVNP

RSKl

LERGF?FVAT

PKBcx

HEPQFSAS

111

FE. 4. Conservation of sequence motif surrounding S6Kl T389 in related kinases. Alignment of S6Kl sequence with those of PKCq RSKl, and PKBa. The conserved residues are underlined and the residues homologous to T389 are shown in bold.

sites: S404, S411, S418, T421, and S424. It is interesting to note that S404 is surrounded by large bulky hydrophobic amino acids in the - 1 and + 1 positions, similar to T229 and T389, whereas the remaining phsophorylation sites are followed by proline at the +1 position, similar to S371. Finally, the extreme carboxy terminus of S6Kl contains a sequence that has been shown to interact with the PDZ domain of neurabin, a neuralspecific F actin binding protein (80, 81). Presumably, S6Kl is localized to nerve terminals through this interaction. However, the physiological meaning of this interaction remains unclear at this point. Structural differences between S6Kl and S6K2 are discussed below.

phosphorylation

C. Mechanisms

of S6Kl

Activation

The mechanism of S6Kl activation is complex, involving the interplay between four different domains and at least seven specific regulatory phosphorylation sites, implying the existence of multiple upstream regulators (68). Current models suggest that the first step in S6Kl activation is the phosphorylation of the ST-P sites in the autoinhibitory domain, which functions in conjunction with the amino terminus to allow phosphorylation of T389. These two events presumably disrupt the interaction between the carboxy and amino termini, allowing phosphorylation of T229 and resulting in S6Kl activation (Fig. 5). This model is supported by the observation that T229 is phosphorylated in a mitogen-independent fashion when both T389 and the S/T-P sites in the autoinhibitory domain are replaced with acidic residues, generating the S6Kl variant T389EDaE (82). The kinase activity responsible for mediating T229 phosphorylation appeared to be constitutive, initially suggested that PDKl may serve as the in vivo T229 kinase. In support of this hypothesis the protein sequence motif surrounding T229 is homologous to the corresponding PDKl-dependent phosphorylation site in protein kinase B (PKB), T308 (83, 84). That PDKl is

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VOLAREVIti

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THOMAS

Mitogen 1 T389 Kinase

L

T389-

s371-P

/

inactlve

I

T229-P

-

active

FIG. 5. Mechanism of S6Kl activation. The S6Kl domains and phosphorylation denoted as in Fig. 1. The model represents sequential steps of S6Kl phosphorylation vation as outlined in the text (68).

sites are and acti-

the physiological T229 S6Kl kinase was based on the fact that PDKl could activate the S6Kl acidic variant in vitro and in vivo, whereas the catalytically inactive PDKl blocked insulin-induced activation of S6Kl in vivo (69). Although the activity of the S6Kl acidic variant is up-regulated following phosphorylation by PDKl, the activity of S6Kl can be increased a further twofold by mitogens (72). The above results suggested the existence of additional sites of phosphorylation involved in S6Kl activation. One such candidate is S3 71 in the linker domain (78). The equivalent site has been found to be a major site of autophosphorylation in members of the PKC family (85-87). However, a transiently expressed kinase dead variant of S6Kl is phosphorylated at S3 71 in the presence of rapamycin (78). This result would strongly imply that S6Kl S3 71 phosphorylation is not regulated by autophosphorylation in either a cis or trans manner (78). The importance of this site for kinase activity is derived

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from the fact that substitution of S371 with either an alanine or aspartic acid blocks kinase activation. In parallel, these substitutions also block mitogeninduced T389 phosphorylation, an effect that cannot be rescued by substituting an acidic residue for T389. These findings would imply that S371 phosphorylation has a dual role-to regulate T389 phosphorylation as well as to contribute to kinase activity (78). It will be of interest to determine whether S3 7 1 is the last link required to bring about kinase activation. In the final analysis it will be necessary to reconstitute S6Kl activation in o&-o. This will require the identification of the kinases that phosphorylate the remaining sites, and complimentary in ciao and genetic studies to confirm their identities.

D. Upstream

Effecters

1. PI3K Because the S/T-P sites in the autoinhibitory domain have motifs similar to the consensus sequence for the mitogen-activated protein kinase (MAPK), and because MAPK can phosphorylate S6Kl in vitro, it was initially speculated that the RASMAPK pathway controlled S6Kl activation (88). However, Ballou et al. (89) d emonstrated that insulin treatment of Swiss 3T3 cells had no effect on MAPK activation, but potently activated S6Kl. Further experiments utilizing both dominant interfering mutants of BASMAPK signaling pathway as well as specific docking site mutants of the platelet-derived growth factor (PDGF) receptor demonstrated that MAPK is neither necessary nor sufficient to elicit S6Kl activation (90). As importantly, such studies demonstrated that S6Kl was a downstream effector of the phosphatidylinositide-30H kinase (PI3K) signal transduction pathway, rather than the RASMAPK signal transduction pathway (90, 91). Support for this conclusion came from studies employing PI3K inhibitors, such as wortmannin and LY294002, which blocked the activation of S6Kl (91), as well as membranetargeted, constitutively active forms of PI3K that activate S6Kl. Nevertheless, the molecular events leading from PISK activation to that of S6Kl have yet to be elucidated. Indeed, not all the studies employing PDGF receptor mutants are wholly consistent with PI3K mediating S6Kl activation (90). Furthermore, studies have challenged the specificity of wortmannin and LY294002 for PI3Ks (92), and membrane-targeted, constitutively active al leles PI3K may not accurately reflect wild-type PI3K function (see below). 2. EFFECTOR KINASES OF PISK Protein kinase B (PKB) has been established as a downstream effector of PI3K (93, 94). Furth ermore, constitutively activated membrane-targeted alleles of PKB induce reporter S6Kl activation in a wortmannin-resistant manner (94). However, subsequent studies showed that PKB does not directly

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phosphorylate S6Kl. In addition, Corms et al. (1998) have demonstrated that the depletion of intracellular stores of Ca 2+ has no effect on PKB activation, but abolishes S6Kl activation (95). In contrast, they observed that an increase in intracellular Ca2+ induces S6Kl activation in a wortmannin-sensitive manner without stimulating PKB activation. Consistent with these findings recent experiments have shown that dominant interfering alleles of PKB, which block insulin-induced reporter PKB activation, glycogen synthase 3kinase (GSK3) inactivation, and initiation factor 4E binding protein (4EBPl) phosphorylation, have no effect on S6Kl activation (96). Additionally, only activated alleles of PKB that are membrane targeted induce S6Kl activation, whereas the constitutively active cytoplasmic form of PKB, harboring acidic residues at T308D and S473D (97), though sufficient to induce GSK3 and 4E-BP1 phosphorylation, have no effect on S6Kl activation (96). These results indicate that membrane-targeted PKB activation does not reflect wildtype PKB function, as has been suggested by others (98, 99), and that PKB and S6Kl represent distinct branches of the PISK signaling pathway. The demonstration that PKB is not involved in physiological activation of S6Kl has initiated the search for additional kinases that are known downstream effecters of PI3K. Earlier studies had revealed that PKCC is regulated in vitro by phosphatidylinositol3,4,5triphosphate ptdIns(3,4,5)P3] (IOO), and that PKCh as well as PKC< are activated in viva through a pathway involving PI3K (101-103). Studies have shown that both PKCh and PKCL associate with S6Kl in a mitogen-independent manner that kinase-dead mutants of both PKCX and PKC[ partially inhibit S6Kl activation in cotransfection studies (104, 105). In support of these findings membrane-targeted alleles of PKC< increase S6Kl activity (105). These results suggest that the atypical PKCs may regulate S6Kl activation through direct or indirect phosphorylation of S6Kl. It should also be noted that as PDKl has been identified as the activation loop kinase for the atypical PKCs (106), implying that PDKl can regulate S6Kl activation directly by phosphorylating T229 and indirectly via the atypical PKCs (Fig. 6). In addition to the atypical PKCs it has been reported that the Rho family of small G proteins, cdc42 and Racl, associate with the hypophosphorylated forms of S6Kl in a GTP-dependent manner. However, this interaction is not sufficient to activate S6Kl (107). Cotransfection of an activated form of Rat can stimulate reporter S6K1, whereas a dominant negative form can block S6Kl activation in viva However, the mechanisms by which small GTP binding proteins contribute to S6K activation remain unclear. 3. MAMMALIANTARGETOFRAPAMYCIN The observation that the immunosuppressant rapamycin inhibits S6Kl activation and S6 phosphorylation has implicated a mammalian target of ra-

