Adenylyl Cyclase Regulates Signal Onset via the Inhibitory GTP-binding Protein, Gi

Abstract

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, Gs and Gi, respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Gαs and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric Gs (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328–1331). This latter action of ACV would facilitate the rapid onset of signaling via Gs. Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Gαi, we investigated whether the receptor-mediated activation of heterotrimeric Gi was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric Gi such that the amount of peptide required to stimulate Gi in steady-state GTPase activity assays was 3–4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5′-(γ-thio)triphosphate (GTPγS) to Gi was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPγS binding to Gi in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc− cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous Gi and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of Gi. Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, Gs and Gi, respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Gαs and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric Gs (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328–1331). This latter action of ACV would facilitate the rapid onset of signaling via Gs. Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Gαi, we investigated whether the receptor-mediated activation of heterotrimeric Gi was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric Gi such that the amount of peptide required to stimulate Gi in steady-state GTPase activity assays was 3–4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5′-(γ-thio)triphosphate (GTPγS) to Gi was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPγS binding to Gi in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc− cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous Gi and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of Gi. adenylyl cyclase type V adenylyl cyclase dithiothreitol guanosine 5′-(γ- thio)triphosphate Adenylyl cyclase (AC)1, the enzyme that catalyzes the conversion of ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, Gsand Gi, respectively. The binding of hormones or neurotransmitters to their respective receptors that couple to either Gs or Gi results in the activation of these G proteins (reviewed in Refs. 1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar and 2Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (265) Google Scholar). Essentially, receptor-mediated activation of the heterotrimeric G proteins involves the exchange of GTP for GDP on the α subunit, and this results in the dissociation of the α from βγ subunits (reviewed in Refs. 1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar and 2Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (265) Google Scholar). Although the activated Gαs subunit can stimulate the activity of all nine forms of membrane-bound adenylyl cyclases that have been cloned and characterized to date (reviewed in Ref. 1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar), activated Gαi inhibits only type V, VI, and I adenylyl cyclases (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar,3Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Crossref PubMed Scopus (35) Google Scholar, 4Dessauer C.W. Tesmer J.J. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) has two other functions. First, the enzyme can act as a GTPase-activating protein for Gαs and thereby expedite the termination of signaling via activated monomeric Gαs (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar). Second, adenylyl cyclase enhances the ability of activated β-adrenergic receptor mimetic peptide to stimulate GTP-GDP exchange on heterotrimeric Gs (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar). This latter action of adenylyl cyclase would facilitate the rapid onset of signaling. Interestingly, the regions on adenylyl cyclase that interact with Gαs (6Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar) are sufficient to exert the GTPase-activating protein- and guanine nucleotide exchange-enhancing actions of the enzyme (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar). Since we (3Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Crossref PubMed Scopus (35) Google Scholar) and others (4Dessauer C.W. Tesmer J.J. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) have shown that the C1 region of ACV interacts with the inhibitory GTP-binding protein Gαi, we investigated whether the activation of heterotrimeric Gi was also regulated by adenylyl cyclase and its subdomains. Our data demonstrate that adenylyl cyclase and its C1 domain increased the ability of an activated muscarinic receptor mimetic peptide to enhance activation of heterotrimeric Gi. These data and our previous results (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar) demonstrate that adenylyl cyclase regulates receptor-mediated activation of both G proteins that modulate its activity. The recombinant soluble C1-C2 ACV and its subdomains C1 and C2 were expressed in the TP2000 strain of Escherichia coli, which does not contain endogenous AC (7Roy A. Danchin A. Mol. Gen. Genet. 