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Sequence homologies between guanylyl cyclases and structural analogies to other signal-transducing proteins Doris KoesliBg, Otinter Schultz a n d Eycke B 6 h m e

hutimt flir Pharn)akoiogt¢, Frel¢ Unircrsit#t Bethel Thi¢lall¢~ tf7~.7.t.D.IO00BerlOt .13.Germm)y Received 17 Decernb~r 1990; revised version received 26 January 1991 The cyclicOM P-fom~inll cn~-me tluanyl~lc)'clas~existsin cytosoli~and in m~mbranc,boua,d forms diffcrlnl~in slructur~and retlulations.Dct~rmi.

n~ztion of the prln~arystructures or lh~ lluanylylcydas~s r~veal~d lllatih~ c)rtosolie~nxFme form consists or two similarsuhunlts and that membrane.bound guan)'lyl c~¢~lascsr©pre~nl cnxymc forms in which the catalytic part is located in an tntracdlular. C.terminal domain and ts rqtulated by an extracellul~lr. N.tcrminal receptor domain, ^ domain of 2SO amino acids conserved in all tjuanylyl cyclases appears to I~ r~quir©d for 1he formation of cyclic nucleottde, as this homolosous domain is also Gsund tn tire c~tosolic rcl|ions of the adenyl~'l cyclase. The jcn~ral structures

of'8uanylyl cyclases shows similaritieswith other sljnal lransdu¢inj cnxymcs such as prot©in.tyrosinephosphatascs and prot~in.tyroslnekinases. which also ,exist in cylosolic and receptor.linked I'orms,

Guanylyl cyclasc;Proteinqyrosinc kinase; Protcin-|:crosin~phosphatasc: Adcnyl cyclasc;Transmcmhranc signalling

1, INTRODUCTION The cyclic nucleotides, cyclic AMP and cyclic GMP, are intracellular signal molecules involved in communications within cells. Although cyclic GMP was discovered more than 20 years ago, the understanding of its physiological functions was very limited for a long time. Now tl~ere is evidence for regulatory roles of cyclic GMP in smooth muscle tone and platelet function [1]. The effects of cyclic GMP in these and other systems are mainly mediated by cyclic GMP.stimulated protein kinases and possibly by cyclic GMP.modulated cAMP phosphodiesterases. Cyclic GMP-sensitive cAMP phosphodiesterases are either stimulated or inhibited by cyclic GMP; hormones and drugs which increase cyclic G M P levels c a n , thereby, lmve either synergistic or antagonistic effects with cAMP-elevating hormones, depending on whether cyclic GMP-inhibited or cyclic GMP-stimulated cAMP phosphodiesterases are present in a given cell type. In the vision system of vertebrates, cyclic GMP plays a key role by direct regulation e r a cation channel, which is kept open in the darkness by high cyclic GMP, acting as a channel ligand, and which closes during visual excitement as a result of an increased cyclic GMP hydrolysis triggered by illumination [2]. The existence of cyclic GMPregulated ion channels appears nor. to be limited to the retina, as cyclic GMP-regulated ion channels have also been demonstrated in renal collecting ducts, where increasing cyclic GMP levels reduce Na- reabsorption by

Correspondence address: D. Koesling, lnstitut fi~r Pharmakologie,

Freie Universit~it Berlin, Thielallee 67-73, D-1000 Berlin 33° Germany

Published by Elsevier Science Publishers B.V.

