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• W1633274114 abstract "The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Gαs, binds within a cleft in the C2 domain of adenylyl cyclase while Gαi binds within the opposite cleft in the C1domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Gαs or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Gαireduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Gαi to inhibit the enzyme. In addition, Gαi can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Gαi but cannot simultaneously bind Gαi and transition state analogs such as 2′d3′-AMP. Gαi also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein α-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme. The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Gαs, binds within a cleft in the C2 domain of adenylyl cyclase while Gαi binds within the opposite cleft in the C1domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Gαs or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Gαireduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Gαi to inhibit the enzyme. In addition, Gαi can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Gαi but cannot simultaneously bind Gαi and transition state analogs such as 2′d3′-AMP. Gαi also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein α-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme. adenylyl cyclase(s) α-subunit of the inhibitory G protein of adenylyl cyclase α-subunit of the G protein that stimulates adenylyl cyclase guanosine 5′-O-(2-thio)triphosphate α,β-methylene adenosine 5′-triphosphate 2′-deoxyadenosine 3′-monophosphate 2′,5′-dideoxyadenosine 3′-triphosphate The classic mammalian adenylyl cyclases (AC)1 consist of two repeats of a unit that includes six transmembrane spans and a cytoplasmic domain. Nine isoforms of adenylyl cyclase have been cloned that share this common topology. However, each of these enzymes display distinct patterns of regulation (reviewed in Refs. 1Smit M.J. Iyengar R. Adv. Second Messenger Phosphoprotein Res. 1998; 32: 1-21Crossref PubMed Google Scholar and 2Taussig R. Zimmermann G. Adv. Second Messenger Phosphoprotein Res. 1998; 32: 81-98Crossref PubMed Scopus (47) Google Scholar). For example, types V and VI adenylyl cyclase can be activated by GTPγS-Gαs(3Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4669) Google Scholar), forskolin (4Seamon K.B. Padgett W. Daly J.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3363-3367Crossref PubMed Scopus (1454) Google Scholar), and protein kinase C (α and ζ, Ref. 5Kawabe J. Iwami G. Ebina T. Ohno S. Katada T. Ueda Y. Homcy C.J. Ishikawa Y. J. Biol. Chem. 1994; 269: 16554-16558Abstract Full Text PDF PubMed Google Scholar) and inhibited by Gαi (6Taussig R. Iñiguez-Lluhi J. Gilman A.G. Science. 1993; 261: 218-221Crossref PubMed Scopus (321) Google Scholar, 7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar), calcium (8Yoshimura M. Cooper D.M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (201) Google Scholar, 9Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar), cAMP-dependent protein kinase (10Kunkel M.W. Friedman J. Shenolikar S. Clark R.B. FASEB J. 1989; 3: 2067-2074Crossref PubMed Scopus (25) Google Scholar, 11Iwami G. Kawabe J. Ebina T. Cannon P.J. Homcy C.J. Ishikawa Y. J. Biol. Chem. 1995; 270: 12481-12484Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and protein kinase C δ (type VI only, Ref. 12Lin T.H. Lai H.L. Kao Y.Y. Sun C.N. Hwang M.J. Chern Y. J. Biol. Chem. 2002; 277: 15721-15728Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Regulation by heterotrimeric G proteins has been the hallmark of adenylyl cyclase activity, but the details of this regulation have remained unclear until recently. In vitro, Gαi can inhibit both Gαs and forskolin-stimulated adenylyl cyclase activities in a non-competitive manner (7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar, 13Hildebrandt J.D. Codina J. Birnbaumer L. J. Biol. Chem. 1984; 259: 13178-13185Abstract Full Text PDF PubMed Google Scholar). But much of our recent knowledge is based upon experiments utilizing the cytoplasmic (or soluble) domains of adenylyl cyclase. The two cytoplasmic domains of adenylyl cyclase (C1 and C2) create a pseudosymmetrical heterodimer that forms the catalytic moiety of the enzyme and is the target for most known intracellular regulators (reviewed in Ref. 14Tesmer J.J. Sprang S.R. Curr. Opin. Struct. Biol. 1998; 8: 