Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, .... cyclase as a member of a novel receptor/enzyme family. EVIDENCE. FROM.
The
guanylate
cyclase/receptor
STEPHANIE SCHULZ,*,t MICHAEL CHINKERS,*? AND *Howard Hughes Medical Institute and tDepartments of Pharmacology Vanderbilt
University
School of Medicine,
Nashville,
ABSTRACT Guanylate
cGMP
cyclase,
which
catalyzes
the
formation
of
from GTP, exists in both the soluble and partic-
ulate fractions of cells. At least two different cellular compartments for the particulate enzyme exist: the plasma membrane and cytoskeleton. The enzyme form found in the soluble fraction is a heterodimer that can be regulated by free radicals and nitrovasodilators, whereas
the
membrane
form
exists
as
a single-chain
polypeptide that can be regulated by various peptides. These peptides include resact and speract obtained from eggs and atrial natriuretic peptides (ANP). The species tions
of guanylate resists
cyclase
solubilization
present with
in cytoskeletal non-ionic
frac-
detergents;
its structural properties are not yet known. cDNAs encoding the membrane form of guanylate cyclase have been isolated from different tissues and species, and in all cases the DNA sequences predict a protein containing a single transmembrane domain. The carboxyl (intracellular) domain is highly conserved from sea urchins through mammals, whereas the extracellular domain (amino terminus) varies considerably. The predicted amino acid sequences demonstrate that the membrane form of guanylate cyclase is a member of a diverse and complex family of proteins that includes a low molecular weight ANP receptor, protein kinases, and the cytoplasmic form of guanylate cyclase. cDNA encoding a membrane form of the enzyme from mammalian tissues has been expressed in cultured cells, and the expressed guanylate cyclase specifically binds ANP and is activated by ANP. The membrane form of guanylate cyclase, then, serves as a cell surface receptor, representing the first recognized protein to directly
catalyze formation of a low molecular weight second messenger in response to ligand binding.-ScHuLz, S.; CHINKERS, M.; GARnERS, D. L. The guanylate cyclase/receptor family of proteins. FASEB j 3: 20262035;
1989.
Woirl.s: guanylate cyclase receptor#{149} egg peptide natriuretic peptide protein Icinase Key
atrial
CYCLASE (EC 4.6.1.2) has been studied extensively since the first reports of such enzyme activity in 1969 (1-3). Initial expectations were that the model GUANYLATE
2026
family
of proteins
DAVID L. GARBERS*t and 1Molecular Physiology
Tennessee 37232-0295,
and Biophysics,
USA
being developed for adenylate cyclase (EC 4.6.1.1) regulation would apply to guanylate cyclase, but continued failure to stimulate the enzyme in broken cell preparations with a wide variety of agents, including those known to elevate cGMP in tissues, led to speculation that guanylate cyclase activity was regulated by hormones, neurotransmitters, and other agents through indirect mechanisms (4). The extensive experimentation that followed demonstrated that guanylate cyclase activity could be assigned to three major cellular compartments: the cytoplasm, membrane, and cytoskeleton (Table 1). It was suggested that the activities in each of the major fractions were due to different proteins based on solubiity properties, immunological cross-reactivity, kinetic parameters, responses to effector molecules, and subunit composition (14). In the mid 1970s, it was reported that the soluble form of the enzyme could be regulated by free radicals, nitrovasodilators, and similar molecules (5). This regulation was subsequently explained by the presence of heme as a prosthetic group (15), and models were then developed whereby cGMP concentrations could be regulated in the cell by receptor-mediated formation of free radicals, lipid oxidation products, or similar molecules, or by the direct diffusion of such molecules across the cell membrane (e.g., endothelial-derived relaxing factor) (16). Although controversy existed as to whether the membrane form of the enzyme could be regulated by the above agents (17), it was clear that these agents did not elevate cGMP concentrations in invertebrate spermatozoa (18), in which a membrane form of guanylate cyclase represented the major and possibly only species of the enzyme (19). Therefore, at least in these cells, cGMP concentrations appeared to be regulated through a mechanism that did not involve the formation of free radicals or specific oxidation products. In 1979, low molecular weight factors found in egg-conditioned media were shown to cause marked elevations of cGMP in sea urchin spermatozoa (20). These substances were then purified and identified as small peptides (6) that could act in a species-specific manner to activate the membrane form of guanylate cyclase (21). The two peptides studied in greatest depth have been speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-ArgLeuNH2), both of which stimulate the metabolism and motility of spermatozoa (22, 23). Subsequently, spermatozoan membranes were shown to bind egg peptides 0892-6638/89/0003-2026/$01.50.
