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• W2017229143 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 EDTA, and 2 mm DTT at −80 °C. Bovine brain βγ subunits of heterotrimeric G proteins were purified to homogeneity as described by Mumby et al. (14Mumby S. Pang I.-H. Gilman A.G. Sternweis P.C. J. Biol. Chem. 1988; 263: 2020-2026Abstract Full Text PDF PubMed Google Scholar) with the modifications of Neer et al. (15Neer E.J. Lok J.M. Wolf L.G. J. Biol. Chem. 1984; 259: 14222-14229Abstract Full Text PDF PubMed Google Scholar). Heterotrimeric Gi was reconstituted by mixing recombinant myristoylated Gαi1 with purified bovine brain βγ subunits (Gαi/βγ molar ratio of 1:5) for 45 min at 4 °C in buffer containing 25 mm Hepes, pH 8.0, 1 mmEDTA, 1 mm DTT, 1 μm GDP, and 0.1% Lubrol. Reconstitution was confirmed by a significant decrease in the steady-state GTPase activity of heterotrimeric Gi versus monomeric Gαi under identical experimental conditions. The reconstituted G protein (24.4 nm active Gαi) was than incubated with or without the peptides of interest at 25 °C in 50 μl of reaction buffer containing 100 nm [γ-32P]GTP, 25 mm Hepes, pH 8.0, 100 μm EDTA, 120 μm MgCl2, and 1 mm DTT. Hydrolysis of [γ-32P]GTP, which was linear for >30 min, was measured as described previously (16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar). Essentially, reactions were stopped after 20 min by transferring the assay mixture into tubes containing 0.75 ml of ice-cold 5% (w/v) Norit A in 50 mm NaH2PO4, pH 8.0. After centrifugation, aliquots (500 μl) of the supernatant were decanted for determination of [32P]phosphate content by scintillation counting. Reconstituted Gi protein (12.2 nm active Gαi) was first mixed with 100 nm GTPγS in 25 mmHepes, pH 8.0, for 20 min at room temperature to bind GTPγS to the α subunits that were not bound to Gβγ subunits. Aliquots of this mixture were then incubated with or without the peptides of interest for 60 min at room temperature in 50 μl of reaction buffer containing 100 nm [35S]GTPγS, 25 mm Hepes, pH 8.0, 100 μm EDTA, 1 mm DTT, 150 μm MgCl2, and 1 mg/ml bovine serum albumin. In parallel incubations, the binding of 1 μm[35S]GTPγS to the same amount of Gi in the presence of 25 mm MgCl2 was also monitored. The latter provided a measure of the total amount of active Gαi in the heterotrimer. Reactions were stopped, and bound and free [35S]GTPγS were separated as described previously (16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar). Nonspecific binding of [35S]GTPγS was also performed in the presence of excess unlabeled GTPγS as described (16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar). S49 cyc− cell membranes were prepared as described by Kassis and Fishman (17Kassis S. Fishman P.H. J. Biol. Chem. 1982; 257: 5312-5318Abstract Full Text PDF PubMed Google Scholar). Where indicated, cells were treated with pertussis toxin (10 μg/ml) for 24 h prior to the isolation of cell membranes. In experiments with MIII-4, membranes (15 μg of protein) were incubated for 30 min at room temperature in the presence or absence of the proteins of interest and with or without the muscarinic receptor mimetic peptide (MIII-4, Alpha Diagnostics). This incubation (50 μl final volume) also contained 100 nmGTPγS, 25 mm Hepes, pH 8.0, 100 μm EDTA, 1 mm DTT, 150 μm MgCl2, 1 mg/ml bovine serum albumin, and a protease inhibitor mixture (5 mm benzamidine, 20 μg/ml lima bean trypsin inhibitor, 20 μg/ml leupeptin, and 20 μg/ml aprotinin). The preincubated S49 cyc− cell membranes were than transferred into the AC activity reactions (100 μl total volume). Assays were performed for 15 min at room temperature in the presence of 1 mmMgCl2 and 10 μm GDPβS as described previously (16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar). Gαs* (50 nm) or forskolin (33 μm) was used to stimulate enzyme activity. To ensure maximal activation of Gαs*, the G protein was incubated with 100 nm GTPγS at room temperature for 30 min. For adenylyl cyclase activity assays with somatostatin, S49 cyc− cell membranes (10 μg) were preincubated for 2 min at room temperature with or without ACV subdomains in the absence or presence of the indicated concentrations of somatostatin in a medium (75 μl final volume) containing 3.3 mm Hepes, pH 8.0, 0.13 mm DTT, 0.13 mm EDTA, 13.3 μm GTP, 1.3 mg/ml bovine serum albumin, and a protease inhibitor mixture (6.7 mm benzamidine, 26.6 μg/ml lima bean trypsin inhibitor, 26.6 μg/ml leupeptin, and 26.6 μg/ml aprotinin). The Hepes, DTT, and EDTA were components in the membrane preparations. The reactions were initiated by the addition of 25 μl of 4× AC assay mixture containing forskolin at a final concentration of 100 μm. Assays were performed as described above in the presence of 1 mm MgCl2. Several studies have shown that peptides corresponding to cytosolic domains of receptors can mimic the actions of activated receptors and can stimulate G proteins and subsequently the