713-719Crossref PubMed Scopus (98) Google Scholar). The cytoplasmic domains contain a 200–250 amino acid region that is ∼50% similar to each other and 50–90% similar to corresponding regions of other adenylyl cyclase isoforms. The C1 and C2 domains can be independently expressed as soluble proteins in Escherichia coli and mixed to reconstitute full adenylyl cyclase activity, including activation by Gαs and forskolin and inhibition by P-site inhibitors and Gαi (15Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 16Yan S.Z. Hahn D. Huang Z.H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). These soluble adenylyl cyclase fragments have proven invaluable in determining the stoichiometry of Gαs, forskolin, and ATP binding. They have also proven invaluable in localizing binding sites for Gαs and Gαi and in discerning the catalytic mechanism of P-site inhibition of adenylyl cyclase. Much of this early work utilized C1 and C2 proteins from dissimilar isoforms of adenylyl cyclase (C1 from type I and the C2 domain from type II adenylyl cyclase), although more recent systems contain both domains from a single adenylyl cyclase isoform (19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 20Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar, 21Yan S.Z. Huang Z.H. Andrews R.K. Tang W.J. Mol. Pharmacol. 1998; 53: 182-187Crossref PubMed Scopus (68) Google Scholar, 22Yan S.Z. Tang W.J. Methods Enzymol. 2002; 345: 231-241Crossref PubMed Scopus (8) Google Scholar). Crystal structures of complexes containing the C1 and C2 domains, Gαs, and forskolin reveal that forskolin and ATP analogs bind at the interface of the C1and C2 domains (23Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar, 24Tesmer J.J. Sunahara R.K. Johnson R.A. Gosselin G. Gilman A.G. Sprang S.R. Science. 1999; 285: 756-760Crossref PubMed Scopus (276) Google Scholar, 25Tesmer J.J. Dessauer C.W. Sunahara R.K. Murray L.D. Johnson R.A. Gilman A.G. Sprang S.R. Biochemistry. 2000; 39: 14464-14471Crossref PubMed Scopus (93) Google Scholar), while the major binding site for Gαs is located on the C2 domain in the cleft formed by the α2′ and α3′ helices (17Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 23Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar, 26Yan S.-Z. Huang Z.-H. Rao V.D. Hurley J.H. Tang W.-J. J. Biol. Chem. 1998; 272: 18849-18854Abstract Full Text Full Text PDF Scopus (51) Google Scholar). A similar groove is formed by the corresponding structural elements of C1 (23Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar). Mutagenesis and binding studies indicate that Gαi binds to this corresponding site in the C1 domain (19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar); however, the mechanism for Gαi-mediated inhibition is still unclear. Activation of adenylyl cyclase by Gαs or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another (15Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 16Yan S.Z. Hahn D. Huang Z.H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). We show herein that Gαi works in opposition to Gαs to reduce domain interaction and thus the formation of the adenylyl cyclase catalytic site. Membranes were prepared from Sf9 cells expressing type V adenylyl cyclase. Cells (2 × 106/ml) were infected with baculovirus (1 plaque-forming unit/cell), harvested after 48 h, and lysed by nitrogen cavitation. After removal of nuclei, membranes were pelleted, washed, and resuspended in 20 mm HEPES (pH 8.0), 2 mmdithiothreitol, and 200 mm sucrose. All G protein α-subunits were synthesized in E. coli as described by Lee et al.(27Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (245) Google Scholar). Gαi was co-expressed with yeast proteinN-myristoyltransferase (27Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (245) Google Scholar, 28Linder M.E. Pang I.-H. Duronio R.J. Gordon J.I. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1991; 266: 4654-4659Abstract Full Text PDF PubMed Google Scholar) for synthesis of myristoylated protein. Purified α-subunits were activated by incubation with 50 mm NaHEPES (pH 8.0), 5 mmMgSO4, 1 mm EDTA, 2 mmdithiothreitol, and 400 μm [35S]GTPγS at 30 °C for 30 min for Gαs or 2 h for Gαi. Free GTPγS was removed by gel filtration. All G proteins were activated with GTPγS unless stated otherwise. The C2 domains of type II and type V adenylyl cyclase (IIC2, VC2) and the C1 and C1a domains of type V adenylyl cyclase were expressed inE. coli and purified by metal affinity chromatography followed by ion exchange as described previously (15Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (116) Google Scholar, 17Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Adenylyl cyclase activity was measured as described (29Dessauer C.W. Methods Enzymol. 