© FASEB
TABLE
1. General forms
of guanylate
Form
Subcellular
cyclase Compartment
Soluble
Cytoplasm
Particulate
Plasma membrane Plasma
membrane
Structure
Heterodimer
Monomer or cytoskeleton
and to retain receptor-mediated responses similar to those seen in the intact cell (24, 25). In 1981, evidence for the presence of natriuretic factors in atrial myocardial extracts was presented (26). These factors were subsequently identified as atrial natriuretic peptides (ANP) and were shown to cause elevations of cGMP in various mammalian tissues (7-9). ANP are derived from a polypeptide precursor synthesized in mammalian cardiac atria and other tissues; their physiological effects include natriuresis, diuresis, vasodilation, and inhibition of aldosterone secretion (27). These various effects of ANP are due to the presence of cell surface receptors for the peptides on the target cells. The presence of ANP-like peptides and binding sites for ANP in mammalian brain has suggested that members of this peptide family may also function in a neuromodulatory manner (28). High densities of ANP binding sites are found not only in regions of the brain associated with the central regulation of blood volume homeostasis, but also in areas associated with sensory perception and coordination and in the limbic system (29). ANP binding has been observed in adipose as well as in many other tissues (30), and has been described in various vertebrates other than mammals (31). As with the sea urchin system, accumulated evidence suggested that the membrane form of guanylate cyclase was regulated in response to ligand binding, which explained the elevations of cGMP concentrations (8, 9). Research on ANP and egg peptides has now merged to provide strong evidence that guanylate cyclase itself is a cell surface receptor. This review will survey the recent biochemical and molecular biological advances that have identified the membrane form of guanylate cyclase as a member of a novel receptor/enzyme family. EVIDENCE FROM CROSS-LINKING EXPERIMENTS THAT GUANYLATE CYCLASE IS A RECEPTOR The membrane form of guanylate cyclase was first purified to apparent homogeneity from sea urchin spermatozoa (32, 33). Because it was not known before 1983 that the enzyme could exist in a highly phosphorylated state (23, 34), it was the dephosphorylated form that was first purified and characterized. The purified enzyme, unlike the enzyme present in unpurified preparations, no longer displayed positive cooperative kinetics as a function of substrate (MeGTP) concentration (32, 33). A single, silver-stained band of 135 kDa
GUANYLATE
CYCLASE
?
Effector
Molecules
Nitrovasodilators,
free
Egg
peptides,
Ca2 Heat-stable
References radicals
5
ANP
6, 7-9
enterotoxin
10-12 13
suggested a single polypeptide chain (33). In later studies, the phosphorylated form of guanylate cyclase, which contains approximately 17 mol phosphate/mol enzyme (35), was purified to apparent homogeneity (36) and was shown to possess a much higher specific activity than the dephosphorylated form of the enzyme; it also retained the positive cooperative kinetic behavior as a function of added MeGTP. From the sea urchin purified enzyme, antibodies were produced that were capable of precipitating mammalian membrane forms of guanylate cyclase but not cytoplasmic forms (37). There was reason to believe that the sea urchin and mammalian membrane forms of guanylate cyclase shared regions of identity. The first evidence that guanylate cyclase might serve as a receptor in the sea urchin spermatozoon came from studies using an analog of resact in cross-linking studies (38). Resact, a peptide found in egg-conditioned media that binds to sperm cells and causes changes in metabolism and motility (23), is also a potent chemoattractant (39). When an ‘251-labeled resact analog was incubated with sea urchin spermatozoa in the presence of disuccinimidyl suberate, and cell extracts were subsequently applied to sodium dodecyl sulfate (SDS)polyacrylamide gels, a major radiolabeled band was evident in the autoradiographs (38). The cross-linked protein was identified as guanylate cyclase based on its immunoprecipitation with antibody to guanylate cyclase, its comigration with 32P-labeled guanylate cyclase, and its shift in apparent molecular weight coincident with that of guanylate cyclase in response to NH4C1 treatment of cells (38).
It needs
to be emphasized
that peptides
other
than
resact are found in the egg-conditioned media of other sea urchin species (6). Of those studied, all elevate sperm cGMP concentrations, yet cross-linking studies with the egg peptide speract under conditions similar to those described for resact resulted in the specific radiolabeling of a membrane protein of 77,000 daltons (40). This cross-linked membrane protein was subsequently purified and a cDNA encoding the protein was isolated (41). The protein bears no significant regions of identity with guanylate cyclase. Because Bentley et al. (42) were able to activate detergent-solubiized guanylate cyclase with speract, it is likely that the 77,000-mol wt protein is closely associated with guanylate cyclase. Whether the 77,000-mol wt protein or guanylate cyclase 1) is the actual receptor, 2) both are subunits of the receptor, or 3) both are closely associated with yet another protein that is the receptor remains unresolved.
2027
At the time cross-linking
these
studies
studies
of spermatozoa
of various
progressed,
mammalian
cells
and
tissues suggested that ANP bound to at least two different receptors with apparent mol wt of 66,000 and 120,000-180,000 (43). The 66,000-mol wt protein appears to exist as a homodimer, resulting in a third apparent receptor of apparent mol wt equal to 125,000140,000 (44, 45). Four groups have reported the copurification of the high molecular weight receptor and guanylate cyclase activity (46-49). It also has been suggested by various groups that only the high molecular weight receptor is
coupled to guanylate cyclase activation (46), although some controversy has existed (51). The specific activity of the purified guanylate cyclases was reported to be similar to that of the purified sea urchin enzyme, and ANP binding in some cases was close to the 1:1 stoichiometry predicted (49). The copurification of the high molecular weight ANP receptor and guanylate cyclase through a number of steps that discriminate among proteins by diverse criteria suggested that these activities resided within a single protein, although low amounts of recovered material prevented rigorous testing for homogeneity. The low molecular weight ANP receptor has been designated as the ANP clearance (ANP-C) receptor, and a cDNA encoding this receptor has been cloned (52). The function of this binding protein has been suggested to be that of clearing ANP from the circulation, without a role in signal transduction (53); it appears to contain only 37 amino acids within the cytoplasm (52). EVIDENCE
THAT
IS A RECEPTOR OF
cDNA
GUANYLATE BASED
ON
CYCLASE EXPRESSION
CLONES
cDNAs encoding membrane forms of guanylate cyclase from two sea urchin species, rat brain, and human placenta and kidney have been cloned and sequenced
(54-57). The predicted amino acid sequences suggest that all encode polypeptides with a single transmembrane domain. The intracellular (catalytic) domains are highly conserved across the species, whereas the extracellular diversity, act with
(putative as would distinctly
binding) domains show greater be expected for receptors that interdifferent peptides (Fig. 1).