G protein effectors (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, 16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar, 18Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 19Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar). In this respect, these peptides can be regarded as constitutively active receptors. Okamoto and Nishimoto (19Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar) have shown that a peptide corresponding to amino acids 382–400 (RNQVRKKRQMAARERKVTR) in the third cytosolic loop of the M4 muscarinic cholinergic receptor (MIII-4) can efficiently activate Gi. Therefore, in our studies, we employed peptide MIII-4 to activate heterotrimeric Gi. The activation of heterotrimeric G proteins involves exchange of GTP for GDP on the α subunit. This GTP-GDP exchange is the rate-limiting step in the activation of G proteins and can be conveniently monitored by measuring the steady-state rate of GTP hydrolysis (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, 18Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 20Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 21Higashijima T. Ferguson K.M. Smigel M.D. Gilman A.G. J. Biol. Chem. 1987; 262: 757-761Abstract 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. Therefore, in initial experiments, we investigated the ability of the C1 and C2 domains of ACV to modulate the steady-state GTPase activity of Gi in the presence of varying concentrations of peptide MIII-4. As demonstrated in Fig.1 A, increasing concentrations of peptide MIII-4 increased the steady-state GTPase activity of Gi. However, in the presence of the C1 domain of ACV, the ability of the peptide to increase steady-state GTPase activity was markedly augmented such that the concentration-response curve was shifted to the left by ∼3–4 log units. In control experiments, in the absence of peptide MIII-4, the C1 domain did not alter the steady-state GTPase activity of Gi (zero peptide concentration in Fig. 1 A), and the C1 domain by itself also did not demonstrate any significant GTPase activity over background levels (data not shown). Moreover, the C2 domain of adenylyl cyclase did not alter the ability of peptide MIII-4 to modulate the steady-state GTPase activity of Gi (Fig. 1 A). Similar to the C1 region, the C2 domain by itself also did not demonstrate any GTPase activity (data not shown). Therefore, these data show that the C1 domain of ACV, but not the C2 region of the enzyme, can augment the ability of the muscarinic receptor mimetic peptide MIII-4 to increase GTP-GDP exchange on Gαi. It is now well established that interactions between the C1 and C2 domains of AC are required to reconstitute enzyme activity, which can be regulated by its modulators (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, 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, 9Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2916-2920Google Scholar). Therefore, to determine whether the C1 domain in the context of the adenylyl cyclase molecule can modulate the ability of MIII-4 to increase 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, 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, 9Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2916-2920Google Scholar), engineered, soluble form of ACV (C1-C2 ACV) was used. The data in Fig.1 B demonstrate that like the C1 domain, in the absence of MIII-4, C1-C2 ACV by itself did not alter the steady-state GTPase activity of Gi. C1-C2 ACV by itself also did not demonstrate any significant GTPase activity over background levels (data not shown). However, similar to the data with the C1 domain, C1-C2 ACV markedly augmented the ability of peptide MIII-4 to stimulate GTPase activity (Fig. 1 B). Thus, the GTP-GDP exchange-enhancing actions of the C1 domain of ACV are also observed when the C2 domain is present to interact with the C1 region. Because the ability of threshold concentrations (0.1 μm) of MIII-4 to activate Gi could be markedly enhanced by the C1 domain and C1-C2 ACV (Fig. 1, A and B), in subsequent experiments, we employed MIII-4 at a concentration (0.1 μm) that by itself does not markedly stimulate the steady-state GTPase activity. Using this concentration of the peptide, we determined that the effects of C1-C2 ACV and its C1 domain to increase the GTPase activity of Gi was concentration-dependent and saturable (Fig. 1 C). On the other hand, in control experiments, the C2 domain of ACV at concentrations up to 2.0 μm did not alter the ability of MIII-4 to activate Gi (Fig. 1 C). Activation of heterotrimeric G proteins also results in increased GTP binding to the Gα 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, 16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar, 18Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar). Therefore, as a second approach to monitoring Gi activation, we measured the binding of the non-hydrolyzable GTP analog GTPγS to Gi. These experiments were conducted under conditions (low Mg2+and 100 nm GTPγS) that are optimized to observe peptide-induced increases in the ability of G proteins to bind 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, 16Sun H. Seyer J.