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Reference: Biol. Bull 183: 155-158. (August, 1992) A Guanylyl Cyclase: Cell-Surface Receptor Throughout the Animal Kingdom STEPHANIE SCHULZ HowardHughes MedicalInstitute andDepartment ofPharmacology, University ofTexas Southwestern Medical Center, Dallas, Texas 75235-9050 Abstract. The enzyme guanylyl cyclase occurs in both ai. 1991). The activity ofthe soluble enzyme is regulated soluble and membrane-associated forms, and catalyzes bydiffiisable substances, nitricoxide forexample (Schulz the formation ofthe second messenger cyclic GMP. The et al., 1991). The particulate enzyme, in contrast, is mo- membrane forms ofguanylyl cyclase encompass a family nomeric (although the active conformation may be mul- ofcell-surface receptorsthat mediatethebiological activity timeric), with a single transmembrane domain (Yuen and ofa variety ofpeptide ligands. Representatives ofthis re- Garbers, 1992). A variety ofstudies have demonstrated ceptor family have been identified in such diverse species that members ofthe particulate guanylyl cyclase family as rats and sea urchins. In echinoderm spermatozoa, are receptors for such diverse ligands as echinoderm egg guanylyl cyclase is a receptor foregg-associated peptides, peptides, natriuretic peptides, and Escherichia coli heat- GMP elicitingspecies-specificeffects,includingchangesinsperm stable enterotoxin. Cyclic generated in response to respiration and motility, and a chemotactic response. In such stimuli isimplicated intheregulation ofion channel mammals, the family ofguanylyl cyclasesincludes recep- function, and the modulation ofcyclic nucleotide-depen- tors for natriuretic peptides, as well as the target ofbac- dent phosphodiesterase and protein kinase activity. This terial heat-stable enterotoxins. The identification of na- review will focus on the current understanding ofthe re- triuretic peptides in amphibians, birds, and fish suggests ceptorguanylyl cyclases in mammals, and their counter- thatguanylyl cyclase receptors playa role in osmotic bal- parts in other species. ance ina varietyoforganisms. Molecularcloningevidence impliesthat homologsofthe mammalian guanylyl cyclase Receptor Guanylyl Cyclases in Echinoderms receptors exist in other vertebrates, and perhaps even in invertebrates.Guanylylcyclasesthusrepresentanexample Particulate guanylyl cyclase was first characterized in, ofreceptorsthat havediversified to serve both asexternal and purified from, the spermatozoa ofsea urchins. This chemoreceptors and as internal hormone receptors. cell type is a rich source of the particulate form of the enzyme, and apparently contains no soluble enzyme. Introduction Studies ofsea urchin spermatozoa were among the first to suggest that guanylyl cyclase might serve as a receptor The production ofcyclicGMP, an intracellularsecond for extracellular signals. Peptides were purified from sea messenger, iscatalyzedbyguanylylcyclases. Thisenzyme urchin egg-conditioned medium that would elicit several family includes both soluble and particulate forms, and biochemical and behavioral changes in spermatozoa of is widely distributed in various cell types throughout the the homologous species. Exposure ofspermatozoa to the animal kingdom. The soluble and particulate forms of peptides resact or speract (purified from Arbacia punc- the enzyme are distinctly different in both their structure tulataorStrongylocentrotuspurpuratus, respectively. Ta- and their mode of regulation. The soluble enzyme is a ble I) increases respiration and motility, elevates intra- heterodimercomposed of and subunits; tissue-specific cellularpH, altersion fluxes,andelevatescyclic nucleotide isoforms of each subunit are known to exist (Schulz el levels(Garbers, 1989). In addition, resact isa potent che- moattractant forArbaciaspermatozoa(Ward etal., 1985). Received 24April 1992;accepted 29 May 1992. Crosslinking studies demonstrated that resact apparently 155 156 S. SCHULZ Table I Aminoacidsequencesofpc; <>activatepaniculateguanylycyclases. LongercirculatingformsofbrainandC-typenatriitreticpeptide havebeen identified I. Atrial Natnuretic Peptide SLRRSSCFGGRMDRI GAQSGLGCNSFRY Human/Porcine SLRRSSCFGGRIDRI GAQSGLGCNSFRY Rat SKSSSPCFGGKLDRI GSYSGLGCNSRK Eel SSDCFGSRIDRI GAQSGMGC-GRRF Bullfrog II. Brain Natnuretic Peptide SPKMVQGSGCFGRKMDRI SSSSGLGCKVLRRH Human SPKTMRDSGCFGRRLDRI GSLSGLGCNVLRRY Porcine NSKMAHSSSCFGQKIDRI GAVSRLGCDGLRLF Rat MMRDSGCFGRRIDRI GSLSGMGCNGSRKN Chicken