The predicted amino from the two sea urchin region
of
greatest
acid sequences of the enzyme species are 77% identical, the
identity
being
the
cytoplasmic
domain most closely apposed to the putative transmembrane domain (55). At the extreme carboxyl terminus, the two amino acid sequences diverge. The more recently evolved sea urchin, Strongylocentrotus purpuratus, possesses an enzyme whose distal carboxyl domain (last
240 amino acids) is 40% identical to the soluble form of guanylate cyclase (58, 59); it is approximately 60% identical to the rat brain membrane form of the enzyme (56). The deduced amino acid sequence of the rat membrane guanylate cyclase is 33% identical to that of the
2028
bovine
Vol. 3
ANP-C
July 1989
receptor
(Fig
2).
The
ANP-C
receptor
possesses
a transmembrane
domain
at
the
same site as the rat brain guanylate cyclase, but contains only a short cytoplasmic segment. In addition, the five cysteine residues found in the extracellular domain of the ANP-C receptor are conserved in the mammalian enzyme. To date, expression of the sea urchin sperm guanylate cyclases in cultured mammalian cells has not resulted
in detectable enzyme activity, although the protein is synthesized (S. Singh, D. S. Thorpe, and M. Chinkers, personal communication). The expression of rat brain cDNA in mammalian cells resulted in substantial increases in enzyme activity and ANP binding activity in transfected cells (Fig. 3) (56). In addition to the data shown in Fig. 3, 1251-ANP could be specifically crosslinked in the presence of disuccinimidyl suberate to an expressed protein of apparent mol wt equal to 130,000 (56). The human cDNA has also been expressed in cultured cells, and guanylate cyclase activity as well as ANP specific binding is elevated in the transfected cells (57). When intact COS cells transfected with the guanylate cyclase cDNA from either rat or human are incubated with ANP, cellular cGMP concentrations are markedly elevated relative to those of nontransfected cells (S. Singh and D. L. Garbers, personal communication) (57). MECHANISMS
OF
GUANYLATE
CYCLASE
REGULATION The particulate placed into at
forms of guanylate least three categories
at
cyclase can be this time (see
Table 1). One group is represented by a form found on the plasma membrane that serves as a cell surface receptor. The mechanism by which ligand binding activates guanylate cyclase remains to be determined, but as will be discussed later, mechanisms of desensitization, at least in sea urchin spermatozoa, have been studied. A second group of guanylate cyclases appears to be regulated by Ca2, and may not serve as receptors for extracellular ligands (although this has not been rigorously tested). A third type of guanylate cyclase is associated with the intestinal brush border membrane and appears to be activated by Escherichia coli heatstable enterotoxin. Toxin binding activity appears to be separate from enzyme activity, indicating that although the toxin receptor is functionally cyclase it is not an intrinsic part
coupled to guanylate of the protein (13).
It
is possible that the toxin receptor is coupled to the membrane form of guanylate cyclase described here or that another form of the enzyme remains to be identified. The regulation of a particulate form of guanylate cyclase by calcium has been documented in Paramecium and Tetrahymena ciliary membranes and in bovine rod outer segments (10, 60, 61). In all three cases, a Ca2-binding protein appears to mediate the enzyme response to the cation. Based on immunological evidence, this protein in Tetrahymena appears to be calmodulin (11). Removal of endogenous calmodulin by La3 treatment of Tetrahymena ciliary membranes results
The FASEB Journal
SCHULZ
El AL.
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Apcyc Spcyc
PIATTRLLFLL
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Apcyc Spcyc Rcyc
VFNTNKEOFD
Apcyc Spcyc Rcyc
LREYTRTDOE RALEANKSVt. IVTGEPVLRS GAWNIYSAIV IDIIALD#{128}PFN GTLELKTDID NASVYIFDAT TELLKALDAT TLEASDYLEQ INQAYEFK IDNALDAPFN GELELRAEID FASVYNFDAT NOLLEALD*T INPOYAILFK NREYTRSDND RALEALKSVI IVTGAPVLKT RNRFSTFV ILDTSEYLNQ P1RGQDR SAIQAFOUK IITYKEPDWP EYLEFLKQLK LLAD KKFNFTVEDG LKJIIIPASFH DGLLL YVQAVTET
a
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#{149}1.