inhibiting an amiloride-sensitive cation channel, an effect which is partially direct by a phosphorylationindependent mechanism and partially indirect through activation of cyclic GMP-dependent protein kinase [31. The diarrhea induced by heat-stable enterotoxin from E. coli, which is known to elevate cyclic GMP levels in the intestinal epithelia (for refs. see [1]), is probably also caused by cyclic G M P changing the open probability of ion channels. In contrast to the formation of cyclic AMP, which in higher organisms is restricted to the plasma membrane, biosynthesis of cyclic GMP occurs in cytosolic and particulate cell fractions. Most tissues contain both enzyme forms, but the relative distribution varies from tissue to tissue, with extraordinary situations in intestinal mucosal cells, where most of the guanylyl cyclase is bound to plasma membranes, and in platelets, where almost all of the enzyme is cytosolic (for refs see [1,4]). Some structural properties and activators of cytosolic and membrane-bound guanylyl cyclases are summarlsed in Table I. 2. REGULATION OF CYTOSOLIC GUANYLYL CYCLASE Since the early seventies, regulations of intracellular cyclic GMP concentrations have been demonstrated by various hormones and neurotransmitters in intact cells (for refs see [1]). The continued failure of hormonal stimulation of the enzyme in broken cell preparations led to the hypothesis that cytosolic guanylyl cyclase is regulated by hormonal factors through indirect mechanisms, e.g. b y intermediates of the Ca 2÷- and O2-dependent biosynthesis of eicosanoids [5]. I n the

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late seventies, NO.containing c o m p o a n d s were demonstrated to be potent activators o f cytosolic guanylyl cyclase [6,7]. As the cytosolic enzyme form was characterized as a heme-containing prc~tein, it was proposed that NO-containing c o m p o u n d s or liberated NO interact with the heme component, thereby stimulating the enzyme [8]. Nevertheless, no physiological activators o f the cytosoli¢ guanylyl cyclase were identified, until vasodilatory factors such as acetylcholine, histamme and bradykinin ~ e r e shown to induce the formation of a labile substance distinct from prostacyclin in endothelial cells ([9] and refs therein). This c o m p o u n d , called endothelium-derived relaxing factor (EDRF), stimulates cytosolic guanylyl cyclase and causes vasodilation via elevated cyclic G M P concentrations. E D R F was identified as NO or as an NO-containing c o m p o u n d (R-NO) [10], which is formed in a Ca2+/calmodulin -, N A D P H - and tetrahydrobiopterin-dependent m a n n e r from L-arginine [11-131. Therefore, an increased cytoplasmaic Ca 2 + concentration appears to be the hormonally induced signal causing a subsequent increase in R-NO synthesis and stimulation o f cytosolic guanylyl cyclase. As the biosynthesis of R-NO is not restricted to endothelial ceils but has also been demonstrated in various other tissues, it is now assumed that NO or RNO is a widespread signal molecule with cytosolic guanylyl cyclase as an effector system. 302

3. R E G U L A T I O N O F M E M B R A N E - B O U N D GUANYLYL CYCLASES Research on membrane-bound guanylyl cyclase was greatly stimulated by the discovery that peptide hormones activate the m e m b r a n e - b o u n d form of guanylyl cyclase but not the cytosolic one. The membrane-botmd guanylyl cyclase of sea urchin sperm was shown to be stimulated by the chemotactic peptide resact, whereas mammalian membrane-bound guanylyl cyclases are stimulated by atrial natriuretic (ANP) and related peptides (for refs see [4]). The effect o f ANP can be markedly potentiated by the addition o f ATP or its analogue adenosine-5'-O-(3'-thiotriphosphate) [14]. Isolation o f the membrane-bound guanylyl cyclase resulted in copurification with an A N P receptor subtype and suggested a rathe r tight coupling between the membrane-bound guanylyl cyclase and the ANP receptor (for refs see [11). A membrane-bound guanylyl cyclase, which i s activated by the heat-stable enterotoxin o f E. coli, occurs in the intestinal mucosa (for refs see [1]). No physiological activator o f the enzyme is known. The occurrence o f the heat-stable enterotoxin-activated guanylyl cyclase may not be limited to intestinal mucosa, as in kidneys o f opossum a n d kangeroo and in certain colon carcinoma cells the heat-stable enterotoxin leads to elevation of cyclic G M P levels [15]. T h e