III. C-Type Natnuretic Peptide GLSKGCFGLKLDRI GSMSGLGC Porcine GWNRGCFGLKLDRI GSMSGLGC Killihsh GWNRGCFGLKLDRI GSLSGLGC Eel IV. Heat-Stable EnterotoxinsandGastrointestinal Peptides PNTCEICAYAACTGC Guanylin SSNYCCELCCNPACTGCY EschericiliacoliSTh N NTFYCCELCCNPACAGCY EscherichiacoliSTP SSDWDCCDVCCNPACAGC YersiniaenterocoliticaST IDCCEICCNPACFGCLN I'ibriocholeraenon-01 ST V. Echinoderm Egg Peptides GFDLNGGGVG Speract(Strongylocentrotuspurpiiratux} CVTGAPGCVGGGRL- Resact(Arbaciapunctulata) DSDSAQNLIG NH, Mosact(Clypeasterjaponicus) binds to a 160,000 molecular weight protein that was bindingdomain, and an intracellular regulatory/catalytic shown to be guanylyl cyclase (Shimomura et a/., 1986). domain. The cDNA encoding the intracellular, presum- Speract, on the other hand, could be crosslinked to a ably conserved, regulatory/catalytic domain was used as 77,000 molecular weight protein (Dangott et a/.. 1989). a probe to screen various cDNA libraries, and the first The relationship between the two crosslinked binding mammalian guanylyl cyclase(GC-A)wascloned from rat proteins is unclear, because both sea urchin species con- and human (Chinkers et a/.. 1989; Lowe et al, 1989). tain both proteins. The functional receptor may be com- Reportsintheliterature haddemonstrated thestimulation posed ofmultiple subunits, only one ofwhich is labeled ofguanylyl cyclase activity by atrial natriuretic peptide bychemical crosslinking. The respectiveguanylyl cyclases (ANP), and the copurification ofANPbindingandguan- and 77 kDa proteinshavebeencloned from bothArbacia ylyl cyclase activities (reviewed in Schulz el ai, 1989a). and Strongylocentrotus (Singh et ai. 1988; Dangott etai. Expression ofcloned GC-A allowed direct testing ofthe 1989; Thorpe and Garbers, 1989; L. J. Dangott, pers. hypothesis that guanylyl cyclase was an ANP receptor, comm.). However, none ofthe clones conferred peptide and this was shown to be the case (Chinkers el al., 1989; binding or cyclase activity upon transfected mammalian Loweelal., 1989). A numberofnatriuretic peptides have cells. Incorrect processingofechinoderm transcripts may now been discovered, and the cloning ofa second mam- be responsible for the lack of functional expression in malian guanylyl cyclase (GC-B) hasdemonstrated thedi- COS cells, or both proteins may be necessary for the versity within this receptor-enzyme family (Chang et al., expression ofa peptide-binding complex. 1989; Schulz et a/., 1989b). GC-B also binds and is acti- vated by natriuretic peptides. But although GC-A pref- Recemor Guanylyl Cyclases in Mammals erentially bindsand respondstoANP,GC-Bpreferentially responds to C-type natriuretic peptide (CNP) (Roller et The cloning o -T urchin guanylyl cyclase provided a ai, 1991). Whether additional natriuretic peptide recep- valuable probe i- .ing mammalian forms ofthe en- tors exist is not yet known, but the existence of brain zyme. The amino acid sequence predicted by the sea ur- natriuretic peptide (BNP) suggests that at least one ad- chin guanylyl cyclase cDNA contained a single putative ditional member ofthe family remains to be identified. transmembrane domain, suggestingatopography likethat Heat-stable enterotoxins (ST) produced by several of the growth factor receptors: an extracellular ligand strains ofpathogenic bacteria have long been known to GUANYLYL CYCLASE-COUPLED RECEPTORS 157 Table II Alignmentofpartial-lengthguanylylcyclase-receptorsequences with thehomologous regionsofthemammalian natriureticandheat-stable enterotoxin receptors Speract Receptor AYMLVSGLPLRNGDRHAGQIASTAHHLLESVKGFIVPHKPRVFLKLRIGIHSGSCVAGVVGLT Natriuretic Peptideand Related Receptors RatGC-A AYMVVSGLPVRNGQLHAREVARMALALLDAVRSFRIRHRPQEQLRLRIGIHTGPVCAGVVGLK RatGC-B AYMVVSGLPGRNGQRHAPEIARMALALLDAVSSFRIRHRPHDQLRLRIGVHTGPVCAGVVGLK Fish AYWVSGLPVRNGKLHAREIAGMSLALLEQVKTFKIRLRPNDQLRLRIGIHTGP Xenopus AYMVVSGLPVRNGKLHAREIARMSLALLDAVKSFKIRHRPDQQLGLRIGI Sea urchin AYMCVSGLPIRNGDFHAREIARMSLALLHEITSFRIRHRPEERLRLRIGVHTG Earthworm AYMVVSGLPVRNGKLHAREIARMSLALLDAVRSFKIRHRPNQQLRLRIGIHTGPVCAGVVGLK Heat-Stable Enterotoxin and Related Receptors RatGC-C AYVVASGLPMRNGNRHAVDISKMALDILSFMGTFELEHLPGLPVWIRIGVHSGPCAAGVVGIK Fish AYMVASGLPKRNGDRHAVDIALMALDMLAGVGTFELQHLPGIPLWIRIGVHSGPCAAGVVGNK Xenopus AYMVVSGLPNRNGNNHAVDISRMALDILCFMGSFELRHLPGLPVWIRIGIHSGPCAAGVVGIK Sequencesobtained usingthepolymerasechainreaction. Thedegenerateprimerswereused inthe PCRtoamplifyaminoacids894-956. 