LQAGISYDG SQVVSNLFNT SYISKAICADY QFDENGVK SYVLLHRIPI NMGIYDG EEWSTLLNS TYRS1CTDTFYQFDENGDGVKPYVLLHLIPI j.AQGGTYTDG EN1TQMIR SF QGVT GYLKlDN
Apcyc Sp
Rcyc
PPLDNPVCOF HGELCTNUGLYLGT L IPAFI II FGG GLGYYIYRKR AYEMLDSLV CItET RESETNSQG. . . . FSIESNV LSAISVISNA PPLO*PCGF NGELCTNIML YLGAS IPTFLIIFGG LIGFFIYNKR AYEMLDSLV 1CVDWSEVQT KATDTNSQG. .. . FSISOINV NSAISVIS$IA SSLERHLRSA GSRLTLSGRG SNYGSLLTTE PPPOVPCGF DIIEDPACNOD HFSTLEVLAL VGSLSLISFL IVSFF1YR QLEKELVSEI. IVRDLQP
Apcyc Spcyc Icyc
EKOQIFATIG TY*GTICAIH AVHKNHIDLT RAVRTELKLN RDNRHDWIP FIGACIDRPH ICILNHYCAK GSLINEND FIGACIDRPH ISILNHYCAK GSLILEND EKOQIFATIG TYRGTVCALH AV1$KNHIDLT RAVRTELKIM RDIHDN1P GQFQVFAKTA YYKGNLVAVK RVNRKRIELT RKVLFELKNN RDVQSIEHLTR FVGACTDPPII ICILTEYCPR GSLILENE
Ap Spcyc Rcyc
VYLNSSEIKS NGNLKSSNCV VDNRWLQIT DYGLHEFRKG QKEDVDLGEH AKLARKLWTA PEHLREGKSN HPGGTPK1 VYLNSSEIKS NGHLKSSNCV.VD$IRWLQIT DYGLNEFKKG QKVDLGDH AKLARQLWTS PEHLRQEGSN PTAGSPQ1 LFLHNGAICS NGNLKSS$ICV VDGRFVLKIT DYGLESFRDP EPEG.. . .GH TLFN(KLWTA PELL.SP PA*GSQAV
Apcyc Spcyc Rcyc
DLELA..Dll MVSKGEVPP YRPVLNAVNE AAPDCVLTAI
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RAcWEDPNE IPHI IEVRTN LAPLQKGLKP NILDIINIAIN ERYTN$LEEL VDERTQELQK EJOLA. .Dll GRVKSGEVPP YRPILNAVNA UPOCVLSAI RAQEDPAD RPNINAVRTNLA LQKGLKPNILDNNIAIN ERYTNMLEELVDERTQELQK GLDLSPIEI I ERVTRGEQPP FRPSILQSH LEELGQL. .N QR1MEDPQE RPPFQQIRLA LRKFNIENSS NILDNLLS*N EQYAIINLEELVEERTQAYLE a. * a a. a. a * a EKAICTEQLLH RNLPPSIASQ EKTKTEQLLH LPPSIASQ #{163}KAKAEALLY QILPHSVAEQ
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a. a * aa * * a * * .a... a a a.a. a. *aaa.a* aaa.*aaa** a... aa a. * a a. * a.. a HSHSCSALHS S RNRMGOI ASTAHNLLES VKGFIVPHKP EVFLKLRIGI HSGSCVAGW GLTNPRYCLFGDTVNTASRN ESNGLALRIN VSPWKQVLD KLGGTELEDR RNGQUIAREV ARNALALLDA VRSFRIRHRP QEQLRLRIGI HTGPVCAGWGLKNPQYCLF GDTVNTASRM ESNGEALKIH LSSETKAVLE EFDGFELELR aaa
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GLVPNNGKGEIHTFWLLGQD PSYKITKVKP PPQKLTQ#{128}A1 E!AANRVIPD DV GDVENKGKGK VRTYWLLGER GCSTRG 1. Alignment
purpuratus
(Spcyc),
of the deduced amino acid sequences and rat (rcyc). Amino acids common
of membrane guanylate cyclases from Arbacia punctulata (Apcyc), St rongylocent rot us to the rat enzyme and the enzyme from either sea urchin species are marked
with an asterisk. in a loss of guanylate cyclase activity, which can be restored by either protozoan or mammalian calmodtilin (11). In Paramecium and rod outer segments, the nature of the regulatory protein is less clear. Although antisera against both Tetrahymena and soybean calmodulin inhibit Paramecium guanylate cyclase, and enzyme activity can be restored by the addition of homologous or nonhomologous calmodulin (12), other evidence suggests that calmodulin may not be the endogenous regulatory protein of guanylate cyclase in Paramecium. 1) Trifluoperazine, which inhibits Ca2-calmodulin-dependent processes, does not affect Ca2-stimuIated guanylate cyclase activity in Paramecium (60). 2) The endogenous Ca2-binding protein is tightly bound to guanylate cyclase. However, when La3-treated ciiary membranes are reconstituted with calmodulin, added calmodulin
GUANYLATE
CYCLASE
associates only weakly with the enzyme (12). 3) The optimal reactivation of guanylate cyclase activity by homologous or nonhomologous calmodulin requires concentrations 10- or 100-fold higher, respectively, than those necessary for activation of calmodulin-dependent phosphodiesterase (62). One-half maximal activation of Paramecium and Tetrahymena guanylate cyclases by Ca2” occurs well within the intracellular concentrations expected during the C#{227}2/K” action potential that regulates ciliary beat direction (60, 61). Studies of behavioral mutants of Paramecium have demonstrated a direct link between voltage-gated Ca2” influx and elevation of cGMP concentrations (63), and it has been proposed that Ca2” entry into the cilium during an action potential activates both the ciliary reversal mechanism and guanylate cyclase. It is then
2029
RAT GC BOA. ANP.C
SDLTVAVVLPLTNT$YPWSWARVGPAVELALAR!KARP.
