A Guanylyl Cyclase from Paramecium with 22 Transmembrane Spans

Abstract

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His6-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu1681 and Ser1748 in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Gαs/GTP or forskolin, and P site inhibitors were only slightly effective. Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His6-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu1681 and Ser1748 in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Gαs/GTP or forskolin, and P site inhibitors were only slightly effective. nitrilotriacetic acid heterotrimeric guanine nucleotide-binding protein, GTPγS, guanosine 5′-O-(3-thiophosphate) guanylyl cyclase fromParamecium Adenylyl and guanylyl cyclases are key proteins in intracellular signaling of essentially all eukaryotic cells. In the freshwater protozoans Paramecium and Tetrahymena cAMP and cGMP levels depend on the ion composition of the extracellular milieu. Whereas adenylyl cyclase activity in vivo is stimulated by membrane hyperpolarization (1.Schultz J.E. Klumpp S. Benz R. Schürhof-Goeters W. Schmid A. Science. 1992; 255: 600-603Crossref PubMed Scopus (128) Google Scholar, 2.Schultz J.E. Schönborn C. FEBS Lett. 1994; 356: 322-326Crossref PubMed Scopus (17) Google Scholar), cGMP formation is enhanced by a depolarizing Ca2+ inward current (3.Schultz J.E. Pohl T. Klumpp S. Nature. 1986; 322: 271-273Crossref Scopus (48) Google Scholar). Recently, we reported on a group of guanylyl cyclases of 280 kDa that is present in the ciliates Paramecium and Tetrahymena but also in the genome of the malaria parasite Plasmodium (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). This novel signal transduction protein is bifunctional. It has a 155-kDa N-terminal P-type ATPase-like domain and a 115-kDa C-terminal guanylyl cyclase domain. Both are linked by a cytosolic loop of about 110 amino acids (see Fig. 1 A). Surprisingly, the guanylyl cyclase domain is topologically identical to mammalian adenylyl cyclases,i.e. it is composed of two pseudosymmetric modules of six putative transmembrane spans (M1 and M2) that serve as membrane anchors for the cytosolic, catalytic segments C1 and C2 (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). The sequences of both cytosolic subdomains are similar to each other and resemble the catalytic regions of metazoan adenylyl and, to a lesser extent, guanylyl cyclases. Sequence comparisons show that the order of the protozoan C1a- and C2-positioned loops is reversed compared with the corresponding mammalian adenylyl cyclase loops (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). Data based on the crystal structure of a soluble adenylyl cyclase type VC1a/type IIC2 chimera demonstrated that Lys938 and Asp1018 in the catalytic pocket of the C2 domain (adenylyl cyclase type II numbering) are responsible for ATP substrate specificity (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, 6.Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar). In theParamecium guanylyl cyclase, those two amino acids have evolved as Glu1681 and Ser1748 in the C1-positioned domain, i.e. specify GTP as a substrate, whereas the surrounding amino acid residues are more akin to metazoan adenylyl cyclases (see Fig. 1 B for an abbreviated sequence alignment). The expressed membrane-bound Paramecium guanylyl cyclase domain uses MgGTP as a substrate; it accepts MnATP as a substrate only slightly and is inactive with MgATP (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). Following successful strategies by others to produce soluble heterodimeric adenylyl cyclases (7.Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 8.Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (117) Google Scholar, 9.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google Scholar, 10.Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (109) Google Scholar, 11.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12.Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar, 13.Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (111) Google Scholar), we wished to assemble a protozoan heterodimeric guanylyl cyclase by expression inEscherichia coli of the catalytic domains either separately or covalently connected by a peptide linker. Here, we report on the expression and activity of such constructs. We then changed the purine nucleotide substrate specificity in this ciliate guanylyl cyclase with mammalian adenylyl cyclase topology by site-directed mutagenesis of two amino acids in the C1-positioned cytosolic loop. The inversion of the cytosolic loops was definitively proven by the successful generation of active adenylyl cyclase chimeras, which consisted only of cytosolic C2 cyclase domains, i.e. the mammalian adenylyl cyclase IIC2 or IXC2 and the C2-positioned domain of the Paramecium guanylyl cyclase. Radiochemicals were purchased from ICN or Amersham-Pharmacia Biotech; pQE vectors and nickel-NTA1 resin were from Qiagen. All enzymes were from Roche Molecular Biochemicals and New England Biolabs. To obtain the soluble constructs of theParamecium guanylyl cyclase cytosolic loops, we used a clone as a template in which all TAA/TAG-Gln codons had been mutated to the universal CAA/CAG-Gln triplets (GenBankTM accession numberAJ238859; Ref. 4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). For