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AND

s6

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115

I

\ J Pl3K

PKB

amino acids autophagy

atypical

PKCs

E2Fcell cycle

insulin transcription

5’TOP mRNAs translation

general translation

FIG. 6. Schematic representation of mitogen-induced S6K signaling cascade. The mitogeninduced activation of S6K is mediated by PI3K. The involvement of other molecules in S6K activation is presented as described in the text. Downstream effects of S6Kl activation are also shown.

pamycin (mTOR) as an upstream effector of S6Kl (108-U). The effects of rapamycin on mTOR are exerted through a gain-of-function inhibitory complex between rapamycin and the immunophilin, FKBP-12 (6, 7). Sequence comparisons revealed that mTOR shares homology with the phosphatidylinositide kinases, although no lipid kinase activity has yet been detected. In contrast, mTOR autophosphorylates (112) and has been shown to phosphorylate a bacterially expressed S6Kl on T389 (113), the principal site of rapamycin-induced S6Kl inactivation and dephosphorylation (77). However, mTOR was also shown to phosphorylate 4E-BP1 (Fig. 6) (114), although the extent to which it phosphorylates all of the initially identified sites has been questioned (115). Rapamycin-resistant and kinase-dead alleles of mTOR either protect S6Kl from the inhibitory effects of rapamycin or block the effects of mitogens on S6Kl activation (113). That mTOR directly regulates S6Kl T389 phosphorylation, however, is in conflict with a number of observations. First, a rapamycin-resistant form of S6K1, lacking the amino and carboxyltermini, is unimpaired in insulin-induced T389 phosphorylation in cells that are pretreated with rapamycin. Nevertheless, T389 phosphorylation is still blocked by wortmannin pre-

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treatment (72). This latter finding also argues against a model that places PI3K upstream of mTOR (92). Second, the T389 phosphorylation site is flanked by large, bulky aromatic amino acids, whereas the phosphorylation sites in 4E-BP1 are followed by a proline at the + 1 position. Finally, only the bacterially expressed form of S6Kl but not a mammalian expressed form serves as a substrate for mTOR in vitro (113). In contrast to the model above, independent studies had led to the hypothesis that mTOR may regulate S6Kl activation by inhibiting a phosphatase rather than directly phosphorylating S6Kl (72). In addition, earlier experiments had shown that S6Kl is selectively dephosphorylated in vitro by protein phosphatase 2A (PPZA) (116), consistent with the finding that treatment of cells with insulin leads to a general inhibition of PPSA activity, which can be blocked by either rapamycin or wortmannin treatment (217). More recent studies have demonstrated that PPBA directly associates with S6Kl (118) and that th is association requires the presence of an intact S6Kl amino terminus (119). It is this domain that is known to impart rapamycin sensitivity to S6Kl (71- 73). The existence of an mTOR-regulated phosphatase activity in mammals has been supported by studies in yeast whereby TOR was observed to mediate the interaction of the SIT4 phosphatase, the yeast homolog of mammalian PP6, with Tap42, an essential yeast protein (120). Genetic evidence indicates that Tap42 positively regulates both PPBA and PP6 and mutations in Tap42 confer rapamycin resistance (120). The closest relative to Tap42 in mammals is a4 (120). Consistent with the results in yeast, rapamycin prevented association of PPBA and cx4 in mammalian cells (121). Additionally, PPBA activity was increased in vitro toward myelin basic protein and phosphorylase A when complexed with a4. However, with regard to a potential in vivo substrate, 4E-BPl, the data are less straightforward. In one case treatment with rapamycin increased PPBA activity toward 4E-BP1 (129), whereas in a second study PPBA activity toward 4E-BP1 in vivo was inhibited by (~4 in a rapamycin-independent manner (122). One possibility that may explain some of these seemingly disparate results is that mTOR may function as a scaffold, tightly associating with both the kinases and the phosphatases responsible for regulating S6Kl activity (6). At this juncture more information will be required to evaluate these possibilities. In addition to mitogenic stimulation, it has been demonstrated that essential amino acids also modulate S6Kl activation, in a PISK-independent manner (123). The first suggestion for a role for essential amino acids in S6Kl activation came from studies on autophagy. Autophagy is distinct from ubiquitin-mediated proteolysis in that it utilizes an invaginated membrane structure, termed an autophagosome, which fuses with the lysosomes targeting proteins for degradation (124). D uring fasting, autophagic degradation of

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proteins is stimulated in order to produce amino acids and nucleotides for gluconeogenesis and other essential metabolic pathways (124). It had been shown that amino acid deprivation of isolated liver cells induced autophagy and blocked S6 phosphorylation, leading to the suggestion that these events may be linked (125). A possible connection between S6Kl activation and amino acids levels was shown in experiments in which amino acid removal blocked S6Kl activation by mitogens, whereas readdition of amino acids reversed this response (123,126-129). That this response is mediated through mTOR was suggested from the finding that rapamycin prevented activation by amino acids of wild-type S6K1, but not a rapamycin-resistant form of S6Kl (123). The above results suggest that mTOR is directly or indirectly involved in regulating S6Kl activation by amino acids. In support of this hypothesis, rapamycin induces autophagy in yeast, even in the presence of nutrients (130). Indeed, a TORl-deficient yeast strain bearing a TOR2 temperature-sensitive allele, when grown at the permissive but not the nonpermissive temperature, induces autophagy, consistent with TOR regulating autophagy (130). As pointed out, amino acid activation of S6Kl does not require PI3K, because it is not affected by wortmannin (128). Moreover, Patti et al. found that amino acids inhibit the early steps of insulin signaling, such as increased tyrosine phosphorylation of IRS-l and IRS-2, increases in ~85 and Grb-2 binding to IRS-l and IRS-2, and stimulation of PISK (127). Consistent with these results, amino acids in the same cells blocked insulin-stimulated proliferation (127). It is tempting to speculate that amino acids may have some role in the stimulation of cell growth in the absence of proliferation-for example, in liver cells during feeding following fasting. Further studies will be necessary to determine the role of mTOR and S6Kl activation in the regulation of balance of protein synthesis and protein degradation by autophagy.