1982; 188: 465-471Crossref PubMed Scopus (52) Google Scholar, 8Beauve A. Boesten B. Crasnier M. Danchin A. O'Gara F. J. Bacteriol. 1990; 172: 2614-2621Crossref PubMed Google Scholar). Expression of protein and cell lysis were performed as described previously (9Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2916-2920Google Scholar), except that the induction with isopropyl-β-d-thiogalactopyranoside (0.1 mm) was performed at 23 °C for 15 h. The hexahistidyl-tagged proteins were then purified as described for C1-C2 ACV. Briefly, after cell lysis, the supernatant was batch-bound for 1 h at 4 °C to metal-chelating resin (Talon, CLONTECH), which was equilibrated in buffer containing 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 1 mm β-mercaptoethanol. The resin was then poured into a column and washed with 10 column volumes of buffer containing 50 mm Tris-HCl, pH 8.0, 500 mm NaCl, and 1 mm β-mercaptoethanol, followed by a second wash with 10 column volumes of buffer containing 50 mm Tris-HCl, pH 8.0, 20 mm NaCl, and 1 mm β-mercaptoethanol. The proteins were eluted with 3 column volumes of 50 mm Tris-HCl, pH 8.0, 20 mmNaCl, 1 mm β-mercaptoethanol, and 125 mmimidazole. The eluted proteins were then applied to a Mono Q 5/5 fast protein liquid chromatography column and eluted with a 30-ml gradient of NaCl (100–450 mm) in 50 mm Tris-HCl, pH 8.0. Fractions containing the proteins were pooled and concentrated in buffer containing 20 mm Tris-HCl, pH 8.0, 1 mmEDTA, and 5% glycerol and stored at −80 °C. All of the G protein α subunits were expressed in the JM109(DE3) strain ofE. coli as described by Lee et al. (10Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (245) Google Scholar). Gαi1 was coexpressed with yeast proteinN-myristoyltransferase to ensure synthesis of myristoylated G protein as described by Linder et al. (11Linder M.E. Pang I.-H. Duronio R.J. Gordon J.I. Sternweis P.J. Gilman A.G. J. Biol. Chem. 1991; 266: 4654-4659Abstract Full Text PDF PubMed Google Scholar). Purification of the recombinant myristoylated Gαi1 protein was achieved by the method of Mumby and Linder (12Mumby S.M. Linder M.E. Methods Enzymol. 1994; 237: 254-268Crossref PubMed Scopus (112) Google Scholar). For the purification of the hexahistidyl-tagged, constitutively active Q213L mutant of Gαs (Gαs*) (13Masters S.B. Tyler Miller R. Chi M.-H. Chang F.-H. Beiderman B. Lopez N.G. Bourne H. J. Biol. Chem. 1989; 264: 15467-15474Abstract Full Text PDF PubMed Google Scholar), the harvested cells were lysed as described by Lee et al. (10Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (245) Google Scholar). The supernatant was than batch-bound to a nickel-nitrilotriacetic acid resin in 50 mm Tris-HCl, pH 8.0. After pouring the resin into a column, it was first washed with 5 column volumes of 50 mmTris-HCl, pH 8.0, and then with 10 column volumes of 50 mmTris-HCl, pH 8.0, and 5 mm imidazole. Gαs* was eluted with a 50-ml gradient of imidazole (5–250 mm) in 50 mm Tris-HCl, pH 8.0. The fractions containing Gαs* were pooled, concentrated, exchanged with buffer (10 mm Tris-HCl, pH 8.2, 2.0 mm DTT, and 10 mm potassium phosphate), and applied to a hydroxylapatite HPHT column (Bio-Rad). This column was washed with 2 column volumes of the same buffer and eluted over a 40-ml gradient of increasing potassium phosphate concentration (10–300 mm) in the same buffer. The fractions containing Gαs* were pooled, concentrated, and stored in 50 mm Hepes, pH 8.1, 1 mm and 2 mm at −80 °C. βγ subunits of heterotrimeric G proteins were purified to as described by Mumby et al. S. Pang I.-H. Gilman A.G. Sternweis J. Biol. Chem. Full Text PDF PubMed Google Scholar) with the of et al. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Gi was by recombinant myristoylated Gαi1 with purified βγ subunits of for at 4 °C in buffer containing mm Hepes, pH 8.0, 1 mmEDTA, 1 mm DTT, 1 and was by a in the steady-state GTPase activity of heterotrimeric Gi monomeric Gαi The G protein active was than with or without the of at °C in 50 of buffer containing 100 mm Hepes, pH 8.0, 100 and 1 mm of which was for was as described previously H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Essentially, were after 20 by the into containing of 5% in 50 mm pH 8.0. After of the supernatant were for of by Gi protein active was first with 100 GTPγS in pH 8.0, for 20 at to GTPγS to the α subunits that were not to of this were then with or without the of for at in 50 of buffer containing 100 mm Hepes, pH 8.0, 100 1 mm DTT, and 1 In the binding of 1 to the same amount of Gi in the presence of mm was also The latter a of the amount of active Gαi in the were and and were as described previously H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). binding of was also performed in the presence of GTPγS as described H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). S49 cyc− cell were as described by and S. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar). cells were with (10 for h to the of cell In with of were for at in the presence or of the proteins of and with or without the muscarinic receptor mimetic peptide This also 100 mm Hepes, pH 8.0, 100 1 mm DTT, 1 and a mm 20 20 and 20 The S49 cyc− cell were than into the AC activity were performed for 15 at in the presence of 1 and 10 as described previously H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Gαs* or was to stimulate enzyme activity. ensure activation of the G protein was with 100 GTPγS at for For adenylyl cyclase activity assays with S49 cyc− cell (10 were for 2 at with or without ACV subdomains in the or presence of the of somatostatin in a containing mm Hepes, pH 8.0, mm DTT, mm and a mm and The Hepes, DTT, and were in the The were by the of of AC containing at a concentration of 100 were performed as described in the presence of 1 mm have shown that to of receptors can the actions of activated receptors and can stimulate G proteins and the G protein (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar, H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. Y. Y. M. E. 1991; Full Text PDF PubMed Scopus Google Scholar, T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In this these can as constitutively active and T. J. Biol. Chem. Full Text PDF PubMed Google Scholar) have shown that a peptide to in the of the muscarinic receptor (MIII-4) can activate Gi. in our we peptide MIII-4 to activate heterotrimeric Gi. The activation of heterotrimeric G proteins involves exchange of GTP for GDP on the α This GTP-GDP exchange is the in the activation of G proteins and can by the steady-state of GTP (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar, T. Y. Y. M. E. 1991; Full Text PDF PubMed Scopus Google Scholar, T.M. Gilman A.G. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Recently, we (3Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Crossref PubMed Scopus (35) Google Scholar) and others (4Dessauer C.W. Tesmer J.J. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) have demonstrated that Gαi interacts with the C1 domain of ACV and inhibits enzyme activity. in we investigated the ability of the C1 and C2 of ACV to modulate the steady-state GTPase activity of Gi in the presence of of peptide MIII-4. demonstrated in increasing of peptide MIII-4 increased the steady-state GTPase activity of Gi. in the presence of the C1 domain of the ability of the peptide to steady-state GTPase activity was markedly such that the was to the by In in the of peptide the C1 domain not alter the steady-state GTPase activity of Gi peptide concentration in 1 and the C1 domain by also not demonstrate GTPase activity over not the C2 domain of adenylyl cyclase not alter the ability of peptide MIII-4 to modulate the steady-state GTPase activity of Gi 1 to the C1 the C2 domain by also not demonstrate GTPase activity not these data show that the C1 domain of not the C2 region of the can the ability of the muscarinic receptor mimetic peptide MIII-4 to GTP-GDP exchange on is well that the C1 and C2 of AC are required to enzyme which can regulated by its (3Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Crossref PubMed Scopus (35) Google Scholar, 4Dessauer C.W. Tesmer J.J. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar, K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2916-2920Google Scholar). to whether the C1 domain in the of the adenylyl cyclase can modulate the ability of MIII-4 to the steady-state GTPase activity of Gαi, the previously described (3Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Crossref PubMed Scopus (35) Google Scholar, K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar, K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2916-2920Google Scholar), soluble of ACV was The data in demonstrate that the C1 in the of C1-C2 ACV by not alter the steady-state GTPase activity of Gi. C1-C2 ACV by also not demonstrate GTPase activity over not to the data with the C1 C1-C2 ACV markedly the ability of peptide MIII-4 to stimulate GTPase activity 1 the GTP-GDP exchange-enhancing actions of the C1 domain of ACV are also the C2 domain is to interact with the C1 Because the ability of (0.1 of MIII-4 to activate Gi markedly enhanced by the C1 domain and C1-C2 ACV and in we MIII-4 at a concentration (0.1 that by does not markedly stimulate the steady-state GTPase activity. this concentration of the we that the of C1-C2 ACV and its C1 domain to the GTPase activity of Gi was and 1 the other in the C2 domain of ACV at to 2.0 not alter the ability of MIII-4 to activate Gi 1 of heterotrimeric G proteins also results in increased GTP binding to the subunit (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar, H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. Y. Y. M. E. 1991; Full Text PDF PubMed Scopus Google Scholar). as a second to Gi we the binding of the GTP GTPγS to Gi. These were 100 that are to in the ability of G proteins to GTPγS (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar, H. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. Y. Y. M. E. 1991; Full Text PDF PubMed Scopus Google Scholar, T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). demonstrated by the data in the of MIII-4 (0.1 the of C1-C2 ACV or its subdomains and C2) in the of MIII-4 GTPγS binding to Gi. the presence of both MIII-4 and C1 or C1-C2 ACV increased GTPγS binding to the G protein the C2 domain of which not modulate the ability of MIII-4 to stimulate the steady-state GTPase activity of Gi also to GTPγS binding to Gi These data demonstrate that C1-C2 ACV and its C1 domain enhance the ability of MIII-4 to activate Gi and to GTPγS binding to the G the of the ability of the C1 domain of ACV to the actions of MIII-4 for the in were Essentially, we the ability of the C1 domain of ACV to modulate adenylyl cyclase activity via MIII-4-mediated activation of endogenous Gi in S49 cyc− S49 cyc− cells not Gαs Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and a to Gi activation without from Gs activity. S49 cyc− cells type AC R. PubMed Google Scholar), which is by Gαi (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar, 2Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (265) Google Scholar). In these the cyc− were with or without peptide MIII-4 (0.1 in the presence and of the C1 or C2 domain of ACV at AC activity assays were then performed in the presence of demonstrated by the data in 3 peptide MIII-4 (0.1 the C1 domain of ACV AC activity in cyc− both MIII-4 and C1 were the AC activity was by the of AC activity in the presence of MIII-4 and the C1 domain of ACV was to activation of endogenous Gi, were performed in of S49 cyc− cells with Gαi, and this of the G protein its activation by receptors T. M. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). shown in in from the of AC activity in the presence of MIII-4 and the C1 domain was These data demonstrate that the of AC activity in the presence of MIII-4 and the C1 domain in of S49 cyc− cells not to is to activation of endogenous Gi. Furthermore, in with the C2 domain of AC activity was not the C2 domain was with MIII-4 These data show that the C1 (but not the C2) domain of ACV enhances the ability of MIII-4 to activate endogenous Gi in cyc− cell and to inhibit AC activity. The was by in which AC activity in cyc− cell was with the constitutively mutant of Gαs and was to the GTP GTPγS to in AC activity in cyc− to in the active of shown in 4 AC activity in was and markedly by to the with 3 C1 of peptide MIII-4 (0.1 by AC activity that was by Gαs* 4 the of MIII-4 and C1 AC activity by 4 This was for the C1 domain of ACV the C2 domain of ACV by or in with MIII-4 to alter AC activity in cyc− cells 4 the in 4 was to activation of endogenous Gi was demonstrated by the that GTPγS was from the of S49 cyc− with or without MIII-4 the C1 the shown in was not not the C1 region of ACV of adenylyl cyclase activity by activation of endogenous were performed with S49 cyc− cell the with the muscarinic receptor mimetic peptide the ability of the muscarinic receptor to inhibit the activity of adenylyl cyclase in cyc− was at to 1 mm not inhibit adenylyl cyclase activity in not it would that cyc− cells not muscarinic Because et al. T. M. Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar) have shown that somatostatin via Gi activation inhibits adenylyl cyclase activity in cyc− cell membranes, were performed with shown in in the of ACV somatostatin adenylyl cyclase activity in a of the C2 domain of ACV not alter the of adenylyl cyclase by somatostatin the C1 domain of ACV the somatostatin to the that the of enzyme activity at of somatostatin was enhanced in the presence of the C1 domain In the C1 domain in the of not inhibit adenylyl cyclase activity also 3 and in of cyc− cells with somatostatin the of somatostatin and C1 domain adenylyl cyclase activity not These data demonstrate that the that we have described employing a Gi protein and a muscarinic receptor mimetic peptide is to of adenylyl cyclase by endogenous in the presence of the C1 region of of and of active receptors are required to inhibit adenylyl the data in 5 demonstrate that ACV and its C1 which interacts with Gαi (3Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Crossref PubMed Scopus (35) Google Scholar, 4Dessauer C.W. Tesmer J.J. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), enhance the ability of the muscarinic receptor mimetic peptide (MIII-4) and somatostatin receptors to activate Gi. These with our previous that the C2 domain of which interacts with enhances the guanine nucleotide exchange activity of the receptor mimetic peptide for Gs (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar), that the of AC the ability of receptors to GTP-GDP exchange on G proteins is to both G proteins that its activity. In this it that and S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar) have shown that does not alter receptor-mediated GTP-GDP exchange on Because our in cyc− with MIII-4 and activation of endogenous somatostatin receptors are with the in data it is that the S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar) and AC K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar and this are to the of receptor S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar) the of receptor mimetic and without K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar and this it is that modulate the activity of their respective G proteins in in the with of heterotrimeric G proteins this of the of our is that the C1 domain of which interacts with Gαi, the guanine nucleotide exchange activity of a receptor mimetic peptide that Gi, the C2 region of which interacts with the guanine nucleotide exchange activity of receptors that couple Gs (5Scholich K. Mullenix J.B. Wittpoth C. Poppleton H.M. Pierre S.C. Lindorfer M.A. Garrison J.C. Patel T.B. Science. 1999; 283: 1328-1331Crossref PubMed Scopus (58) Google Scholar). it would that the of the ACV with their respective subunits in the of the is for the from receptors to the G proteins that are are to the of the Gilman for the Gαs for the Gαi1 for the TP2000 strain ofE. Gordon for the S. and for the ACV