890-952, and854-916 in thecatalyticdomainsofGC-A,GC-B, andGC-C, respectively. increase the activity ofa paniculate guanylyl cyclase in RNA was reverse transcribed to cDNA which was used the intestinal brush-border. Elevated cyclic GMP is cor- as a PCR template with degenerate primers similar to related with increased intestinal secretion, which results those usedtoidentifyGC-C. Theseprimersamplifya250 in the diarrhea associated with enterobacterial infection base pair region in the highly conserved cyclase catalytic (Field el al., 1978). Reports in the literature suggested domain. Natriuretic peptide receptor-like sequences (ho- that toxin binding and guanylyl cyclase activities reside mology > 70%) were amplified from frog, fish, and sea in separatemolecules, butchromatographic separation of urchin; sequences homologous with GC-C were amplified thetwoactivitieswasnotcomplete. Todeterminewhether from frog and fish (Table II). Additional sequences with a guanylyl cyclase is the ST receptor, degenerate primers homology to the membrane guanylyl cyclases, but with were designed based on conserved sequences in the cat- no strong homology to any particular receptortype, were alyticdomain ofallclonedguanylylcyclases. These prim- amplified from both sea urchin and fish. Additional ers were used in the polymerase chain reaction (PCR) to members of the receptor family may yet remain to be amplify guanylyl cyclasesequencesin thecDNA from rat identified in mammals. Severalguanylylcyclasesequences small intestine. A newmemberofthecyclasefamily(GC- were also amplified from earthworm. Although none of C) was identified by this method, and its cDNA subse- thesesequenceswasstrongly homologouswithaparticular quentlycloned(Schulzetal., 1990). GC-Cconferredboth mammalian cyclase, they were clearly ofthe membrane, ST binding and guanylyl cyclase activity on transfected rather than ofthe soluble type. An ANP-like peptide has COS cells (Schulz el al. 1990). Recently, a 15 residue been identified in earthworm heart by immunological peptide, guanylin, was purified from acid extracts ofrat cross-reactivity with mammalian ANP(Vesely and Gior- small intestine (Currie et al.. 1992). Guanylin is 47% dano, 1992). This suggests that a homolog ofGC-A may identical with the active portion ofST, and elevated cyclic exist in invertebrates outside the Deuterostomia. Crus- GMP levels in T84, an intestinal cell-line rich in GC-C tacean hyperglycemic hormone (CHH) seems to activate (Currie et al., 1992). While the effects of guanylin on membrane guanylyl cyclase (Goy, 1990). CHH is a large cloned GC-Chave not yet been assessed, thispeptide may, peptide without apparent sequence homology to the na- in fact, be its endogenous ligand. triuretic peptides or guanylin, although it does contain multiple cysteine residues. Thus, the Protostomia may Guanylyl Cyclase-Linked Receptors have evolved an independent set ofguanylyl cyclase re- in Other Organisms ceptors and ligands. Natriureticpeptides havebeen identified insuchdiverse Summary organisms as amphibians, fish, and birds (Table I), sug- gesting that receptors for these ligands are also present Guanylyl cyclase is a receptor for diverse intracellular throughout the animal kingdom. We have used a PCR and extracellularsignalling molecules. A growingbodyof approach to identify guanylyl cyclases in a variety ofspe- evidence suggests that multiple forms of this receptor- cies representing several phyla. RNA was prepared from enzyme exist throughout the animal kingdom. Some re- various tissues of frog, fish, sea urchin, and earthworm. ceptortypesareapparently conserved across species, oth- SCHULZ 158 S. ers may be unique. Studying the interaction between the Roller, K. J., D. G. Lowe, G. L. Bennett, N. Minamino, K. Kangawa, guanylylcyclasesandtheirrespeGctMivPeligandswill bevalu- Hna.trMiautrestuioc,peapntdidDe.rVe.ceGpoteodrdbeyl.C-1t9y9p1e.natSreiluercettiivcepaecpttiivdaeti(oCnNPo)f.thSeciB- ableindenningtheroleofcyclic incellularfunction. ence252: 120-123. Lowe,D.G.,M.S.Chang,R.Hellmiss,E.Chen,S.Singh,D.L.Garbers, Acknowledgments andD.V.Goeddel. 1989. Humanatrialnatnureticpeptidereceptor defines a new paradigm for second messenger signal transduction. I am grateful to David L. Garbers in whose laboratory EMBOJ. 8: 1377-1384. this work was done. I thank Cecelia Green and Rustico Schulz, S., M. Chinkers, and D. L. Garbers. 1989a. The guanylate Ramos forperforming the PCR and sequence analysis of cyclase/receptorfamilyofproteins. 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