2002; 345: 112-126Crossref PubMed Scopus (24) Google Scholar). All assays were performed for 7–10 min at 30 °C in a final volume of 50–100 μl with 5 mmMgCl2 and 100 μm ATP for the membrane-bound enzyme. In assays containing C1 and C2 domain proteins (reconstitution assays), limiting concentrations of the C1 domain protein were first incubated with Gαi for 15 min on ice followed by addition of Gαs and VC2 prior to initiation of the assay. The final concentration of C2 was at least 0.5 μm to promote interaction between the C1 and C2 proteins except where indicated otherwise. The final concentration of ATP was 1 mm for reconstitution assays, unless stated otherwise. All determinations were performed in duplicate and are representative of at least two experiments. Proteins (100-μl sample volumes) were applied to a Superdex 200 column (HR10/30; Amersham Biosciences). Samples were eluted with 20 mm NaHEPES (pH 8.0), 5 mm MgCl2, 1 mm EDTA, 2 mm dithiothreitol, 15 μm forskolin, and 100 mm NaCl. The first 7 ml were collected in a single fraction; 200-μl fractions were collected thereafter. Samples were run on SDS-PAGE and detected by Coomassie Blue staining or Western blotting. Binding of Ap(CH2)pp by equilibrium dialysis was performed as described in Ref. 18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar. Filter binding was performed using HA, 0.45-μm cellulose ester plates on a 96-well vacuum manifold unit (Millipore). Reactions contained 20 mm HEPES (pH 8.0), 2 mm MnCl2, 5 mmβ-mercaptoethanol, 2–20 μm[3H]Ap(CH2)pp, and VC1a (10 μm), IIC2 (10 μm), and GTPγS-Gαs (15 μm) in the presence or absence of 15 μm GTPγS-Gαi in a final volume of 25 μl. G protein subunits were activated in the absence of radiolabeled GTPγS. The reactions were incubated for 10 min on ice, then rapidly filtered on prerinsed HA membranes and washed twice with 200 μl of ice-cold wash buffer (20 mmHEPES (pH 8.0), 2 mm MnCl2, 1 mmMgCl2, and 50 mm NaCl). Filter membranes were dried in a vacuum desiccator, and the bound ligand was quantified by liquid scintillation counting. We have previously shown that Gαi binds within the cleft formed by the α2 and α3 helices of the C1a domain of type V adenylyl cyclase. Using [35S]GTPγS-Gαi a clear shift in molecular weight of Gαi is observed upon addition of the C1a domain as determined by gel filtration. However, the tight binding of the C1a domain and Gαi is partially disrupted by formation of a C1-C2complex in the presence of forskolin (Fig.1 A). Furthermore, the complex of C1a and Gαi is completely abolished in the presence of both Gαs and forskolin. Gαsdoes not compete for binding of Gαi to the C1domain; however, full activation of adenylyl cyclase by Gαs and forskolin does limit the extent of inhibition by Gαi (7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar, 19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Therefore we examined the formation of a potential Gαi-C1-C2-Gαscomplex in the absence of forskolin (Fig. 1 B) using the full-length C1 domain, which has a 10-fold higher affinity for Gαi (19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Even in the presence of high concentrations of C1, C2, Gαs, and Gαi, complexes between C1-Gαiand C2-Gαs are formed, but no heterotetramer is ever observed. Note that myristoylated Gαi and complexes containing the myristoylated protein tend to be slightly retarded on gel filtration columns most likely because of the hydrophobic nature of the myristate group. Both forskolin and Gαs have been shown to increase the affinity of C1 and C2 by 100-fold more than basal, and the combined action of the activators increases the apparent affinity of C1 and C2 by more than 1000-fold. Our gel filtration results suggest that Gαi may work in opposition to these regulators to decrease the affinity between C1 and C2. We therefore examined the ability of Gαi to inhibit adenylyl cyclase with increasing concentrations of C2 protein to drive the interaction between the two domains (Fig.2 A). At low concentrations of C2, Gαi greatly inhibits adenylyl cyclase activity. However, as the concentration of C2 increases, the interaction between C1 and C2 increases, reducing