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eharact~rbalion o f ~nteroto.~tn,activated 8uany|yl cycla~¢ is w r y poor, as attempts to purify the enzyme I~ave failed so far, R~tinal r o d outer Scl~mcnts contain a n'~mbraneb o u n d gunnylyl cyclase ~, which in ¢ontra.~t to other 8uanylyl cyclascs is activated by decreasht~ con¢cntr|t, lions of Ca = ' ; Visual excitation in retl;,,a; rod cells i.~ mediated by a cascade o f cvenes which lead io incrc~scd hydrolysis o f cyclic G M P and the consequent closure o f cyclic GMP-activnted cation.specific channels, whereby the influex o f Ca" = and olher cations is blocked, T h e lowering o f cytosc~lic Ca"* stimulatc~ the rclinal 8uanylyl cyclase, and through the resynthcsis of ~yclic G M P the clark stt~te is recovered, The inhibitory effect o f Ca" " on guanylyl cyclase activity is n~ediated by a Ca" ".sensitive regulatory proteil~ (for rers sec [21), A n o t h e r Ca = "-re~,uhtted form o f a membrane-hot=hal 8uanylyl cyclasc has been found in Paramecium and Tetrallymena plasma tnembrancs. In contrast to the effect on the enzyme in retina, Ca ~"" leads to stimulation o f this enzyme; the C a " " .sensitive protein mediating the response to the cation appears to be calnaodulin (for refs see lilt; however, Ca"*/calmodulin-activated, guanyly| cyclascs have so far not been detected i~ vertebrates. 4. S T R U C T U R E S O F C Y T O S O L I C AND MEMB R A N E - B O U N D G U A N Y L Y L CYCLASES Cytosolic guanylyl cyclase was purified to apparent h o m o g e n i t y in several laboratories and was shown to consist o f two different subunits. Two groups developed imn~unoaffinity purification procedures, which yielded enzymes with subunits of 70 and 82 kDa from rat lung [16] and 70 and 73 kDa from bovine lan~ [17] (apparent molecular masses on SDS-PAGE). Sequencc analyses o f the 73 kDa [18] and 82 kDa [19] subunit revealed that both subunits consist of 691 amino acids, have a calculated molecular mass of 77,5 kDa and share 84.6°70 o f the amino acids. Tile variations observed in t he sequences are probably due to the differences in species, although the bovine [20] and rat [21] 70 kDa subunits are more conserved, showing 97.4°;0 identical a m i n o acids. A comparison o f the amino acid sequences o f the bovine 70 kDa and 73 kDa subunit revealed strong similarities between the subunits with about 32°/0 identical amino acids over the whole sequences [ 18]. Tile homology is most prominent in the central part o f the molecules and continues towards the C-terminus. In the N-terminal part o f the sequences, which show rather low homology, four conserved cysteine residues are found, which may be imp o r t a n t for redox regulation o f the enzyme, Expression experiments with soluble bovine and rat guanylyl cyclase subunits revealed that coexpression of both subunits ~s necessary for the cat~dytic function of the enzyrnc [19,22]. This finding and the high structural