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2229-2233Crossref PubMed Scopus (60) Google Scholar, 18Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 19Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar). As demonstrated by the data in Fig.2, neither the threshold concentrations of MIII-4 (0.1 μm) nor the addition of C1-C2 ACV or its subdomains (C1 and C2) in the absence of MIII-4 altered GTPγS binding to Gi. However, the presence of both MIII-4 and C1 or C1-C2 ACV increased GTPγS binding to the G protein (Fig. 2). Notably, the C2 domain of ACV, which did not modulate the ability of MIII-4 to stimulate the steady-state GTPase activity of Gi (Fig.1 A), also failed to increase GTPγS binding to Gi (Fig. 2). These data (Fig. 2) demonstrate that C1-C2 ACV and its C1 domain enhance the ability of MIII-4 to activate Gi and to increase GTPγS binding to the G protein. To determine the functional significance of the ability of the C1 domain of ACV to augment the actions of MIII-4 for Giactivation, the experiments depicted in Fig.3 were performed. Essentially, we monitored the ability of the C1 domain of ACV to modulate adenylyl cyclase activity via MIII-4-mediated activation of endogenous Gi in S49 cyc− membranes. S49 cyc− cells do not express Gαs (22Sternweis P.C. Gilman A.G. J. Biol. Chem. 1979; 254: 3333-3340Abstract Full Text PDF PubMed Google Scholar) and therefore provide a convenient model to study Gi activation without interference from Gs activity. Moreover, S49 cyc− cells express predominantly type VI AC (23Premont R.T. Jacobowitz O. Iyengar R. Endocrinology. 1992; 131: 2774-2784Crossref PubMed Google Scholar), which is inhibited 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 experiments, the cyc− membranes were incubated with or without peptide MIII-4 (0.1 μm) in the presence and absence of the C1 or C2 domain of ACV (each at 1.5 μm). AC activity assays were then performed in the presence of forskolin (33 μm). As demonstrated by the data in Fig. 3 A, neither peptide MIII-4 (0.1 μm) nor the C1 domain of ACV altered AC activity in cyc− membranes. However, when both MIII-4 and C1 were present together, the AC activity was inhibited by 50%. To determine if the inhibition of AC activity observed in the presence of MIII-4 and the C1 domain of ACV was due to activation of endogenous Gi, similar experiments were performed in membranes of S49 cyc− cells treated with pertussis toxin. Pertussis toxin ADP-ribosylates Gαi, and this modification of the G protein precludes its activation by receptors (24Katada T. Bokoch G.M. Smigel M.D. Ui M. Gilman A.G. J. Biol. Chem. 1984; 259: 3586-3595Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.3 A, in membranes from pertussis toxin-treated cells, the inhibition of AC activity in the presence of MIII-4 and the C1 domain was abolished. These data demonstrate that the inhibition of AC activity in the presence of MIII-4 and the C1 domain observed in membranes of S49 cyc− cells not exposed to pertussis toxin is indeed due to activation of endogenous Gi. Furthermore, in experiments with the C2 domain of ACV, AC activity was not affected even when the C2 domain was present along with MIII-4 (Fig.3 B). 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 membranes and to inhibit AC activity. The conclusion noted above was further substantiated by experiments in which AC activity in cyc− cell membranes was stimulated with the constitutively active, GTPase-deficient mutant of Gαs (Q203L, Gαs*) (Fig.4, A and B). Gα2 was bound to the non-hydrolyzable GTP analog GTPγS prior to experimentation (see “Experimental Procedures”). Therefore, changes in AC activity in cyc− membranes cannot be attributed to alterations in the active form of Gαs*. As shown in Fig. 4 A, basal AC activity in cyc−cells was very low and markedly stimulated by Gαs*. Similar to the findings with forskolin (Fig. 3 A), neither C1 nor threshold concentrations of peptide MIII-4 (0.1 μm) by themselves significantly altered AC activity that was stimulated by Gαs* (Fig. 4 A). However, the combination of MIII-4 and C1 inhibited AC activity by 60% (Fig. 4 A). This effect was specific for the C1 domain of ACV since the C2 domain of ACV by itself or in combination with MIII-4 failed to alter AC activity in cyc− cells (Fig. 4 B). That the inhibition observed in Fig. 4 A was due to activation of endogenous Gi was demonstrated by the findings that when GTPγS was omitted from the preincubations of S49 cyc− membranes with or without MIII-4 and/or the C1 domain, the inhibition shown in Fig.4 A was not observed (data not shown). To determine if the C1 region of ACV could augment inhibition of adenylyl cyclase activity mediated by activation of endogenous receptors, experiments were performed