III. Downstream Effecters of S6K A. S6 Phosphorylation

and 5’TOP

mRNA Translation

As previously stated, the mitogen-stimulated increase in the biogenesis of translational components is a critical requirement for cells to increase protein synthetic capacity, grow, and ultimately proliferate. S6K1, presumably through S6 phosphorylation, is involved in translational up-regulation of 5’TOP mRNAs (Fig. 6) (45), wh ic h encode for components of the translational apparatus. In support of this conclusion, rapamycin and dominant interfering mutants of S6K1, both of which prevent S6 phosphorylation (46), block the selective translational up-regulation of 5’TOP mRNAs, whereas ra-

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pamycin-resistant forms of S6Kl protect mitogen-induced 5’TOP mRNA translation from the inhibitory effects of the macrolide (45). To determine the involvement of S6 phosphorylation in this response will require both an in vitro and a genetic approach. Studies employing purified protein synthesis initiation factors and defined mRNA transcripts have made it possible to measure the formation of 40s and 80s preinitiation complexes in a process termed “toe printing” (131). Employing such strategies should eventually allow the identification of those components required to form competent initiation complexes with 5’TOP mRNAs. To resolve this issue a genetic approach in theory also could be applied in both the mouse and in Drosophila. In both cases it should be possible to generate mutants in which the wild-type S6 is replaced with an S6 mutant harboring specific changes in identified phosphorylation sites. In the mouse this can be approached by generating an inducible-targeted deletion of the S6 gene in specific tissues, and introducing S6 mutant alleles by either a similar strategy or with viral vectors. A parallel approach could be used in Drosophila, by taking advantage of the described P-element mutations in the S6 gene. One could initially attempt to compliment these mutants by generating transgenic flies harboring a wild-type allele. If such a strategy were effective the next step would be to employ S6 mutants. The advantage of the mouse system would be the potential to carry out follow-up biochemical studies, whereas with Drosophila the genetic manipulations could be more readily carried out. An additional advantage of Drosophila would be the possibility to employ genetic tools, such as genetic interacting screens with other potential regulatory components. It should also be noted that rapamycin and dominant interfering mutants of S6Kl do not completely suppress the translational up-regulation of 5’TOP mRNAs, suggesting the existence of an additional regulatory mechanism involved in controlling the expression of this family of mRNAs (42, 45). Th is mechanism by definition is independent of rapamycin, S6K1, and S6 phosphorylation.

B. S6Kl

and Cell Cycle Progression

Earlier studies have shown that microinjection of inhibitory antibodies against S6Kl significantly represses the progression of cells from G, to S phase of cell cycle (132). Similar results have been obtained in some cell types with rapamycin (108). The most logical explanation for this finding would be that by inhibiting S6Kl activation, and consequently S6 phosphorylation, synthesis of nascent translational components also would be repressed. Work in T lymphocytes suggests that S6Kl activation could also regulate the activity of a critical cell cycle components. In T lymphocytes interleukin-2 (IL2) uses PI3K to regulate E2F transcriptional activity, which is a critical step in cell cycle progression (133). IL-2 stimulation of E2F transcriptional acti-

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vation is made up of both rapamycin-resistant and -sensitive components (134). Expression of a rapamycin-resistant S6Kl could restore rapamycin-inhibited E2F responses (Fig. 6) (134). H owever, the same S6Kl mutant could not rescue an inhibitory effect of rapamycin on cell cycle progression, suggesting additional rapamycin-sensitive effecters in the control of IL-2-mediated T cell cycle progression. Whether the effects of S6Kl on E2F transcriptional activity are mediated through a translational-independent or -dependent step has not been established. However, given the fact that rapamycin suppresses retinoblastoma protein phosphorylation in this system it should be possible to dissect the mechanism by analyzing known upstream cell cycle regulators.

C. Insulin

Production

S6Kl has also been implicated in the regulation of insulin-induced gene transcription in pancreatic R cells (Fig. 6). Leibiger et al. have shown that following glucose-induced release of insulin, insulin acts via its R cell receptor to up-regulate its own transcription in primary pancreatic R cells (135). The analysis of this pathway implicated S6Kl as one of the critical components regulating transcription of the insulin gene. This conclusion was based on the observation that rapamycin and a specific PI3K inhibitor, LY294002, blocked insulin transcription, However, more convincingly, rapamycin-resistant alleles of S6Kl rescued the rapamycin-suppressed transcription of the insulin gene. Interestingly, proliferation of rhabdomyosarcomas is driven by an intracellular growth factor (IGF-2) autocrine-loop, which is blocked by rapamycin (136). Furthermore, the IGF-2 transcript that has been implicated contains an oligopyrimidine tract in its 5’UTR and rapamycin suppresses its translation (137). It will be of interest to determine whether these effects on IGF-2 are mediated through S6Kl. The observation that S6Kl is involved in autocrine regulation of insulin secretion suggests that the mutation in the S6Kl gene may be responsible for at least some cases of metabolic dysfunction in noninsulin-dependent diabetes mellitus (NIDDM) (138). Analysis of S6Kl- and S6K2-deficient mice will aid in determining the impact of these signaling components on this complex disease.

IV. Physiological Importance A. S6Kl

Deletion

in Mice

and Discovery

of S6K2

Studies addressing the function of S6Kl in mammalian cells have been largely performed in tissue culture model systems. To obtain corroborative in T&O data in the animal to support a role for S6Kl in cell growth, the S6Kl

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catalytic domain was targeted in embryonic stem (ES) cells and the mutation transferred through the germ line to generate heterozygous and homozygous mutant mice lacking S6Kl protein (65). Unexpectedly, homozygous mice deficient for the S6Kl protein were viable and fertile, although they had a reduced body size. The size reduction was most pronounced during the embryonic stages, with heterozygous mice displaying an intermediate phenotype, suggesting that the absence of S6Kl gene product effected an early stage of embryogenesis. The mouse embryonic fibroblasts (MEFs) and splenic T lymphocytes from either mutant or wild-type mice showed similar proliferative responses when stimulated with serum or anti-T cell receptor antibodies, respectively. Rapamycin treatment inhibited the proliferative response equally in both wild-type and S6Kl mutant mice. These findings were supported by the observation that full stochiometric S6 phosphorylation was still observed in the livers of fasted S6Kl deficient mice after refeeding, a treatment known to induce S6Kl activation and S6 phosphorylation. Serum stimulation of SGKl-deficient MEFs also induced S6 phosphorylation and the translational up-regulation of 5’TOP mRNAs in a rapamycin-sensitive manner. The above results suggested the existence of an additional rapamycinsensitive S6 kinase, which was subsequently confirmed by the identification of S6K2 (65). In all tissues examined, compensatory up-regulation of the S6K2 transcript was found in SGKl-deficient mice, consistent with its putative role as an S6Kl homolog (65). S e q uence comparison of S6K2 revealed a kinase with almost identical domain organization and all the same regulatory phosphorylation sites found in S6Kl. The overall identity between S6Kl and S6K2 is 670/o,with the catalytic domains sharing 82% identity (Fig. 2) (65- 67). The major significant difference between the two kinases resides in the carboxy terminus of the molecules, where a proline-rich domain and a potential nuclear localization sequence are found in S6K2, but not in S6Kl. The proline-rich domain could, through its interaction with SH3-containing proteins, affect cellular localization and function of S6K2, allowing nonoverlapping functions with S6Kl. The importance of S6K2 and S6Kl as in viva S6 kinases will be tested following the deletion of the S6K2 gene in mice. Additionally, comparison of the phenotypes of S6Kl- and S6K2-deficient mice could aid in the elucidation of potential differential functions in vivo for the two highly homologous kinases.