Nlar~:l~ 1991

I~omology bctweel| t h e two s~lbuni[~ su88¢Sl that botl~ ,~ubunit~ are involved in the formation o f cyclic G M P . Very re~:ently, the .settucn~:e of anotller ~2y.losoli¢ 8uanylyl e y c l a ~ subunil, which is. preferentially expressed in rat kidney, was reported 1231i, Th~ existence o f different forms o f subunizs demands, a unifying .and consislent subtmit i~omenclatnre. As proposed by Garbers rind associates [-~31, the subunii.~ o f soluble 8nanylyl cyclase (.GC-S) should bc referred to as t~ and t;~subunits, whereby ¢~ refers to the "/3182 kDa subtlnit (apparent molecular masses on S D S . P A G E ) and i:~ to the 70 kDa o f the enzyme characterisetl from lung. The new subunit {23], which was identified using the polymerasc chain rellction with olilonudeotides c o l responding to amino acid sequences con.~erved between all 8uanylyl cyclases, has a calculated molecular mas~ o f "76,3 kDo :rod shows tl~e highest ttcgree of similarity to the i~ subtmit with 3 4 % of identical amino acids over the whole lengttt o f the sequences. This new subunit, designated tJ.,, lacks the first 62 amino acids of tile 3~ subunit but extends with C.terminal 86 amino acids beyond the C.termiaus o f i~ and contains a consensus sequence for isoprenylation/carboxymethylation [231. The diversity irt subunits of soluble guanyly/ cyclase suggests possible differences In regulations, As the expression o f the t~.~subunit by itself did n0[ yield an enzymaticaily active guanyly[ cyclase, the question remains whether the ¢3., subunit c a n form a catalytically active heterodimer with the cj subunit or whether another c subunit ~s necessary for catalytic activity, In addition the mechanism o f activation of the resulting heterodinaer has to be clarified. In contrast to the cytosolic gual~ylyl cyclase, n'~embrane-bound guanylyl cyclases consist o f one subunit With a M,. of about 120--180 kDa. All membrane-bound enzymes purified so far are glycosylated. The mammalian enzyme was shown to bind ANP; however, tiffs binding was taot associated with increased enzymatic activity o f tile purified enzyme (for refs see [41), Tile primary structures of two manlnmlian, type A and type B, membrane-bound guanylyl cyclases and a membrane-bound guanylyl cyclase from sea urchin sperm have been determined (for refs see [4]). The amino acid sequences predict proteins that are structurally related to tile protem-tyrosine kinase-linked growth factor receptors, with an N-terminal extracellular part, one traas-membrane domain and a Cterminal cytosolic region (Fig. 1). The membraneb o u n d guanylyl cyclases show weak homology to the cytosolic region of the growth factor receptors, which is supposed to be responsible for the tyrosine kinase activity; for membrane-bound guanylyl cyclases tyrosine ki~ase activity has, however, not been demonstrated. The N-terminal parts o f the ANP-stimulated guanylyl cyclases show homologies to another, smaller A N P receptor which is n~3t coupled to a guanylyl cyclase, and 303

Volume 2t0, humor 2

FEB$ LETTERS

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Fig. I. Schemafi~ representation of struclurally related enzyme families. Shown are the membra.e-boand and eytosolic forms of protem-tyroxine ~hospllatases, protein.tyrosine kina~es. 8.anylyl cyclasesand tile membraf~e.bound adenylyl cyclone. Labelled boxes indicate sequence tlomoloBies, PTP, putative catalytic domain o f protein.tyro.~ine phosphata~es; PTK, putative catalyl;ic domain of protein.tyro~ine kina~e~; cyc. putative catalytic domain of guanylyl and adenylyl cyclascs; CD 45. leakocyte common antigen; LAR, leukocyte common aluigen.related molecule; EC3F. epidermal growth factor; PDGF, plalele| derived growlll factor; ANP, atrial natriuretic peptlde; ST, heat-stable ¢nterotoxin ofE. co~i, For further cxpiana¢ions See text,

the C.terminal region of all membrane.bound guanylyl cyclases contains a stretch of about 250 amino acids homologous with the subunits of the cytosolic enzyme (see below). Expression of the mammalian membranebound guanylyl cyclases proved that the proteins serve as a receptor for ANP and contain guanylyl cyclase activity and that catalytic activity is increased by hormone binding (for refs see [4]). Deletion of the region homologous to the tyrosine kinases resulted in a permanent ANP- and ATP-independent activation of membrane-bound guanylyl cyclase (for refs see [4]). The ANP-stimulated guanylyl cyclases type A and B share an overall homology of 62% identical amino acids; the intracellular regions appear more highly conserved than the extracellular domains (78°7o versus 43°70 identity). This may explain the differences in potencies of various natriuretic peptides on guanylyl cyclase A and B activities; the relatively high ANP concentrations required to elicit a response of the guanylyl cyclase B suggests the possible physiological stimulation by a more potent, unidentified natural ligand (for refs see

[41. A comparison of the sequences of the subunits of the cytosolic guanylyl cyclase and the sequences of all membrane-bound guanylyl cyclases sequenced so far