with S49 cyc− cell membranes. To complement the experiments with the muscarinic receptor mimetic peptide MIII-4, the ability of the muscarinic receptor agonist carbachol to inhibit the activity of adenylyl cyclase in cyc− membranes was assessed. However, carbachol at concentrations up to 1 mm did not inhibit forskolin-stimulated adenylyl cyclase activity in cyc−cells (data not shown). Therefore, it would appear that cyc− cells do not express muscarinic receptors. Because Katada et al. (24Katada T. Bokoch G.M. Smigel M.D. Ui M. Gilman A.G. J. Biol. Chem. 1984; 259: 3586-3595Abstract Full Text PDF PubMed Google Scholar) have shown that somatostatin via Gi activation inhibits adenylyl cyclase activity in cyc− cell membranes, further experiments were performed with somatostatin. As shown in Fig. 5, in the absence of ACV subdomains, somatostatin inhibited forskolin-stimulated adenylyl cyclase activity in a concentration-dependent manner. Addition of the C2 domain of ACV did not alter the inhibition of adenylyl cyclase by somatostatin (Fig. 5). However, the C1 domain of ACV shifted the somatostatin concentration-response curve to the left so that the inhibition of enzyme activity at lower concentrations of somatostatin was enhanced in the presence of the C1 domain (Fig. 5). In control experiments, the C1 domain alone, in the absence of somatostatin, did not inhibit adenylyl cyclase activity (Figs. 5; also see Figs. 3 and 4). Moreover, in membranes of cyc− cells treated with pertussis toxin, neither somatostatin nor the combination of somatostatin and C1 domain altered adenylyl cyclase activity (data not shown). These data (Fig. 5) demonstrate that the paradigm that we have described employing a reconstituted Gi protein and a muscarinic receptor mimetic peptide (Figs. Figure 1, Figure 2, Figure 3, Figure 4) is applicable to inhibition of adenylyl cyclase by endogenous Gi-coupled receptors. Thus, in the presence of the C1 region of ACV, lower amounts of agonist and therefore lower amounts of active receptors are required to inhibit adenylyl cyclase. Taken together, the data in Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 demonstrate that ACV and its C1 subdomain, 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 M4 muscarinic receptor mimetic peptide (MIII-4) and somatostatin receptors to activate Gi. These findings, along with our previous demonstration that the C2 domain of AC, which interacts with Gαs, enhances the guanine nucleotide exchange factor activity of the β2-adrenergic receptor mimetic peptide (βIII-2) 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), indicate that the novel concept of AC modulating the ability of receptors to increase GTP-GDP exchange on G proteins is applicable to both G proteins that regulate its activity. In this context, it should be noted that Mukhopadhyay and Ross (25Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (151) Google Scholar) have shown that phospholipase Cβ does not alter receptor-mediated GTP-GDP exchange on Gq. Because our experiments in cyc− membranes with MIII-4 and activation of endogenous somatostatin receptors are consistent with the in vitro reconstitution data experiments, it is unlikely that the differences between phospholipase Cβ (25Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (151) Google Scholar) and AC (Ref. 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 and this study) are related to the use of lipid vesicles and/or full-length receptor (25Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (151) Google Scholar) versus the use of receptor mimetic peptides and reconstitution without lipid vesicles (Ref. 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 and this study). More likely, it is possible that different effectors modulate the activity of their respective G proteins in different manners. Additional studies in the future with various effectors of heterotrimeric G proteins should help resolve this possibility. One of the most interesting aspects of our studies is that the C1 domain of ACV, which interacts with Gαi, increases the guanine nucleotide exchange activity of a receptor mimetic peptide that activates Gi, whereas the C2 region of ACV, which interacts with Gαs, increases 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). Therefore, it would appear that the interaction of the ACV domains with their respective Gα subunits in the context of the heterotrimer is important for augmenting the signals transduced from receptors to the G proteins that they are coupled with. We are deeply indebted to the following individuals who supplied several of the reagents: Dr. Alfred G. Gilman for the Gαs cDNA, Dr. Randall Reed for the Gαi1 cDNA, Dr. Wei-Jen Tang for the TP2000 strain ofE. coli; Dr. Jeffrey I. Gordon for providing the plasmid pBB131 encoding S. cerevisiae N-myristoyltransferase, and Dr. Yoshihiro Ishikawa for the canine ACV cDNA." @default.
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