the ability of Gαi to inhibit the enzyme. At maximal concentrations of C2 protein, no inhibition by Gαi is observed. Therefore, the interaction between the C1 and C2 domains not only decreases the binding of Gαi to C1 but also decreases the ability of Gαi to inhibit the enzyme. A similar phenomenon is observed with a mutant of C1 (E418A), which has a 6-fold higher affinity for Gαi (19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Once again, maximal inhibition occurs at low C2 concentrations, and the effect of Gαi decreases with increasing C2(Fig. 2 B). In this case, however, the effect of Gαi is not eliminated at maximal concentrations of C2. This mutation is within the Gαi binding cleft and has no effect on the apparent affinity of the C1and C2 domains (Fig. 2 C) and does not increase dimerization of C1 as measured by gel filtration (data not shown). The inability to eliminate the effect of Gαi at high C2 concentrations is most likely caused by the increased efficacy of Gαi-mediated inhibition of both the membrane-bound and cytoplasmic domains of adenylyl cyclase when this mutation is present (19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). These data suggest that the mutation of Glu-418 to alanine induces a conformational change that facilitates Gαi inhibition independent of changes at the C1-C2 interface. We have also examined the interaction between the C1 and C2 domains utilizing a mutant within the C2domain (K1014N in type II AC) with an increased affinity between the C1a and C2 domains in the presence and absence of Gαs (30Hatley M.E. Benton B.K. Xu J. Manfredi J.P. Gilman A.G. Sunahara R.K. J. Biol. Chem. 2000; 275: 38626-38632Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). When paired with the full-length C1 domain from type V, the C2-K1014N mutant shows a similar 10-fold shift in affinity for C1 (Fig.3 A). This point mutant is located 16–28 Å from Gαi-interacting residues and on the opposite domain from which Gαi binds. Thus it is not predicted to directly interfere with Gαi binding. However, the inhibition of C1 and the mutant C2domain by Gαi is remarkably diminished as compared with wild type (Fig. 3 B). Therefore, once again as the affinity of C1 and C2 is increased, the ability of Gαi to inhibit adenylyl cyclase is decreased. The Gαi binding cleft is located in close proximity to the catalytic site, particularly to residues contacting the magnesium ions at the active site and the phosphate moieties of substrate or P-site molecules. Therefore, Gαi may affect binding of molecules to the active site. To address this possibility, kinetic analysis of inhibition by Gαi and P-site inhibitors or substrate analogs was performed. P-site inhibitors bind at the active site and are postulated to mimic a product-like transition state. Classic P-site inhibitors (such as 2′d3′-AMP) require the product pyrophosphate for binding to the enzyme and show uncompetitive inhibition with respect to MgATP upon activation with Gαs(18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The P-site inhibitor 2′d3′-AMP and Gαi behave as mutually exclusive inhibitors of adenylyl cyclase giving rise to a family of parallel lines on a Dixon plot obtained at different Gαi concentrations (Fig.4 A). This kinetic pattern is true for both the recombinant membrane-bound enzyme and the cytoplasmic domains of type V adenylyl cyclase (Fig. 4 B). Therefore any complex of adenylyl cyclase containing Gαi has significantly reduced ability to obtain a conformation capable of binding this type of transition state analog. This same pattern of inhibition is also observed for the membrane-bound adenylyl cyclase with the more potent P-site inhibitor, 2′5′dd3′-ATP (data not shown) (31Desaubry L. Shoshani I. Johnson R.A. J. Biol. Chem. 1996; 271: 2380-2382Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This is an important feature of catalysis because product release is one of the rate-limiting steps along the reaction coordinate for adenylyl cyclase (18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Ap(CH2)pp is a non-hydrolyzable analog of ATP that competitively competes for ATP binding (18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). As shown in the Dixon plot for the membrane-bound adenylyl cyclase (Fig.5 A), Ap(CH2)pp also shows a nearly parallel family of curves obtained at different Gαi concentrations with respect to Ap(CH2)pp, indicating that each of these two inhibitors