304

revealed a homologous domain of about 2S0 amino acids in the C-terminal regions of all molecules [18]. Two of these homologous domains are also found in the cytosolic parts of adenylyl cyclase from bovine brain [24] and rat olfactory neurons [25] (see Fig. l). The primary structures of the sequenced mammalian adenylyl cyclases predict proteins with 12 membrane spanning regions and two large hydrophilic domains, which are similar to each other and share homologies with the C-terminal part of all guanylyl cyclases [I 8]: As the regulation of all these cyclases is very different (coupling to G-proteins, direct hormone binding and NO-heme interaction for adenylyl cyclase, plasma membrane-bound and cytosolic guanylyl cyclases, respectively), but the principal reaction catalyzed by the enzymes is the same, we presume that the homologous region common to all sequences represents a domain involved in the catalytic function of the cyclases. Out of the adenylyl cyclases of lower organism, i,e. bacteria and yeast, sequenced so f a r ([26] and refs therein), only the adenylyl cyclase of yeast shows some rather low homoiogies to the hydrophilic domains of the mammalian adenylyl cyclase; the homologies to guanylyl cyclases are even less. Among the group of adenylyl cyclases o f lower organism, only the

Volunw 21tO, mtmber 2

FEllS L~TTERS

ealmodulin=sensitlve adenylyl cy¢lases from Bacillus an. thra¢i$ and from Bordere/la pertus.vis sinew significant homologies between each otl~er. 5, S T R U C T U R A L SIMILARITIES O F T H E G U A N Y L Y L CYCLASES TO T H E FAMILIES OF P R O T r ' I N . T ¥ ROSINE K INASES A N D P R O T E I N . T ¥ ROSINE PHOSPFIATASES Occurrence o f the membrane.bound guanylyl cyclases type A and B and the resact-receptor coupled guanylyl cyclase with highly conserved intracellular domains but relatively distinct receptor domains suggests the existence o f more isoforms of guanylyl cyclases, which may all share the catalytic domain, but are linked to different receptor domains and, thereby, are activated by different Iigands. In the course of tile preparation of this review, Garbers and associates published the primary structure of another membranebound form of guanylyl cyclases (OC type C), which had been obtained by the polymerase chain reaction using template eDNA from small intestine mucosa [27]. Tile sequence revealed strong similarities to the other membrane-bound guanylyl cyclases in the intracellular part of the molecule while the extraceltutar ligand bin. dh'~g domain showed almost no homologies. The cyclic GMP-forming activity of the expressed membranebound guanylyl cyclase type C was enhanced by the preincubatioa with the enterotoxin of g. co/i. These data demonstrate that indeed the guanylyl cyciase stimulated by the enterotoxin of E. coil is an isoform of the membrane-bound guanylyl cyclases type A and B with a different hormone binding domain. The principle of coupling various extracellular receptor domains to a common intracellular catalytic domain and the existence of a cytosolic enzyme form containing a homologous catalytic domain appears to be analogous to the families of protein-tyrosine kinases [28] and protein-tyrosine phosphatases [29]. In the membrane-bound form of tyrosine kinases, the catalytic intracellular domain shared by all members of the family is coupled to different growth factor receptors represented by the extracellular part o f the molecules. The cytosolic enzyme forms, e.g. the c-src protoncogene product pp60=-src are not coupled to a receptor, and the mechanism of regulation appears to be very complex and is not fully understood. In the leukocyte common antigen, CD45, which comprises a broad family of structurally related membranespanning molecules in hematopoietic cells, a tandem repeat of the putative protein-tyrosine phosphatase domain is linked to large, different external N-terminal domains. In the leukocyte common antigen-related molecule (LAR), again two catalytic phosphatase domains are connected to an external domain displaying structural characteristics of the N-CAMs. Nothing is known about the ligand of CD45, but N-CAM is known