RAT GC
GYCSDTAAPLAAYDLKWEHSPAVFLGPGCVYSAAPYGRFTAIINRVPLI.TAGAPALSIGVK
. . DLLPGW1VRNVLGSSENAA
57
OKIEYLVLLP.ODDULF$L*RVRPAIEYALRTVEGNATGRRLI,PA.GTHFQVAYEDSDC 63 117
BOA. ANP.C GNRALFS1vDRYAAARGAKPDLII.apvcEYAAapVAHLAsIINoLpNLsaSaLAASFaHk
122
RAT GC BOy. ANP.C
O.EYALTT*TGPSHVKLSOFVTALHIRLGWEHQALVLYAIRI.GDDNPC?PIVIOLYMRVR DTETSHLTRVAP$YARNGEMMLALFNHHOERAVLYYSDDKI. EINFTLESVHEVFO
176 180
BOy. ANP.C
ERLNITV000EPVIGDPDHYPKLLRAVRRKGIVIYICSSPDAFNNLELALNAQLTGEDY EE#{149}GLHTSAYNFDETKDLDLEDIVRHIQASERVVINCA$SDTIIGIILAAHRHIMTSGSY
236 239
RAT CC BOA. AHP.C
VFFHLDVFGOSLKSAQQLVPOKPWERUG009SARGAFOAAKI ITYKEPDNPIYLEFLKO AFFNIELFNSBF.YGDG SVKISDKHDFEAKQAYSSLQTITLLRIVKLIFEKFSME
296 293
RAT CC BOA, ANP.C
LKLLADKKFNFTVEDCLKWI IPASFN0GLLLYVQAVTETLAQETYTNENITORMSS AESSVEKQ.GLS.EEDYVIOIFAEGFHOAILLYVLALREVI.RASYSKK$GKI a071
356 351
RAT CC
FQeyTaYLKIofiNQsoTeFSLwDu.pPETeAFRvVLNyNeToELNAVSEHRLYw-n.o
414
BOA. ANP.C
FEGIA$OASIOANQOAYGDSFAIAITITEAQTOETIGD?FSKEGRFENRPNV*YPSGPtK
411
RAT CC
YPPPDVPKCGFDNEDPACNQDHFSTLEVLALVGSLSLISFLIVSFFIYIXNQLEKELVSE
414
BOA. ANP.C
DRIDETRMVEHTNSSPCKASGGLEESAYTGIYVGALLGAGLLMAFYFFUKYRITIERRN
471
RAT CC
LWRVRWEOLOPSSLERHLRSAG$RLTLSGRGSNYGSLLTT000FOVFAKTAYYKGNLVAA
534
BOA. ANP.C
QOEESNYGKHRELREDSI*$HF$VA
496
RAT
OC
RAT CC
KRVNRKRIELTRKVLFELKHMRDVONEHLTRFVGACTDPPNICILTEYCPRGSLQDILEN
594
ESITLDFRYSLTNDIVKGMLFLI4NGAICSHGNLKSSNCAVDGRFV1KITDYGLESFRD PEPE009TLFAKKLW1APELLRMASPPARGSOAGDVYSFGI lICE IAIRSGVFYVEGLDL SPKEI IERATRGEOPPFRPSMOLQSHLEELGQINQRCWAEDPOERPPFOOIRLAIRKFNK ENSSNIIDNLLSRMEOYANNLEEIVEERTOAYLEEKRKAEALLYOILPHSYAEOLKRGET
654 714 774 834
AQAEAFDSVTIYFSDIYGFTALSAESTPMQVATLLNDLYTCFDAVIDNFDVYKYETIGDA YNVVSGLPVR000LHAREAARMAIALIDAVRSFRIRHRPOEOLRIRIGIHTGPVCAGVVG LKMPRYCLFGDTVNTASRMESNGEALKIHLSSETKAVLEEFDGFELELRGOVEN6060KV RTYWILGERCCSTRG
894 954 1014 1029
Figure 2. Comparison of the deduced amino acid sequences of rat membrane guanylate cyclase and the bovine ANP-C receptor. The alignment begins with residue 1 of the mature rat protein (56), which corresponds to residue 6 of the mature bovine ANP-C receptor (52). Identical amino acid residues are enclosed in shaded boxes, and the predicted transmembrane domains are underlined.