binds with greatly reduced affinity in the presence of the other. The intersection of these lines well below the x axis provides a reduction in the K i for Ap(CH2)pp of at least 6-fold in the presence of Gαi. However, the cytoplasmic domains of type V show an intersection of lines on thex axis, suggesting no significant reduction in the affinity of the enzyme for Ap(CH2)pp in the presence of Gαi (Fig. 5 B). This non-competitive interaction is consistent with the formation of a Gαi-C1-C2-ATP complex. To further understand the binding of Ap(CH2)pp to the cytoplasmic domains of adenylyl cyclase, we measured the direct binding of Ap(CH2)pp to C1, C2, and Gαs in the presence or absence of Gαi. These measurements were made using Mn2+, which greatly increases the affinity of adenylyl cyclase for Ap(CH2)pp as compared to Mg2+ (18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). An identical pattern of non-competitive inhibition was obtained for the soluble type V domains with Ap(CH2)pp and Gαi in the presence of Mn2+ as previously shown for Mg2+ (Fig.6). Gαs is required to form a complex at reasonable concentrations of C1 and C2, but Gαs will reduce the effectiveness of Gαi in this assay by reducing the binding of Gαi to the C1 domain. Therefore a complete loss of binding is not anticipated, but clearly a 50% reduction in Ap(CH2)pp is observed by both filter binding assays (Fig.6 B) and equilibrium dialysis (data not shown). Scatchard plot analysis of the filter binding data would suggest non-competitive binding of Ap(CH2)pp, although we were unable to fully saturate binding. One unusual aspect of Gαi-mediated inhibition of adenylyl cyclase is the inability of Gαi to inhibit the membrane-bound adenylyl cyclase in the presence of manganese. This has been reported previously by Hildebrandt and Birnbaumer (32Hildebrandt J.D. Birnbaumer L. J. Biol. Chem. 1983; 258: 13141-13147Abstract Full Text PDF PubMed Google Scholar), but it was unclear at that time if this was caused by an effect of Mn2+ on the G protein or catalytic subunit. We show that Gαi is unable to inhibit type V adenylyl cyclase in the presence of Mn2+ alone, Mn2+ and Gαs, or Mn2+ and forskolin (Fig.7 A). In fact, Gαi actually increases forskolin-stimulated activity in the presence of Mn2+. This may be due to the weak ability of Gαi to compete at the Gαs binding site (7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar). The inability of Gαi to inhibit adenylyl cyclase in the presence of Mn2+ is not caused by an inactivation of Gαi, since Gαi can inhibit the Mn2+-Gαs-stimulated cytoplasmic domains of adenylyl cyclase activity under identical conditions (Fig.6 A). In addition to 3 mm Mn2+, these assays all contain 0.5 mm Mg2+, which should be sufficient to maintain activation of the G proteins. The inclusion of an additional 1 mm Mg2+ has no effect on the membrane-bound enzyme in the presence of Mn2+ (Fig.7 B). The mechanism for this effect of metals on Gαi-mediated inhibition and the difference between the soluble and membrane-bound adenylyl cyclase is discussed below. Preliminary kinetic analysis suggests that Gαs and Gαi bind simultaneously to adenylyl cyclase; however, a complex of C1-C2-Gαs-Gαi has not been observed (7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar, 19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This suggests a model in which Gαi inhibits the enzyme by decreasing the affinity of one domain for another. This is in stark contrast to Gαs and forskolin stimulation where the affinity between C1 and C2 increases with increased activation. We show that binding of Gαi to the C1 domain is weakened or lost in the presence of a C1-C2-forskolin or a C1-C2-Gαs-forskolin complex (Fig. 1). However, Gαi inhibits the fully stimulated Gαs-forskolin-activated enzyme very poorly, and we would not expect to observe a complex with both Gαs and Gαi under these conditions. Therefore, we also examined whether a heterotetrameric complex could be formed with Gαi, Gαs, and adenylyl cyclase in the absence of forskolin. Even under conditions of high protein concentrations, a complex of C1-C2-Gαs-Gαi is never observed. A direct test of our hypothesis is the measurement of C1/C2 affinity in the presence of Gαi. It is clear that increased C1-C2 complex formation, driven by increasing C2 concentrations, decreases the ability of Gαi to inhibit adenylyl cyclase activity (Fig. 2). This is consistent with the limited ability of Gαi to