B. Drosophila

S6K Mutant

The mammalian genome is thought to have been duplicated two times (139), which in some cases makes phenotypic characterization of a specific gene deficiency difficult, as exemplified by SGKl-deficient mice. Therefore, in some instances it is more attractive to study gene function in less complex

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organisms, such as Drosophila melanogastw, in which individual genes are usually represented in a single copy. Additionally, the mammalian signaling pathways are largely conserved in Drosophila and Drosophila offers a powerful genetic system to uncover novel components in signaling cascades. As a first step in the use of Drosophila to study S6Kl signaling and function, a Drosophda S6Kl homolog, dS6K was cloned (140, 141). The dS6K protein has an overall identity of 57% with either S6Kl or S6K2 and 78% identity in the catalytic domain. The organization of functional domains and key phosphorylation sites is also conserved between the two enzymes. The observation that dS6K phosphorylated rat S6 in both a rapamycin- and wortmanninsensitive manner further suggested that upstream elements and downstream effecters are conserved in mammals and flies (140, 141). In order to study the functional role of dS6K, Montagne and colleagues characterized a female sterile mutation (142), which they showed contained a P-element insertion in the dS6K 5’UTR (143). Only 25% of the expected number of homozygous flies emerged as adults, with a 3-day delay in development and reduced body size. That mutant phenotype observed was due to insertion of the P-element into the dS6K gene, demonstrated by showing that all aspects of the mutant phenotype were rescued by either excision of the Pelement or by expression of a dS6K transgene or transgenes expressing either of the two mammalian S6Ks. Because the P-element insertion into the dS6K gene resulted only in decreased expression of S6K, a more severe allele was obtained by generating imprecise excisions that removed part of the gene. Only a few of those homozygous flies survived, emerging after a 5-day delay in development, living a maximum of 2 weeks and showing an almost 50% reduction in body size. Morphologically, all body parts were reduced in size to the same extent (143). Given that growth is dominant over proliferation (2), it was rationalized that the reduction in body size of homozygous dS6K mutant flies was the result of decreased cell number. This possibility was tested by counting the number of cells in the wings and in the eyes. Surprisingly, the results revealed that the cell size was reduced, but not the cell number. Furthermore, analysis of the developing wing disc revealed that that the effect on cell size was displayed throughout development. Further experiments demonstrated that mutant cells were proliferating at half the rate of wild-type cells and that that the decrease in the rate of proliferation was exhibited equally throughout all stages of the cell cycle. Because S6Kl has been implicated in the regulation of insulin and IGF-2 (136, 137), it raised the possibility that the effect on cell size was a humoral response. However, the generation of genetically marked homozygous mutant dS6K somatic clones in a heterozygous background revealed that the effect was cell autonomous. Consistent with this conclusion, expression of an extra copy of the dS6K gene in the dorsal compartment of

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the wing caused only those cells and not the cells in the ventral compartment to grow larger. Thus &6K regulates cell size, growth, and proliferation in a cell autonomous manner without impinging on cell number. Because S6K is implicated in the regulation of translational components that make up the protein synthetic apparatus, the dS6K mutant was speculated to mimic ribosomal protein mutants, termed Minutes, a name derived from the strong reduction in bristle size (144). Surprisingly, the phenotype of the BS6K mutant was quite distinct from that of Minutes. Although both were delayed in development, consistent with a reduced ribosome content, dS6K mutants did not exhibit a strong reduction in bristle size and Minutes displayed a wild-type cell and body size (143). Taken together, these data imply that the defect in the number of ribosomes is not the only reason for the observed phenotype in dS6K mutants. It is possible that dS6K is involved in the modulation of a putative cell size regulator. Alternatively, the difference between dS6K mutant cells and Minute cells could be that the &6K-deficient cells, in addition to a decreased number of ribosomes, have a decreased level of phosphorylated S6, which could affect cell size through favoring the translation of mRNAs lacking a 5’TOP. If such mRNAs encoded cell cycle regulators, such regulators could reach a critical concentration at a smaller cell size. Evidence for such a model has been presented for CDC25A in Xenopus mitotic cell cycles (59).

V. Future Perspectives A great deal of information about the mechanism of S6Kl activation has been obtained by dissecting the primary structure of the enzyme into domains and identifying critical regulatory phosphorylation sites. However, less progress has been accomplished in identifying upstream regulators, which are responsible for phosphorylation of critical regulatory sites of S6Kl. Only PDKl kinase has been identified as a direct in vivo and in vitro modulator of S6Kl activity. The genetics of Drosophila will certainly be an important tool in elucidating new components of this signaling cascade. Of particular interest is the molecular mechanism by which mTOR functions to bring about S6Kl activation and 4E-BP1 phosphorylation. Another important field of endeavor will be the identification of signaling components involved in amino acid activation of S6K and determination of the relationship of this signaling pathway to insulin signaling and the regulation of autophagy. Mice deficient in S6Kl and S6K2 will certainly aid in the determination of differential functions of these two isoforms in vivo. Finally, a major task will be to determine the role of S6 phosphorylation in mediating S6K response, both in mammals and in other model organisms such as Drosophila melanogaster.

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Inhibition of the mTOR/SGK signaling pathway by rapamycin has shown great promise as an immunosuppressant for use in organ transplantation. Selective pharmacological manipulation of this signaling cascade, which is intimately involved in cell growth, may be useful in modulating the proliferative response associated with such diseases as cancer.

ACKNOWLEDGMENTS We thank P. B. Dennis for a critical reading of the manuscript. We are also grateful to the European Economic Community (Grant Number BI04-CT97-2071) and Novartis Research Foundation for their financial support.

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KWAN”

*LX&ion

of Medicinal Chemistry College of Pharmacy *The lnstitute for Neuroscience ?he Institute fw Cellular and Molecular Biology University of Texas Austin, Texas 78712

I

I. Gene Cloning and Structure ........................ A. Cloning and Sequencing of MAO A and B cDNAs ... B. Chromosomal Location and Gene Organization ..... II. FunctionalRegionsofMAOB ....................... A. Strategy for Identifying Crucial Amino Acid Residues B. Dinucleotide-Binding Site ....................... C. Second FAD-Binding Site ........................ D. Fingerprint Site ................................ E. Covalent-Binding Site ........................... E Active Site ..................................... G. Other Potential Targets .......................... III. Tissue and Cell Distribution ........................ . Physiological Functions ............................ V FuturePerspectives ................................ References .......................................

Monoamine degrading

A and B) are the major neurotransmitter-

enzymes in the central nervous system and in peripheral

A and B cDNAs deduced

oxidase A and B (MAO

131 131 132 135 135 136 141 144 14-i 148 149 149 150 152 152

..

tissues. MAO

from human, rat, and bovine species have been cloned and their

amino acid sequences

enzyme shows approximately

compared.

Comparison

70% sequence

of A and B forms of the

identity, whereas comparison

A or B forms across species reveals a higher

sequence

of the

identity of 87%. Within

these sequences, several functional regions have been identified that contain crucial amino acid residues participating

in flavin adenine dinucleotide

strate binding. These include a dinucleotide-binding

(FAD) or sub-

site, a second FAD-binding

site, a fingerprint

site, the FAD covalent-binding

brane-anchoring

site. The specific residues that play a role in FAD or substrate

binding were identified by comparing with those in soluble flavoproteins

Progress in Nucleic Acid Research and Molecular Biology, Vol. fi5

site, an active site, and the mem-

sequences in wild-type and variants of MAO

of known structures. The genes that encode

129

Copyright 0 2001 IJ~ Academic Prraa. All rights ofrqxoduction in my form resewed. 0079-6603~01 $35.00

130

CREED

W. ABELL

AND SAU-WAH

KWAN

MAO A and B are closely aligned on the X chromosome (Xp11.23), and have identical exon-intron organization. Immunocytochemical localization studies of MAO A and B in primate brain showed distribution in distinct neurons with diverse physiological functions. A defective MAO A gene has been reported to associate with abnormal aggressive behavior. A deleterious role played by MAO B is the activation of I-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a proneurotoxin that can cause a parkinsonian syndrome in mammals. Deprenyl, an inhibitor of MAO B, has been used for the treatment of early-stage Parkinson’s disease and provides protection of neurons from age-related decay. 8 zoooAcademic press.