suggested that the resulting increase in intracellular cGMP is responsible for recovery of forward ciiary beat, probably through the activation of cGMPdependent protein kinase (64). The guanylate cyclase of retinal rod outer segments (which are evolutionarily derived from cilia) resists detergent solubiization and remains associated with the axoneme during partial purification, suggesting a cytoskeletal linkage (65). As in the ciliated protozoans, the activity of the retinal enzyme is regulated by Ca2 concentration. In contrast to the protozoan guanylate cyclase, however, the retinal enzyme is activated by a decrease in intracellular Ca2. Light activation of rhodopsin leads to a stimulation of phosphodiesterase (EC 3.1.4.1) activity mediated by the GTP-binding protein transducin. The increased hydrolysis of cGMP results in the closure of cGMP-gated cation channels, which leads to a reduction in the intracellular Ca2” concentration, membrane hyperpolarization, and a proposed compensatory activation of guanylate cyclase (66). The activation of guanylate cyclase would then replenish cGMP and result in the reopening of the cation channel and recovery of the dark state. A loosely bound accessory protein confers Ca2” sensitivity on retinal guanylate cyclase, which in the absence of this protein exhibits low specific activity (10). This acces-
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sory protein is distinct from calmodulin as guanylate cyclase is insensitive to calmodulin inhibitors and calmodulin is unable to substitute for the accessory protein in activating retinal guanylate cyclase (10). Although the mechanism of ligand activation of the membrane receptor form of guanylate cyclase has yet to be studied, desensitization mechanisms are tinderstood to some extent. In the sea urchin spermatozoon, phos-
phorylation
dramatically
affects
guanylate
cyclase
ac-
C
Figure activity
3. Bar graph depicting the stimulation of guanylate cyclase and the increase in specific ANP binding in mammalian
cells transfected
with rat guanylate
procedures
as described
The FASEB Journal
were
cyclase
by Chinkers
cDNA.
Experimental
et al. (56).
SCHULZ
EF AL.
tivity (Fig. 4). In 1983, Ward and Vacquier (67) reported that egg jelly caused a rapid dephosphorylation of a protein with an apparent mol wt of 160,000. Subsequently, this protein was identified as guanylate cyclase (23, 34), and the component of egg jelly that caused the rapid dephosphorylation was identified as the egg peptide resact (23). When the activity of the phosphorylated form of guanylate cyclase in detergent extracts was compared with that of the dephosphorylated form, the phosphorylated form was found to have approximately 10-fold higher activity (68). These results were unexpected, as resact causes marked (although transient) increases in sperm cGMP concentrations (23). The apparent paradox was resolved, however, by subsequent experiments in which sperm plasma membranes were isolated under conditions where guanylate cyclase remained in the phosphorylated form (21). The addition of resact to these membranes caused a dephosphorylation of guanylate cyclase, a response also seen in intact cells, but large increases in guanylate cyclase activity were observed prior to the loss of phosphate (21). As the enzyme was dephosphorylated, its activity returned to basal levels, thereby desensitizing the system to the presence of the egg peptide. The loss of phosphate from guanylate cyclase also explains the loss of positive cooperative kinetic behavior as a function of MeGTP concentra-
Guanytate Cyclase
gggggg
gggggggggg
P
PP/P/
GTP
Peptide cGMP
p
Ppppp Guanylate
Cyclase
lion. The purified phosphorylated form of guanylate cyclase has a specific activity approximately fivefold higher than that reported for the purified dephosphorylated form of the enzyme, and added protein phosphatases can cause a rapid decrease in enzyme activity (36). The added protein phosphatases also cause a shift in kinetics from positively cooperative to linear as a function of added MeGTP (36), which suggests that the phosphorylation state alters interactions between substrate binding sites. Whether multiple nucleotide binding sites exist on a single polypeptide chain or multiple molecules of guanylate cyclase interact in the phosphorylated state is not known. The mechanisms by which guanylate cyclase is regulated in mammalian cells that respond to ANP are much less clear, although several observations suggest that the mammalian enzyme may be regulated in a manner similar to that of the sea urchin. The activation of mammalian guanylate cyclase by ANP is transient (9), as is the activation of sea urchin sperm guanylate cyclase by egg peptides (22, 23). In the intact mammalian cell, guanylate cyclase exhibits cooperative behavior with respect to the substrate MeGTP (69); however, during purification, there is a shift to typical Michaelis-Menten kinetics (47)- again, a response also seen with the sea urchin sperm enzyme. Purification of the giianylate cyclase-coupled ANP receptor is also associated with a loss of enzyme stimulation by ANP (46-49). Although the biochemical basis for the loss of response to ANP is unknown, sea urchin sperm guanylate cyclase shows a similar loss of response to egg peptides (21). Evidence suggests that mammalian guanylate cyclase contains an allosteric ATP binding site. Such a site could exist on the enzyme itself or on an associated regulatory protein. The addition of ATP to membranes prepared from ANP.-sensitive cells potentiates ANP activation of guanylate cyclase activity in a concentration-dependent and saturable manner (70). ATP enhances the maximal rate of cGMP production without affecting the affinity of the enzyme for
GTP or ANP (70). The ATP effect is probably to phosphorylation, logs also enhance o
ggg
ggggggggg
GTP cGMP
p
4. Model for the regulation of Arbacia punctulata guanylate cyclase activity after resact binding. The enzyme exists in a highly phosphorylated state under normal conditions. The binding of resact markedly enhances enzyme activity, followed by a rapid dephosphorylation of guanylate cyclase and a return of activity to the basal state despite the continued presence of the egg peptide. Figure
GUANYLATE
CYCLASE
activity
not due
since nonhydrolyzable ATP anaANP-stimulated guanylate cyclase
(70).