inhibit the most stimulated forms of adenylyl cyclase that display the highest C1/C2 affinity (7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar, 19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In fact, a mutant C2 protein (K1014N, Ref. 30Hatley M.E. Benton B.K. Xu J. Manfredi J.P. Gilman A.G. Sunahara R.K. J. Biol. Chem. 2000; 275: 38626-38632Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) with increased affinity for C1 displays dramatically decreased inhibition by Gαi (Fig. 3). Although the membrane spans of adenylyl cyclase physically link C1 and C2 in close proximity, the structural changes that we measure through a change in affinity are still occurring in the native enzyme. Supporting a structural change at the interface of C1/C2 is the fact that residues on one face of helix α2 interact with Gαi, whereas residues on the opposite face of α2 interact with the C2 domain (19Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar). A conformational change at the C1/C2 interface and the close proximity of the Gαi binding site to the catalytic site might also suggest that Gαi influences the binding of molecules to the active site of adenylyl cyclase. Kinetic analysis of inhibition by P-site inhibitors and Gαireveals that the actions of these inhibitors are mutually exclusive. Therefore, binding of Gαi prevents formation of specific conformations at the active site, particularly those capable of binding these transition state analogs. This is true for both the soluble and membrane-bound enzymes, and this pattern of inhibition is observed with both uncompetitive (2′d3′-AMP) and non-competitive (2′5′dd3′-ATP) types of P-site inhibitors. It is clear that the key to regulation of adenylyl cyclase is the conformational state of the C1/C2 domain interface. The change in affinity is a measurement of the changes in conformation at the active site located at the domain interface. This type of movement is mimicked somewhat with P-site inhibitors. Even with bound Gαs and forskolin, the unliganded active site maintains an open conformation. But upon addition of P-site inhibitors that mimic a product-like transition state, the active site clamps down on the inhibitor as one might observe in an induced fit model of regulation (33Dessauer C.W. Tesmer J.J. Sprang S.R. Gilman A.G. Trends Pharmacol. Sci. 1999; 20: 205-210Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In the absence of activator, it is speculated that an even more open active site would be observed, similar to the structure obtained for the C2 homodimer (34Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (321) Google Scholar). This open to closed transition involves the inward collapse of structural elements around the active site. The Gαi binding site is directly adjacent to these regions and may affect binding of molecules to the catalytic site. In fact, the most flexible regions of the C1 domain are located on the back side of the Gαi binding site. We put forth evidence that Gαi mediates its effects by reducing the ability of the enzyme to obtain a closed conformation. This is observed as a reduced affinity between the C1 and C2 domain and a reduced ability to form a transition state conformation necessary for binding P-site inhibitors relative to the ground state. The kinetic analysis of inhibition by the substrate analog Ap(CH2)pp reveals a different pattern. Kinetic and binding data of the soluble domains and the membrane-bound enzyme show non-competitive aspects between Gαi and the substrate analog Ap(CH2)pp, as displayed by intersecting lines on a Dixon plot. However, the soluble domains have no reduction in theK i for Ap(CH2)pp in the presence of Gαi, while the membrane-bound enzyme displays a reduced affinity for Ap(CH2)pp and Gαi in the presence of each other. Despite this difference, clearly both Gαi and a substrate analog can bind simultaneously to adenylyl cyclase. However, it is not clear whether a Gαi-ATP-bound enzyme can lead to cAMP production. Gαi binding may allow limited binding of substrate to an inactive enzyme. Alternatively, Gαi binding may produce a catalytically competent enzyme but with greatly reduced activity. The latter rationale might explain why Gαi inhibition of either the soluble domains or membrane-bound type V AC never leads to zero or basal activity levels. Generally, the maximal inhibition by Gαi is 50–70% when stimulated with modest levels of Gαs (7Taussig R. Tang W.