Monoamine oxidase A and B (MAO A and B) are the major intracellular enzymes in the brain and in peripheral tissues that degrade neuroactive and vasoactive amines (1). They can also metabolize potentially harmful substances found in the environment (xenobiotics), including protoxins that enter the central nervous system (2). MAO A and B are thought to be linked to psychiatric and neurological disorders such as depression and Parkinson’s disease (PD), respectively. For example, MAO A and B are the targets of drugs that are used to treat depression (moclobemide, an MAO A inhibitor) (3) and PD (deprenyl, an MAO B inhibitor) (4 - 6). C urrent interest in the A form of the enzyme has been intensified by the discovery of a defective MAO A gene, which encodes a tnmcated enzyme in some members of a Dutch family that exhibit abnormal aggressive behavior (7). Several strains of knockout mice, each harboring a different inactivated gene (encoding MAO A, 5-HTlB receptor, o-calmodulin-dependent protein kinase II, or nitric oxide synthase), exhibit increased aggressive behavior (8-9). The existence of defective genes other than that for MAO A illustrates the complex nature of the determinants associated with aggressive behavior. Furthermore, MAO A deficiencies in the general population and in “putative” high-risk groups (e.g., males with adult attention deficit hyperactivity disorder) appear to be highly uncommon (IO). An alternate form (allele) of the MAO B gene has been identified in patients with PD (II), suggesting that an inherited variant form of MAO B may be associated with a predisposition for this disorder. However, other allelic association studies of PD for both MAO A and B failed to find polymorphisms (12). Other recent genetic studies have identified a defective gene on chromosome 4 that is responsible for a relatively rare form of hereditary PD (13). This gene encodes c-w-synuclein,a presynaptic protein that is thought to be involved in neuronal plasticity. Interestingly, ol-synuclein was identified as the precursor protein for the non+-amyloid component of plaques in Alzheimer’s disease. An inverse relationship between cigarette smoking and risk of developing PD has been observed (14). A possible explanation for neuroprotection

MAO STRUCTURE

131

AND FUNCTION

is that one or more components in cigarette smoke inhibit MAO B activity. In fact, cigarette smokers have been found to have 28 and 40% lower MAO A and B activities, respectively, than either nonsmokers or former smokers (15, 16). Because dopamine is degraded by MAO B, elevated levels of dopamine could augment the addictive effect of nicotine in smokers. A recent study has shown that a genetic polymorphism of MAO B eliminated this protection (17). This finding supports the concept that MAO B is linked to PD, but the definitive relationship remains unknown. The purpose of this review is to focus on the structural and functional relationships of these two important neurotransmitter-degrading enzymes. Because it is not possible to reference the many publications on different aspects of this topic, additional review articles on MAO A and B are included (18-22).

I. Gene Cloning and Structure A. Cloning

and Sequencing

of MAO

A and B cDNAs

MAO A and B are integral proteins of the outer mitochondrial membrane (22) and can be distinguished by differences in substrate preference (23), inhibitory specificity (24), tissue and cell distribution (25, 26), and immunological properties (27, 28). Despite these differences in the characteristics of MAO A and B, the true identity of their fundamental nature was unresolved until we and our collaborators (29) isolated and characterized the human liver cDNA clones that encode these enzymes and determined their nucleotide and deduced amino acid sequences. Comparison of sequences of MAO A and B shows that these enzymes have a very high degree of identity (approximately 700/o),but different amino acid residues occur at the same place throughout the polypeptide chains. This indicates that MAO A and B are derived from at least two separate genes rather than by a splicing mechanism. Cloning and sequencing studies (see Fig. 1) in other laboratories and by us have yielded the deduced amino acid sequences for human liver MAO A and B (29), human placental MAO A (30), bovine adrenal MAO A (31), rat liver MAO A and B (32-34), and bovine liver MAO B (Kwan and Abell, Fig. 2). Direct amino acid sequencing of about 30% of bovine liver MAO B has been reported (31). We have cloned and sequenced the entire MAO B cDNA from a bovine liver cDNA library (in the Uni-Zap XR vector, from Stratagene). All oligonucleotide primers were custom synthesized by National Biosciences. The nucleotide and deduced amino acid sequences are shown in Fig. 2. The latter sequence is also included in Fig. 1 to provide easy comparison with MAO B from rat and human tissues.

132

CREED

W. ABELL AND SAU-WAH KWAN

MAO A or B from bovine, rat, and human species (Fig. 1) will show strikingly high similarity in their amino acid sequences, and they contain motifs that are found in the vast majority of flavin-requiring enzymes (e.g., a dinucleotide-binding site) (35-37). Comparison of MAO A or B across species (human, rat, and bovine) shows 86-880/o identity, with the exception of bovine versus human MAO B, which is slightly higher (910/o).Sequence identity between MAO A and MAO B is approximately 70% across all species. Human platelet and frontal cortex MAO B were also compared and were found to have amino acid sequences identical to those of MAO B from human liver (38).

B. Chromosomal

Location and Gene Organization

Earlier work using somatic cell mouse/human hybrid lines indicated that the MAO A and B genes are located on the X chromosome (39, 40). Use of cDNA probes and in situ hybridization studies has confirmed that MAO A and B are located in close proximity within Xp11.23-11.4 (41, 42). Linkage analysis in reference pedigrees placed the MAO A locus between DXS14 and OTC (43). Furth ermore, the MAO A and B genes were found to be proximal to each other and deleted in patients with Norrie disease, a neurological disorder that is a result of a microdeletion on the short arm of the X chromosome (41, 42). Altered MAO genes that are associated with specific diseases have not been identified in human subjects, with the exception of the Dutch pedigree having defective MAO A associated with aggressive behavior (7). However, the pentameric distribution of platelet MAO B activity in healthy men (44) supports the notion that genetically determined variations in MAO occur in the human population. Identification and characterization of these variants await future genetic studies. For studies on gene organization, the human MAO A and B genes were isolated from X chromosome genomic libraries (45-47). Both genes were found to contain 15 exons, and they exhibited identical exon-intron organization. Examination of the promoter sequences in these genes revealed that fragments responsible for transcriptional activation contained GC-rich sequences and potential Spl binding sites, and had 60% sequence identity (48). The promoter region of the MAO A gene has extensive repetitive structures, including two 90-bp tandem repeats, but no TATA or CCAAT box sequences

FIG. 1. Comparison of deduced amino acid sequences of bovine (b), rat (r), and human (h) MAO A and B. Identical amino acids at the same site are indicated by an overline. The C residue that binds FAD covalently is indicated by an underline. The different groups of amino acids are color coded: red, charged; blue, polar: green, hydrophobic; and black, neutral. Sources: bMA0 A (3I); rMA0 A (32, 34); hMA0 A (29, 30); hMA0 B (29); rMA0 B (33); bMA0 B (Fig. 2).

s~IPPT~TAKI~F~~ELPS~~~QLTQXLPY SAIPPILTAKIBPRPPLPPERNQLIQRLPN NAIPPTLTAKIBPRPBLPAPRNQLIQRLPM SAIPPTLQYKIEFNPPLPMMRKQMITRVPL SAIPPVLQYKIBESPPLPILRNQLITRVPL SAVPPVLQMKIBFNPPLPYYRNQLITRVPL

300 300 300 291 291 291

-GAVIKCYYYYKPAFWKKKDYCQCMIIBD~E

-

-

-

-

330 330 330 321 321 321

QAVIKCYVYYKBABWKKKDYCQCMIIEDKK QAVIKCYYYYKEAFWKKKDYCQCMIIBDBD GSVIKCIVYYKPPPWRKKDYCQT~IIDGEB GSVIKCYVYYKBPFWRKKDPCGT~VIl?GE~ GSVIKSIVYYKPPFWRNYDYCQSMIIBGBE WROA -A -A

bUAOB -B ki43OB

-A MAOA -A

-

~ZISITLDDTKPDQSLP~MQFILARKADR APIAITLDDTKPDQSLPAIYQFILARKADR APISITLDDTKPDGSLPAIYGFILARKADR APVAYTLDDTKPBQNYAAIMQFILABKARK APIAYTLDDTKPDAGCAAIYGFILABKARK APVAYALDDTKPDGSYPAIIQFILABKARK -