In addition to short-term changes in enzyme activity, the guanylate cyclase-coupled ANP receptor exhibits down-regulation in response to agonist exposure. Pretreatment of smooth muscle cells with ANP results in a time-dependent decrease in the total number of ANP receptors with no change in apparent affinity (71). Recovery of down-regulated receptors is inhibited by actinomycin D or cycloheximide, which suggests that receptor recovery requires both RNA and protein synthesis (71). Prior treatment with ANP also causes a dose-dependent decrease in ANP-induced cGMP accumulation (72). GUANYLATE Peptide hormones, latory molecules
CYCLASE
RECEPTOR
MODEL
neurotransmitters, and other moduinteract with cell surface receptors to 2031
set in motion a cascade of intracellular events. Cell surface receptors may be divided into two broad categories: those containing single transmembrane domains and those spanning the membrane multiple times, which include receptors composed of multiple membranespanning subunits. Within these broad categories, receptors may be subdivided into classes on the basis of the mechanism by which ligand occupancy of the receptor is transduced to an intracellular signal (Fig. 5). Molecular cloning and sequencing of a variety of cell surface receptors demonstrate that members within a receptor class also share primary sequence and structural homologies, which suggests a common evolutionary pathway. Receptors that span the membrane multiple times include receptors that modulate their respective effector systems through the action of an intermediary GTP-binding protein and ligand-gated ion channels, which are composed of multiple transmembrane subunits. Cell surface receptors with protein-tyrosine kinase activity, which are members of the large protein kinase family, represent a class of receptors with a single transmembrane domain. The membrane form of guanylate cyclase, now shown to be a cell surface receptor, can also be included in this category. Receptors that act through GTP-binding proteins have been identified in evolutionarily diverse organisms, and many, if not all, have retained a conserved struc-
A
Agonist
G-Protein
B Agonist
0=
C
D
Agonist
0=
Protein-Tyr
emTyrP
Agonist-. cGMP+PPI
Figure 5. Schematic diagram depicting interactions of agonists with various receptor classes. A) Agonist interacts directly with an ion channel. B) Agonist interacts with receptor to release the ct-
subunit of a GTP-binding (controversy
protein from the inhibitory
‘y
subunits
as to whether y itself also functions to regulate various G,. in turn interacts with an enzyme or ion channel. C) Agonist interacts with receptor to stimulate the proteintyrosine kinase activity of the same protein. D) Agonist interacts with receptor to stimulate guanylate cyclase activity of the same protein. exists processes).
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July 1989
ture similar to that of bacteriorhodopsin, a prokaryotic membrane protein that uses light energy to drive an ATP-generating proton pump (73). Receptors identified in this class are predicted to contain seven a-helical transmembrane domains. Many of these receptors share sequence similarity, but it is the retention of secondary structure and the fact that all mediate their effects through GTP-binding proteins that unite them as a class. However, seven transmembrane domains may not necessarily be required for receptor interaction with a GTP-binding protein (74). The ligands for the seven-domain receptors, which include yeast peptide mating factors (75), chemotactic cyclic nucleotides in Dictyostelium (76), adrenergic and cholinergic agonists, other neurotransmitters, and light (77), show little similarity. The effector molecules and second messenger systems involved are also diverse. Signal transduction may be accomplished by modulation of enzyme activity (e.g., adenylate cyclase, phosphodiesterase, or phospholipases) or by direct interactions with ion channels (77). Several neurotransmitters mediate target cell response by binding to receptor ion channels. Receptor-ligand interaction results in the opening of the ion channel, leading to an alteration in cell membrane potential. Channel-gating ligands include ‘y-aminobutyric acid, glycine, and acetylcholine; the ion channels regulated by these three ligands are hetero-oligomeric glycoproteins (78). The amino acid sequences deduced from the cloned and sequenced cDNAs encoding these receptors demonstrate subunit homology both within and between receptors, which suggests that ligand-gated ion channel proteins may have evolved by repeated duplication of a common ancestral gene (78). The receptors with protein-tyrosine kinase activity bind polypeptide ligands, such as epidermal and platelet-derived growth factors, and insulin. In response to ligand binding, protein kinase activity is increased and phosphorylation of tyrosine residues on the receptor itself and other cellular substrates is observed (79). Most data suggest that it is the protein kinase activity of the receptor that mediates the cellular response to ligand binding (80), but as indicated above, data also exist to support an involvement of GTP-binding proteins (74). The growth factor receptors that have been sequenced contain a single transmembrane domain, although these receptors may function as multimeric complexes of disulfide-linked subunits (81). The intracellular portion of the molecule contains a highly conserved catalytic domain as well as tyrosine residues that are autophosphorylated during receptor activation, whereas the extracellular domains have diverged to accommodate various ligands, although they show characteristic spacing or clusters of cysteine residues (81). The membrane form of guanylate cyclase defines a new receptor family in which ligand binding results in the apparent direct activation of a catalytic domain to produce a low molecular weight second messenger. This form of the enzyme binds and is activated by a diverse family of ligands that includes sea urchin egg and mammalian atrial natriuretic peptides. Membrane guanylate cyclase shares some features with other
The FASEB Journal
SCHULZ
Er AL.