-J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar), suggesting that an AC-Gαi complex might retain low activity and hence bind substrate. Additional experiments are required to differentiate between these two possibilities. The effects observed with manganese may also point to an inability of Gαi to interact with a closed conformation. Residues from both C1 and C2 domains are required for binding ATP and ATP analogs (23Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar, 35Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (109) Google Scholar, 36Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In fact, P-site analogs bound at the active site can stabilize complex formation between C1 and C2 (30Hatley M.E. Benton B.K. Xu J. Manfredi J.P. Gilman A.G. Sunahara R.K. J. Biol. Chem. 2000; 275: 38626-38632Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Manganese acts to increase the affinity of adenylyl cyclase for ATP analogs. This is observed as a 22-fold decrease in the K d for Ap(CH2)pp and a 2.5-fold decrease in the K m for ATP with the Gαs-stimulated cytoplasmic domains of type V adenylyl cyclase in the presence of Mn2+ versusMg2+ (36Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The difference between Mn2+ and Mg2+ is even more dramatic for the basal or forskolin-stimulated soluble enzyme (36Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This phenomenon is also present in the membrane-bound enzyme yielding a 4–7-fold reduction in the K m for ATP in the presence of Mn2+(37Garbers D.L. Johnson R.A. J. Biol. Chem. 1975; 250: 8449-8456Abstract Full Text PDF PubMed Google Scholar) and a 5-fold reduction in the K i for competitive ATP analogs (38Westcott K.R. Olwin B.B. Storm D.R. J. Biol. Chem. 1980; 225: 8767-8771Abstract Full Text PDF Google Scholar). We measure a 30-fold reduction in the IC50 for Ap(CH2)pp in the presence of 100 μm ATP and manganese versus magnesium (data not shown). A similar reduction in K i is observed in the presence of manganese for all P-site analogs; however, the mechanism of this reduction may be due to the increase in activity of the enzyme rather than the increase in P-site affinity (18Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 39Londos C. Preston M.S. J. Biol. Chem. 1977; 252: 5951-5956Abstract Full Text PDF PubMed Google Scholar). By increasing the affinity of metal-ATP for the enzyme, manganese may be serving to increase the interaction between C1 and C2, driving the enzyme to a more closed conformation. The question is why is this observed only for the membrane-bound enzyme. The soluble enzyme may be more susceptible to Gαiinhibition because of its very nature of two separate proteins. The membrane-bound enzyme holds the C1/C2 domains in the optimal orientation for catalysis. AlthoughV max is comparable to the soluble domains, theK m for metal-ATP and the affinity for ATP analogs are considerably lower for the membrane-bound enzyme (∼13-fold difference in the K m for Mg-ATP, data not shown). This may be part of the reason that Gαi reduces the affinity of Ap(CH2)pp for the membrane-bound enzyme and not for the soluble domain. The increased affinity for metal-ATP, particularly Mn-ATP, may reflect an inability of Gαi to inhibit this enzyme as compared with the C1/C2domains that have substantially weaker affinity for substrate. For the C1/C2 domains an increase in affinity for Mn-ATP is not sufficient to significantly reduce the inhibition by Gαi. As with all model systems, the cytoplasmic domains have been an invaluable tool but may have their limits in faithfully reproducing the kinetic features of adenylyl cyclase. In summary, these data suggest a model where Gαidecreases the interaction of C1 and C2 for each other and also decreases catalytic activity by decreasing the formation of the active site. This is directly opposite to the actions of Gαs and highlight the pseudosymmetry of the adenylyl cyclase structure that is poised for bidirectional regulation. We thank Mark Hately for providing the IIC2K1114N mutant clone, Travis Vaught for purification of IIC2K1114N, Roger Johnson for providing the P-site inhibitor 2′5′dd3′-ATP, Kathy Graves for technical assistance, and Dr. Roger Barber for critical review of this article." @default.
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• W1633274114 title "Mechanism of Gαi-mediated Inhibition of Type V Adenylyl Cyclase" @default.
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