-

-

360 360 360 351 351 351

---

LAKVRKDIRKR~ICBRYAKVLG~QEALH~

390 390 390 361 361 361

mB IHAOB bYK)B

LAKLBKDIRKRKICELYAKVLGSQZALYPV LAKLBKBIRKKKICELYAKVLGSQEALHPV LARLTKPERLKKLCELYAKVLGSLEALEPV LVRLTKPERLRKLCELYAKVLNSQEALQPV LARLTKEBRLKKLCDLYAKVLQSQXALHPV

bURGA WA bMAOA MJAQB IZNAOB kWAOB

BYPEKNWCQPQYSGQ~YTAYFPPDIYTPYe BYPBKNWCBBQYSGQ~YTAYRPPGIMTQYG RYPEKNNCPBQYSQG~YTAYFPPQIKTQYQ HYEEKNWCEBQYSQG~YTTYFPPGILTQYQ RY~BKNWCPBQYSQQ~YTAYFPPQILTQYG BYEEKNWCEEQYSQQ~YTSYFPPQIMTQYQ

420 420 420

-A -A -A

RVIRQPVQR~YFAQTBTATQ~SQY~KQA~R RVIRQPVGRIYFAGTETATQWSQYMEGAVB RVIRQPVQRIFFAGTBTATKWSGYMEQAVE RVLRQPVDRIYFAQTETATHNSGYMEQAVA RVLRQPVQKIFFAQTETASEWSQY~EQAVE RVLRQPVQRIYFAQTETATHWSGYMEGAVE

450 450 450 441 441 441

hM4OB HAOB bMKIB BUAOA -A blRoA

h?AOB dRL)B kUAOB

AQERAARBVLNALQKLSARDIWIQEPEAE~

411 411

411

--

480

AGPRAAREVLNALGKVAKKDIWVRAPBSKD 460 471 471 471

AGERAARPVLNQLQKVTBKDIWVQEPESKD AQERAAREILBANGKIPEDEIWQSBPESVD AQBRAARPILHAIGKIPEDEIWQPEPESVD AQBRAARPILBAYQKIPBDEIWLPEPl3SVD --VPAVBITPSFWERNLPSVSQLLKIVQ~STVPAIBITRTFLPRNLPSVPQLLKITGVSTVPAVZITBTFIPRNLPSVSQLLKIIQPSTVBAQPITTTBLERELPSVPQLLRLIGLTTI VPARPITNTBLBRRLPSVPQLLKLLGLTTI VPAKPITTTFLQRBLPSVPQLLKLIQLTTI

-

-

509 509 509 501 501 501

-SITALWFVYYRFRLLSRS -SVALLCFVLYKIKKLPC -SVTALQFVLYKYKLLPRS FSATALQFLAAKRQLLVRV LSATALQFLAEKKQLFVRB FSATALQYLAAKRQLLVRI

527 521 527 520 520 320 FIG.

1

continued

WAOA WOA BNAOA hlAOB MAOB M4AOB

30 30 30 21 21 21

YESLQKTSDAQQ~FDVV;*GGQISGL~**K

MTDLEKPNLAQHYFDVVVIQQQISQLAAAK MENQPKASIAQHYFDVVVIQQQISQLSAAK MSNKCDVVVVQQQISGMAAAK MSNKCDVIVVQQQISGYAAAK HSSKCDVVVVQGQISGHAAAK -

-LLAEHEVNVLVLEARERVGQRTYTVRNES~ LLSRYKINVLVLEARDRVGGRTYTVRNERV LLTPYQVSVLVLEARDRVGQRTYTIRNEHV LLHDSQLNVVVLEARDRVGGRTYTLRNQKV LLHDCGLSVVVLEARDCVQGRTYTIRNKNV LLHDSGLNVIVLEARDRVGQRTYTLRNQKV

60 60 60 51 51 51

k44AOA rMAOA hMAoA lMAOB z?dAOB bMAOB

DYVDV=AYVQPTQNRILRLS;Q~LETYK KWVDVGGAYVQPTQNRILRLSKRLQIETYK DYVDVQQAYVGPTQNRILRLSKELQIETYK KYVDLGQSYVQPTQNRILRLAKRLGLETYK KYVDLQGSYVGPTQNRILRLAKELQLETYK KYVDLGQSYVQPTQNHILRLSKELGLETYK

-A -A mAoA hMAOB ZM?LOB bUAOB

VNVNERLVHYVKQKTIPFRGAGPVWNPIA VNVNBRLVQYVKGKTYPFRGAFPPVWNPLA VNVSERLVQYVKQKTYPFRQAFPPVWNPIA "NEVERLIHHVKQKSYPFRGPFPPVWNPIT VNEVERLIHFVKGKSYAFRQPFPPVWNPIT VNEVBRLIHHTKQKSYPFRGSFPSVWNPIT

MAOA HAOA hNFaA hMAOB SMAOB LMAOB

YLDY~LWRTM;NM(IKEIPADAPWR~PH;V YLDYNNLWRTMDEMGKEIPVDAPWQARHAQ YLDYNNLWRTIDNHQKEIPTDAPWEAQHAD YLDHNNFWRTMDDMQREIPSDAPWKAPLAE YLDYNNLWRTHDEHGQEIPSDAPWKAPLAE YLDHNNLWRTHDDYGREIPSDAPWKAPLAE

150 150 150 141 141 141

bNAoA -A maoA hMAOB r-B bMAOB

-EWDKHTMKD;IEXICWTKTARQF;SLFVNI EWDKHTNKDLIDKICWTKTAREFAYLFVNI KWDKHTMKELIDKICWTKTARRFAYLFVNI EWDNYTMKELLDKLCWTESAKQLATLFVNL EWDYYTMKELLDKICWTNSTKQIATLFVNL QWDLHTYKELLDKICWTESSKQLAILFVNL

180 180 180 171 171 171

N~S;PHEVSALWFLWYVKPCQQTTflF~I NVTSEPHEVSALWFLWYVRQCQGTARIFSV NVTSEPHEVSALWFLWYVKQCQGTTRIFSV CVTABTHEVSALWFLWYVKQCQGTTRIIST CVTAETHEVSALWFLWYVKQCQQTTRIIST CVTAEIHEVSALWFLWYVKQCGQTTRIFST

210 210 210 201 201 201

90 90 90 81 81 81 120 120 120 111 111 111 --

H6AOA HADA MAOA

WAOB WAOB bMAOB

-mmoa MAOA hNAoA hWAOB MAOB UAOB

TNGQQERKFVGQSQQVSER;MQLLQDRVKL TNQQQERKFVQQSQQVSPQIMGLLGDKVKL TNQGQERKFVGGSGQVSERIMDLLGDQVKL TNGQQERKFVQQSGQVSERIMDLLGDRVKL

MAOA -A IINAOA hWAOB -B MAGB

R~PUTY~DQSSRNITVETLNREL~ECRYVI

240 240 240 231 231 231

TNGGQBRKFIGGSGQVSERIKDILGDRVKL

SNGGQERKFVGQSGQVSERIMDLLGDRVKL

270 270 270 261 261 261

SSPVTYIDQTDDNIIVETLNKEHYRCKYVI NHPVTHVDPSSDNIIIETLNHEHYECKYVI ERPVIYIDQTRENVLVETLNHEMYEAKYVI ERPVIHIDQTQENVVVKTLNHEIYEAKYVI

ERPVIHIDQTGENVLVETLNHELYRAKYVI

FIG.