Mar~h 1991

to be involved in cell-cell interaction, and such interaction could modulate the a~:tivity of the C.terminally located tyrosine phosphatas,:, The eytosolie f o r m o f the protein.tyrosine phosphatases contains only one catalytic domain, whose regulation is unknown. 6. CONCLUSIONS Analysis of the sequences of different guanylyl cyelas,:s reveals one region of about 250 amino acids with strong homologies, which probably represents the catalytic domain as this homologous region is also found in the adenylyl cyclas¢, an enzyme whose regulation is quite different from that of the guanylyl cyclases but which in principle catalyzes the same reaction, i.e. formation of a cyclic nueleotifle from nucleoside triphosphate. The fact that cytosolic guanylyl cyclase and adenylyl cyclase contain two putative catalytic do. mains suggests tllat two of these domains are necessary for enzymatic activity of the cyclases, but dimerisation of the membrane-bound guanylyl eyclase has not been demonstrated. This hypothesis is supported by the finding that receptor-linked tyrosine kinases, which are structurally related to the membrane-bound guanylyi cyclases, undergo dimerisation in the process of hormonal stimulation [301. Besides the sequence homology to the adenylyl cyclases, the family o f guanylyi cyclases shows structural analogies to the families of the protein-tyrosine kinases and protein.tyrosine phosphatases. All of these enzyme families consist of membrane-bound enzyme forms, in which different extracellular receptor domains are linked to a common catalytic domain, and of cytosolic enzyme forms containing a homologous catalytic domain. As the number of protein-tyrosine kinases and protein-tyrosine phosphatases has increased dramatically during the last years, the family of guanylyl cyclases is also expected to grow significantly in the near future.

Acknowletlgements: The research of our laboratory cited herein was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen lndustrie.

REFERENCES [1] Walter, U. (1990) Rev, Pl~ysiol, Biochem, Pharmacol, 113, 41-88. [2] Fain, G,L. and Matthews, H,R, (1990) Trends Neurosci, 13, 378-384. [3] Light, D,B,, Corbin, J,D, and Stanton, B,A, (1990) Nature 344, 336:-339. [4] Garbers, D.L. (1990) New Biologist 2, 499-504. [5l Spies, C,, Schultz, K.-D. and Schultz, G. (1980) NaunyoSchmiedeherg's Arch, Pharmacol, 311, 71-77. [6] Murad, F., Mittal, C,K,, Arnold, W,P., Katsuki~ S, and Kimura, H. (1978) Adv. Cyclic Nucleotide Res. 9, 175-204. [7] B6hme, E., Graf, H. and Schultz, G. (1978) Adv, Cyclic Nueleotide Res. 9, 131-143.

305

V~lume 21~O, number 2

FEBS LETTERS

Ilel (Jerker, R.. l-]Ohm~. ~,. I-Iofmann. F. ~md ~i,dtull~. (~i:. (l?llll .I"I~II~l,¢m l,~i].'~1-74, 2OO'/=2t111~. [10l I~lmer, R.,M,J., F©rrille, A.(i. ~md Mo~w.~d,. S, (1~#~) Nnllirr )27, ~14-~126, [111 Knowl¢~, R,¢~, Paln~le~. M,, Pnlmar, R.M.J, ~md Mon~adn, ~, 1121 13r~d~,D.S, nnd Snyd~r, ~,FI. (19901 Pro~, Na~l, A~;~d, S~;|, USA 1t7. 6112-ai1~, ll;I I Mayer, B., John. M, nnd B(~hm¢, E. (1990) FEBS l.~ll, 2"/7, [14] Cha|l~, C',.H., Koh~. K,P,, Chanl!. B., Flir~l~l, .M,. Jt~ml¢, B,. l~)oui~l~, J.E. and Murltd, F, [1990} Biod~htt, I:lioph~'.., A¢ln 10J2, I.~9-16~, II~] i:orl~. I..R., Krau~e, W,J, and Frecm¢m, R,II, (19~8)Am, J, Phy~lol, 2~$, FI0.10-FI046, [16) K~ml~nki, Y,, Sahekl, S., I'qakn~te, M,. Palmier|, J., Ku||o, T,, Clmnlh B., Wnldman, S.A. und Mur~td, F, (19~16}J, Blot, Chem, 261. 7236-7241. [171 Humbert, P.. Niroomantl, F. i-.'i~dter,(L, M~a)'~:r,B,, Koe'~lil~ih D,, Hinsch, K..H,I, G~|u~:pollh H., Fr~tnk, I~.,,Scht|h¢., G, and BOhm~, E. (1990) Eur. J. Bioehem, 190, 27.1-271~, [181 Koeslinlh D,, Harlene~:k, Ch,, Humbefh P., 13o~serhofl', A,, frank, R., Sehuhz, G, a~d BOhme, E. (1990) FEBS Le||, 266. 128-1.12,

306

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