receptor/effector systems, but in other respects it is unique. Despite the obvious parallel between hormonesensitive adenylate cyclase and membrane guanylate cyclase as enzymes that generate cyclic nucleotide second messengers, the mechanisms of regulation of the two molecules seem to have little in common. The lack of primary sequence data on the mammalian form of adenylate cyclase, however, precludes discussion of
whether
or not these enzymes
share common
structural
domains. Membrane direct ligand binding
guanylate cyclase is activated by (56), whereas ligands that regulate the activity of adenylate cydase appear to bind to proteins that are distinct from the enzyme. Receptor-ligand interaction activates a GTP-binding protein to stimulate
or inhibit
the activity
of adenylate
cyclase (77).
In terms of primary sequence, guanylate cyclase contains many of the highly conserved amino acids present in protein kinases (54-57) and is therefore closely aligned in this respect to the cell surface receptors that possess protein kinase activity. Guanylate cyclase also resembles these receptors with its single transmembrane domain. It is likely, then, that these two classes of cell surface receptors have evolved from a common precursor molecule. Many members of the protein-tyrosine kinase family have been identified as cellular homologs of viral oncogenes (81). Will similar results be forthcoming on the guanylate cyclase receptor? Reports suggest that the relative amounts of the membrane form of guanylate cyclase increase in regenerating liver (17), but little strong evidence exists to support an involvement of cGMP in the regulation of cell proliferation or differentiation (4). The diversity of the protein-tyrosine kinase receptors in terms of ligand specificity also raises the question of whether or not mammals contain guanylate cyclase surface receptors that bind ligands other than ANP. Assuming that catalytic domains remain highly conserved, it should be possible to define the number of different family members by recombinant DNA technology. [!J The authors wish to thank Dr. C. S. Ramarao for the artwork and Mrs. Penny Stelling for her secretarial assistance. This research was supported in part by NIH grants HD 10254 and GM 31362.
7. Hamet, P., 1}emblay, J., Pang, S. C., Garcia, R., Thibault, G., Gutkowska, J., Cantin, M., and Genest, J. (1984) Effect of native and synthetic atrial natriuretic factor on cyclic GMP. Bioche,n. Biophys. Res. Commun. 123, 515-527 8. Winquist, R. J., Faison, E. P., Waldman, S. A., Schwartz, K., Murad, F., and Rapoport, R. M. (1984) Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle. Proc. NatL Acad. Sci. USA 81, 7661-7664 9. Waldman, S. A., Rapoport, R. M., and Murad, F. (1984) Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. j BioL Chem. 259, 14332-14334 10. Koch, K. -W., and Stryer, L. (1988) Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions. Nature (London) 334, 64-66 11. Schultz, J. E., Sch#{246}nefeld, U., and Klumpp, S. (1983) Calciuml calmodulin-regulated guanylate cyclase and calcium permeability in the ciliary membrane from Tetra4ymena. Eur. j Biochem. 137, 89-94 12. Klumpp, S., Kleefeld, G., and Schultz, J. E. (1983) Calcium!
calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. j Biol. Chem. 258, 12455-12459 13. Kuno, T, Kamisaki, Y., Waldman, S. A., Gariepy, J., Schoolnik, G., and Murad, F. (1986) Characterization of the receptor for heat-stable enterotoxin from E. coli in rat intestine. j Biol. Chem. 261, 1470-1476 14. Kimura, H., and Murad, F. (1975) Two forms of guanylate cyclase in mammalian tissues and possible mechanisms for their regulation. Metabolism 24, 439-445 15. Gerzer, R., B#{246}hme,E., Hofmann,
16. 17.
18.
19.
20.
21.
F., and Schultz, G. (1981) Soluble guanylate cyclase purified from bovine lung contains heme and copper. FEBS Lell. 132, 71-74 Murad, F. (1986) Cyclic guanosine monophosphate as a mediator of vasodilation. j Clin. Invest. 78, 1-5 Mittal, C. K.; Murad, F. (1982) Guanylate cyclase: regulation of cyclic GMP metabolism. Handbook of Experimental Pharmacology (Nathanson, J. A., and Kebabian, J. W., eds) Vol. 58-1, pp. 225-260, Springer-Verlag, Berlin Garbers, D. L., and Kopf, G. 5. (1978) Effects of factors released from eggs and other agents on cyclic nucleotide concentrations of sea urchin spermatozoa. j Reprod. Fertil. 52, 135-140 Gray,J. P., Drummond, G. I., Luk, D. W., Hardman,J. G., and Sutherland, E. W. (1976) Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm. Arch. Bioc/jem. Biophys. 172, 20-30 Kopf, G. S., and Garbers, D. L. (1979) A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates. j Re/mid. FertiL 57, 353-361 Bentley, J. K., Tubb, D. J., and Garbers, D. L. (1986) Receptormediated activation of spermatozoan guanylate cyclase. j BioL Chem.
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tor/signal
of
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