1

CTGCAGGCGG

GGGCCGAGAT

CCAGACACCG

AAGCAGCTGG

CACCGGGTAG

CCCGGAGAGG

GGCGAGCAAC

ATG AGC AGC AT&IiTGC GAC GTG GTC GTG GTG GGG GGC GGC ATC TCA GGT ATG GCA GCA GCC MSSKCDVVVVGGGISGMAAA

-70 60 20

AAA CTT CTA CAT GAC TCT GGC TTG AAT GTG ATT GTT CTG GAA GCC CGG GAC CGC GTG GGA KLLHDSGLNVIVLEARDRVG

120 40

GGC AGG ACT TAC ACC CTT AGG AAC CAA AAA GTT AAA TAT GTG GAC CTT GGA GGA TCT TAT GRTYTLRNQKVKYVDLGGSY

180 60

GTT GGG CCA ACT CAG AAT CAT ATC TTA AGA TTA TCC AAG GAG CTA GGA TTA GAA ACC TAC VGPTQNHILRLSKELGLETY

240 80

AAG GTG AAT GAA GTA GAG CGT CTG ATT CAC CAT ACA AAG GGC AAA TCC TAC CCC TTC AGG KVNEVERLIHHTKGKSYPFR

300 100

GGC TCA TTC CCG TCT GTG TGG AAT CCT ATC ACC TAC CTA GAT CAT AAC AAC CTC TGG AGG G S F P S V W N P I T Y L D H N N L W R

360 120

ACG ATG GAT GAC ATG GGA CGA GAG ATT CCC AGT GAT GCC CCG TGG AAG GCA CCC CTT GCA TMDDMGREIPSDAPWKAPLA

420 140

GAA CAG TGG GAC CTG ATG ACA ATG AAG GAG TTG CTG GAC AAG ATC TGC TGG ACA GAA TCT EQWDLMTMKELLDKICWTES

480 160

TCA AAG CAG CTT GCT ATT CTC TTT GTG AAC CTT TGC GTC ACT GCA GAG ATC CAT GAG GTC S K Q L A I L F V N L C V T A E I H E V

540 180

TCC GCT CTC TGG TTC CTG TGG TAT GTG AAG CAG TGT GGG GGC ACG ACC AGG ATC TTC TCA SALWFLWYVKQCGGTTRIFS

600 200

ACA TCC AAT GGA GGG CAG GAG AGG AAA TTT GTG GGT GGA TCT GGT CAA GTG AGT GAG CGG T S N G G Q E R K F V G G S G Q V S E R

660 220

ATA ATG GAC CTC CTG GGG GAT CGA GTG F&G CTG GAG AGG CCT GTG ATC CAC ATT GAC CAG I M D L L G D R V K L E R P V I H I D Q

720 240

ACA GGA GAA AAT GTC CTT GTG GAG ACC CTA AAC CAT GAA TTG TAC GAG GCT AAG TAC GTG K Y V T G E N V L V E T L N H E L Y E A

780 260

ATT AGC GCT OTT CCT CCT GTT CTG GGC ATG AAG ATT CAC TTC AAT CCC CCT CTG CCA ATG I S A V P P V L G M K I H F N P P L P M

840 280

ATG AGA AAT CAG CTG ATC ACT CGT GTG CCT TTG GGT TCA GTC ATC AAG AGT ATA GTT TAT MRNQLITRVPLGSVIKSIVY

900 300

TAT AAA GAG CCC TTC TGG AGA AAT ATG GAT TAC TGT GGA AGC ATG ATT ATT GA?+ GGA GAG YKEPFWRNMDYCGSMIIEGE

960 320

GAA GCT CCA GTT GCC TAT GCA TTG GAT GAT ACT AAA CCT GAT GGC AGC TAT CCT GCC ATA E A P V A Y A L D D T K P D G S Y P A I

1020 340

ATA GGA TTT ATC CTT GCC CAC AAA GCC AGA AAG CTG GCT CGT CTT ACC AAG GAG GAA AGG IGFILAHKARKLARLTKEER

1080 360

FIG. 2. The nucleotide and deduced amino acid sequences of bovine liver MAO B. The amino acid numbering starts at the amino-terminal residue, and the nucleotide sequence is shown above. The cysteine residue to which flavin adenine dinucleotide is covalently bound and the polyadenylation signal sequences are underlined. The stop codon is indicated by an asterisk.

134 TTG AA0

AAA

CREED W. ABELL AND SAU-WAH KWAN CTC

TGT GAC CTC TAT GCA AAG GTT CTG GGC TCA CAA

GAA GCT TTG CAC CCA

LKKLCDLYAKVLGSQEALHP

1140 380

GTG CAC TAT GAA GAG AA0 AAC TOG TGT GAG GAG CAG TAC TCC GGA GGC TGC TAC ACT TCC VHYEEKNWCEEQYSGGCYTS

1200 400

TAC TTC CCT CCT GGG ATC ATG ACT CAA TAT GGA AGG GTT CTA CGC CAG CCA GTG GGC AGG YFPPGIMTQYGRVLRQPVGR

1260 420

ATT TAC TTT GCA GGC ACA GAG ACT GCC ACA CAC TGG AGT GGC TAC ATG GAG GGG GCT GTG

1320 440

IYFAGTETATHWSGYMEGAV GAG GCT GGC GAG AGA GCG GCC CGA GAG ATC CTG CAT GCC ATG GGC AAG ATC CCA GAG GAT EAGERAAREILHAMGKIPED

1380 460

GAA ATC TGG CTG CCT GAA CCA GAG TCT GTG GAT GTC CCT GCG AAG CCC ATC ACC ACC ACC E I W L P E P E S V D V P A K P I T T T

1440 480

TTC TTG CAA AGG CAT TTG CCC TCC GTG CCA GGC CTG CTG AAG CTG ATT GGA TTG ACC ACC F L Q R H L P S V P G L L K L I G L T T

1500 500

ATC TTT TCA GCA ACT GCT CTC GGC TAC CTG GCC CAC AAG AGG GGG CTC CTG GTG CGA ATC IFSATALGYLAHKRGLLVRI

1560 520

TAA

1563

??

AGAGAGGGCA

TCTGTAACCA

CACCCTGGTG

TGTGGGTTTG

GGGGAAGGCA

TTGT-

GTTCCACAAA

1633

GATGCAAAGA

ATGTAGAGTG

AGGCGGCGAG

CATGATGATC

AGTCAGACTT

TCTGACCACA

GGATACACAG

1703

TCTCTTTCTC

CATTTTGACA

CCTGTGTATT

GTCTAGTACC

TAGCTTAGCA

CTGTCTCACC

CACTTCCAAG

1773

TTCACTGGCT

CCAGAATCTT

TACAGTAGTT

AAATTGGCTT

GTGAAAGGTC

CTTGCTATCC

TACTATACAT

1843

TGCCCAGGCA

CACACACACA

CACACACACA

CACACACACC

CCCACACACA

CACTACTTTT

TTCTTACCTC

1913

TATGGCTTTG

TGCTTGTCCT

TCCTCTTTCC

TGTAATGTCC

ACAACCTTCC

AGGTTCTCTG

CATTTGTCCT

1983

TAGAATCCCA

TATTGTTACA

GCTGGAAGAA

CTTAGACACC

ATCCAACCCT

TACTTTCTAT

TTTAGAGTTG

2053

AGCAAACTGA

AGCGGAGAGA

GGAGGAACTT

AATG‘XTCAG

TGTCCACAAT

AAGCCACTGA

TATTTTGGTG

2123

ACTAGGACAC

AGGTCCATTG

CTTTATCCCA

TCTCTCTGGA

TGGATTGCCC

AATCACCCTT

CTCTACTCCC

2193

TGCCAAGGTC

GCTGTGTTCC

CTTGGGTAGG

TTTACTCTGT

ACTAAGCTGT

TTTGTGCTGC

TCAGATGCTA

2263

CTACTCAGTA

TATATCCTTA

AGTCTTACCG

TCTTGCGCAG

TGTGCCTTCA

GCTCATTTTA

CTTTTTTTTC

2333

ATGGTAAGAG

TTCTTGTCTT

TTCTTCCTTT

TGTATCCTCC

ACTGAATCTG

GATACAAAGG

TTGGTGCACA

2403

TTTGGGTAAT

TCAA&TAU

AG-

AAAAAAAAAA

AAACTCGAGG

GGG

2466

G TTGATTGACC

FIG. 2. (continued).