cloning purposes the C1a- (ParaGC-C1a) and C2- (ParaGC-C2) positioned cytosolic domains were fitted withSalI, MluI, and AscI restriction sites. The exact segment boundaries including the N-terminal His6-sequence and N- and C-terminal stuffer amino acids added to accommodate respective restriction sites are depicted in Fig.2 A. Conventional experimental protocols in molecular biology were followed to obtain the DNA constructs. A list of oligonucleotide sense and antisense primers and of synthetic mutagenic primers that were used in this study is available on request. The peptide linker AAGGPPAAGG in the construct ParaGC-C1aLParaGC-C2 was generated with two complementary oligonucleotide primers. The correctness of all constructs was verified by double-stranded DNA-sequencing. The constructs were cloned into a pQE30 expression vector that was modified to contain only a 5′-SalI and a 3′-AscI/HindIII cloning site. The C2 clone from rat type II adenylyl cyclase (GenBankTMaccession number M80550; IIC2) and a mouse type IX adenylyl cyclase clone (GenBankTM accession number Z50190) were kindly provided by Dr. C. Kleuss (Berlin) and Dr. F. Antoni (Glasgow), respectively. The C2 regions were cloned the with respective as and specific with and restriction sites for cloning of the into the pQE30 expression vector (see Fig. mutagenesis and was by the of and 1992; PubMed Scopus Google the site-directed mutagenesis and by to the C1a- and C2-positioned loops of the cyclase cloned into II as The constructs in the pQE30 expression were into the were in of containing and and with of were and in For C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google were in of containing a of inhibitors from were and were and were and the was for was by The were in and of was the resin was into a and with A with A with and were with of C containing The of the recombinant proteins with to adenylyl and guanylyl cyclase was Using we were to the protozoan constructs active upon on or by The of activity affected all protozoan constructs and not from to and on adenylyl or guanylyl cyclase were and all were from the affinity of activity was not to the proteins as analyzed by but to The proteins were analyzed by a mouse or a mouse Molecular were for in a of either or and 1 of were used as an of recombinant and The recombinant cyclase domains were on for to the by of and were and chromatography S. Schultz J.E. J. PubMed Scopus Google Scholar, Y. C. M. PubMed Scopus Google Scholar). The membrane-bound guanylyl cyclase from the Paramecium of two domains 1 A). The 155-kDa N-terminal domain resembles a P-type ATPase, and the C-terminal for a guanylyl cyclase of kDa with a structure of canonical mammalian adenylyl cyclases yet with reversed cytosolic C1a- and C2-positioned loops (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). For soluble constructs of cytosolic adenylyl cyclase loops have been (7.Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 8.Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (117) Google Scholar, 9.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google Scholar, 10.Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (109) Google Scholar, 11.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12.Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar, 13.Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (111) Google Scholar, W.-J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.K. Dessauer C.W. R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar, C.W. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We wished to a soluble protozoan guanylyl cyclase from and C2-positioned loops and expressed the recombinant proteins in coli either or linked by a peptide in to W.-J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.K. Dessauer C.W. R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar, C.W. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We used a construct with an molecular of kDa i.e. amino acid in of the which to a slightly for in C2 of rat type II adenylyl cyclase. The protozoan C2-positioned loop with an molecular of kDa consisted of the of the transmembrane A). of the recombinant a the as the proteins and only a of a was present in Guanylyl cyclase activity of catalytic loops was not whereas a of the C1a- and C2-positioned loops had activity either or MgGTP as substrate the protozoan guanylyl cyclase loops had a affinity for each other to a active This is in to with the catalytic domains from mammalian adenylyl cyclases of soluble adenylyl cyclase activity mixing of cytosolic domains from and the of C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google and guanylyl cyclase of type and mutated constructs of Paramecium were with of the respective or and of each recombinant cytosolic domain or of the linked all affinity in were and in the C1-positioned domain and in the C2-positioned domain of the Paramecium guanylyl cyclase. not The of the was Data are from with similar The were of the C2-positioned in a were with of the respective or and of each recombinant cytosolic domain or of the linked all affinity in were and in the C1-positioned domain and in the C2-positioned domain of the Paramecium guanylyl cyclase. not The of the was Data are from with similar The were of the C2-positioned It was demonstrated that the activity of soluble constructs of mammalian adenylyl cyclases is or the catalytic loops are linked by a peptide (7.Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar). we linked the protozoan cytosolic domains a the order of the domains present in the reversed compared with mammalian adenylyl cyclases Fig. A). affinity the was by as a guanylyl cyclase activity was it not that in a of the expressed loops The of the of for MgGTP and were and and not from the with the linked construct and and to for the membrane-bound guanylyl cyclase domain expressed in or for the (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar, S. Schultz J.E. J. PubMed Scopus Google Scholar). of substrate were from to MgGTP and not from a substrate GTP The was to with the In with GTP as a substrate a of ATP guanylyl cyclase a specificity for GTP as a The of was A of was from the of an To the substrate specificity of the soluble guanylyl cyclase, we all constructs for adenylyl cyclase activity Using MgATP as a substrate, adenylyl cyclase activity was or of the respective guanylyl cyclase activity MnATP adenylyl cyclase activity was about of guanylyl cyclase and was similar to that with GTP as a substrate The crystal structure of a rat adenylyl cyclase type IIC2 and of a and rat to and and two or amino acids in the cyclases as responsible for purine nucleotide specificity (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, 6.Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google W.-J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). amino acids are all mammalian adenylyl and guanylyl cyclases and are In adenylyl cyclases Lys938 and the IIC2 catalytic domain with the and of the J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar, Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). In guanylyl cyclases the corresponding amino acids are and to the of rat soluble guanylyl which with the and the of the guanine of amino acid residues the respective substrate of mammalian guanylyl and adenylyl cyclases R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to the Paramecium guanylyl cyclase, the C1-positioned domain is the of the catalytic of soluble guanylyl cyclases. Glu1681 in the C1-positioned loop is to and Ser1748 is to 1 In the a that is in mammalian guanylyl cyclases 1 This not the specific of GTP in the a the of the guanine and the This was a mutation the guanylyl cyclase activity to the of the A. C. J. Schultz G. Biochemistry. 1997; PubMed Scopus Google Scholar). To the of Glu1681 and Ser1748 in theParamecium domain in substrate we mutated to the adenylyl cyclase specific residues Lys and In we generated the the was not The constructs were enriched by affinity chromatography and by All recombinant proteins the molecular and were more or of Ser1748 to all cyclase of ATP or GTP as with as a guanylyl cyclase activity was by more and adenylyl cyclase activity was by more the of the adenylyl and guanylyl cyclase was only which that substrate was The then that the with the amino acid corresponding to to the for ATP or GTP substrate and to substrate The was in a purine nucleotide cyclase. in of two amino acids in mammalian membrane-bound or soluble guanylyl cyclases and in the mammalian had all of cyclase activity R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the guanylyl cyclase the nucleotide specificity was switched from GTP to of guanylyl cyclase were of type whereas adenylyl cyclase activity to and This demonstrated the inversion of the of the cytosolic loops in the ciliate guanylyl cyclase the amino acids were in the domain, whereas in mammalian adenylyl cyclases are in the C2 domain R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it demonstrated that which a that is in all mammalian guanylyl cyclases, is and a to had the In it the that the Paramecium guanylyl cyclase with a mammalian adenylyl cyclase membrane topology is a guanylyl cyclase we substrate specificity by site-directed mutagenesis of the protozoan domain as by the available of the mammalian adenylyl cyclase C2 (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, 6.Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). A novel for a cyclase with a canonical mammalian adenylyl cyclase topology was the in purine nucleotide This has been only for mammalian guanylyl cyclases R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The to the substrate specificity of a soluble mammalian adenylyl cyclase in a purine nucleotide cyclase R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the C1 domain of adenylyl cyclases type adenylyl was as a of for substrate by and by (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In adenylyl cyclases this is of a In soluble guanylyl cyclases the corresponding is an which to in the Paramecium C2-positioned loop. we mutated is in guanylyl in the ciliate C2-positioned domain to the adenylyl cyclase B and The construct was expressed in to a protein on a of the type with the guanylyl cyclase activity to and with MgGTP and the is adenylyl this have been it was to that also the adenylyl cyclase activity was to and with MgATP and MnATP as the of guanylyl to adenylyl cyclase that the of the C1-positioned loop is not all in substrate We that the was inactive which the The in which all residues and specific for were not cAMP formation that with the are in