MAO STRUCTURE

AND FUNCTION

135

(48, 49). The promoter activity was inhibited by upstream sequences (48). However, this negative cis regulation was not observed, but a putative initiator (Znr) element was detected (49). Furth er study showed that the presence or absence of Znr-like sequence in promoter constructs did not appear to affect significantly the negative cis regulation in the MAO A gene (50). For the MAO B gene, the promoter fragment consisted of two clusters of overlapping Spl sites [see Shih et al. (21) for a discussion of transcriptional regulation of MAO A and B]. Further study of these elements should increase our understanding of how MAO A and B are regulated and differentially expressed in catecholaminergic and serotonergic neurons, respectively. Knowledge of gene expression at the molecular level may ultimately lead to identification of new determinants (e.g., hormones, vitamins, calcium) that can alter MAO A and B activities in humans.

II. Functional Regions of MAO B A. Strategy for Identifying Crucial Amino Acid Residues The long-term goal in many laboratories is to determine the three-dimensional structure of MAO A and B in order to facilitate an understanding of the molecular and catalytic properties of these enzymes. Once the structures of MAO A and B are known, computer-assisted approaches can be applied to design novel drugs for the treatment of selected psychiatric and neurological disorders. Unfortunately, because MAO A and B are integral proteins of the outer mitochondrial membrane (22), they have resisted crystallization. An alternative approach, which is less definitive but more accessible, is to use site-directed mutagenesis (SDM) to help identify amino acid residues that reside in putative functional regions of these enzymes or to prepare and test hybrids from chimeric constructs of MAO A/MAO B cDNAs. Earlier work had shown that both MAO A and B are flavoproteins. They contain a pentapeptide with a cysteine residue that is covalently coupled to the required cofactor, FAD, through a thio ether linkage (51). Initially, it was thought that one FAD was covalently bound to an enzyme molecule (llO120 kDa) that is composed of two identical subunits (52). However, reexamination of the ratio showed one coupled FAD per subunit of 60 - 65 kDa (53). Moreover, bovine MAO B can exist as a dimer, but the enzyme preferentially functions as larger oligomeric complexes (54). A useful starting point is to examine data bases of soluble flavoproteins of known structures. Using this approach, a few short sequences (motifs) from 5 to 34 amino acid residues long were identified in several flavopro-

136

CREED

W. ABELL

AND SAU-WAH KWAN

teins, including MAO. Furthermore, structural studies of soluble flavoproteins have identified specific amino acid residues that likely interact with its riboflavin cofactor or substrate. To assess whether analogous interactions occur in MAO, mutants that encode variants of MAO proteins were prepared and transiently expressed in COS-7 cells, a mammalian cell line that permits expression and comparison of variants with the wild type (55). Type-specific antisera and monoclonal antibodies that recognize either MAO A or MAO B (27, 55) were used in Western blots to monitor the level of variant expression and showed that it was approximately the same level as the native enzyme. FAD covalent- and noncovalent-binding studies and catalytic activity assays were also performed in parallel on crude extracts of the enzyme. Because the expression of MAO B protein could be quantitated by enzyme-linked immunoassays (ELISAs) with a type-specific monoclonal antibody (e.g., MAO B-lC2) (27), it is possible to express catalytic activity as enzymatic activity (kmol/min/mg MAO B). Furthermore, results derived from one form of the enzyme (e.g., MAO B) can be used to gain insight into the other form (MAO A) because identical amino acid sequences are found in both enzymes in all the functional motifs identified thus far. By using site-directed mutagenesis (56, 57) to examine selected amino acids that are thought to play a role in function, several FAD-binding sites and a putative substrate-binding site were identified in MAO B. These include a dinucleotide-binding site (DBS), a second FAD-binding site (SBS), a fingerprint site (FPS), the FAD covalent-binding site (CBS), and an active site (ACT). The membrane-anchoring site (MEM) has also been identified (58). A schematic representation of our current knowledge of these functional regions in MAO B is shown in Fig. 3.

B. Dinucleotide-Binding

Site

The crystal structures of glutathione reductase, p-hydroxybenzoate hydroxylase, lipoamide dehydrogenase, and other flavoproteins contain a dinucleotide-binding motif (Br-o-BJ that interacts with FAD (36,59). This motif has a consensus sequence of Asp-Val-Val-Val-Ile-Gly-X-Gly-X-X-Gly-LeuX-X-Ala-X-X-Leu-X-X-X-X-X-X-Val-X-Val-Leu-Glu (60) (see Fig. 4). Highly conserved hydrophobic residues are located at positions 7-10, 17,20,23, 30,32, and 33 in MAO B. In contrast, a highly conserved hydrophilic residue is present at position 6 (Asp), and the motif ends with a glutamate residue at position 34. Within this motif, the Gly11-X-Gly13-X-X-Gly16 sequence in MAO B is identical to a sequence found in many flavoproteins (37), and it is likely to constitute a turn between the first B-sheet and the beginning of the a-helix. The y-carboxylate group of Glu-34 is thought to bind to the 2’-hydroxyribose of the AMP moiety of FAD to align this cofactor for participa-

MAO STRUCTURE

137

AND FUNCTION

H273

a

N I

0

b

d

C

I 40

T42a

I

I 60

120

160

200

240

280

320

e

f

I

I

I

360

400

440

I 460

c

I 620

Amino Acid Residue FIG. 3. Putative functional regions and active residues in human MAO B. (a) Dinucleotidebinding site; (b) second FAD-binding site; (c) fingerprint site; (d) covalent-binding site; (e) active site; (f) anchor to outer mitochondrial membrane.

tion in the oxidation-reduction cycle during the oxidative deamination of amines to their corresponding aldehydes. To test the postulated role of Glu-34 in dinucleotide binding, several mutant cDNAs of human MAO B were prepared and transiently expressed in COS-7 cells (55). Variants E34Q and E34A were devoid of activity, and the E34D variant was only 7% of wild-type activity (see Table I for a comparison of the enzymatic activities of all variants with their corresponding wild type). It was not unexpected that the E34A variant was inactive, because Ala has a more hydrophobic character than Glu and does not carry a negative charge. The Gln-34 residue in E34Q is closest to the wild-type Glu-34 in size, but as expected, this variant had no activity because it also lacks a negative charge. However, the dramatic decrease in activity in the variant E34D was not anticipated because Asp contains a negative charge and is similar in size to Glu. The low activity presumably is due to the shorter side chain of the Asp residue, resulting in a decrease of contact between its B-carboxylate side chain and the 2’-hydroxy group of the ribose in FAD. An additional highly conservative mutant (WOI) was also constructed and expressed (Table I). This mutant encoded a variant protein containing Ile-10 in place of Val-10, which is positioned at the end of the first B-sheet immediately before the Gly11-X-Gly13-X-X-Gly16 turn. Analysis of VlOI showed that it had only moderate activity (approximately 6500) compared to the wild type. These results demonstrate that even the most conservative substitutions of essential residues of the Br-c~-Ba motif had a pronounced effect on enzyme activity. This suggests that the pi-o-B2 motif has a precise conformation that accommodates FAD recognition and/or binding to this functional region (Fig. 4).

138

CREED

S

W. ABELL

AND

SAU-WAH

KWAN

ci”D

G

H

L

L L K

N A

A

V

A