with substrate of the adenylyl cyclase chimera in which the Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). we the that a of the to substrate in other cyclase (see In theParamecium guanylyl cyclase the amino acid residues for substrate to for guanylyl cyclase adenylyl cyclase cyclase activity were with of the respective or Guanylyl cyclase activity was not with either All the domain were residues are in (see for in a were with of the respective or Guanylyl cyclase activity was not with either All the domain were residues are in (see for In the protozoan the molecular of the cytosolic and C2-positioned loops is reversed (see and J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). To the of on we the of the C2-positioned domain for of cyclase activity with the catalytic C2 loop from mammalian adenylyl cyclases of rat type II and mouse type IX Fig. expression and IIC2 as a on a whereas the of IXC2 by of guanylyl cyclase activity was in protozoan C2 chimeras, whereas adenylyl cyclase were in with either IIC2 or IXC2 The were with MnATP as a substrate as compared with MgATP This was in the which was more active with MnATP the inversion of the cytosolic loops in the Paramecium guanylyl cyclase has been on an the C2-positioned domain from the ciliate guanylyl cyclase for a mammalian adenylyl cyclase domain. of the sequence theParamecium C2-positioned and mammalian domains, the which are to a were in the ciliate C2 loop. we used the for adenylyl cyclase with IIC2 or Whereas adenylyl cyclase activity was the for MnATP as a substrate was This that the to the of the C1 and C2 domains in adenylyl cyclases, not the domain not an active cyclase with mammalian domains not All membrane-bound mammalian adenylyl cyclases by the of and the forskolin, and by P site of M. Google Scholar). we the of canonical adenylyl cyclase or affected of the guanylyl cyclase constructs. This was not of the Paramecium guanylyl cyclase sequence that amino acid residues to for cyclase by and for such as (adenylyl cyclase numbering) are J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar). all were also by and In respective an of a was not The are of a chimera of and the of a soluble rat guanylyl cyclase, which also was to the adenylyl cyclase S. Kleuss C. Biochemistry. 1999; PubMed Scopus Google Scholar). adenylyl cyclase by and to constructs the mammalian C1 and C2 P site inhibitors the of the cyclases with C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). is M. Google Scholar). The available P site for adenylyl cyclases is with of 1 for the and in the upon by In a similar guanylyl cyclase is by R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We the of and on the constructs generated In P site soluble Paramecium guanylyl cyclase constructs was 1 not guanylyl cyclase and 1 by about guanylyl cyclase was with as a i.e. in a activity guanylyl cyclase activity was with as an i.e. in a activity was more i.e. was and specific as as the purine was The of the by the in the of a soluble guanylyl cyclase had on the of we the adenylyl cyclase reconstituted from the mutated Paramecium domains and and from the chimeras, i.e. and 1 adenylyl cyclase of all reconstituted enzymes by about was 1 i.e. we that substrate and P site specificity to in constructs as reported for mammalian constructs R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the of the P site inhibitors compared with mammalian cyclases. The then of this was the The protein that is in for the of the group of the nucleotide in with the of two Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). The amino acids are of the C1 and in the domain and and IIC2 Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). from respective sequence all of amino acid residues are present in the corresponding C2- and C1-positioned domains of the protozoan guanylyl cyclase. are of the C2-positioned and in the C2-positioned loop and and in the catalytic C1-positioned loop (see the abbreviated in we that the amino acid for the site is present in the protozoan guanylyl cyclase and that the of of P site inhibitors has a In mammalian adenylyl cyclases loops a substrate ATP to a specific and active site and the heterotrimeric proteins either or such a which the substrate group and the catalytic residues Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). all of the amino acid residues for such a are present in theParamecium guanylyl cyclase, we that it in a similar to that of a mammalian adenylyl cyclase. it is that it also an to the into a activity which then affected by P site inhibitors mammalian adenylyl cyclases have to for the of of the heterotrimeric protein in Paramecium U. T. U. and J. In in the of a and a protein that the substrate, have been we have to for the of a protozoan protein that the Ca2+ in the guanylyl cyclase S. Schultz J.E. J. PubMed Scopus Google Scholar). In this the of the 155-kDa N-terminal P-type domain the more The of a P-type with a guanylyl cyclase as a mammalian adenylyl cyclase in a sequence to novel as M. Science. 1999; PubMed Scopus Google of cyclase by with 1 cyclase cyclase and was used in all residues are in (see for in a and was used in all residues are in (see for We C. Kleuss and (Berlin) for a clone of the adenylyl cyclase type IIC2 and a of and Dr. F. for a clone of the mouse type IX adenylyl cyclase.