Identification of a Giα Binding Site on Type V Adenylyl Cyclase*
- From the Departments of ‡Pharmacology and§Biochemistry, University of Texas Southwestern Medical Center, and the ¶Howard Hughes Medical Institute, Dallas, Texas 75235
Abstract
The stimulatory G protein α subunit Gsα binds within a cleft in adenylyl cyclase formed by the α1-α2 and α3-β4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory α subunit Giα could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5′-3-O-(thio)triphosphate-Giα1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Giα1. These mutations suggest binding of Giα within the cleft formed by the α2 and α3 helices of C1, analogous to the Gsα binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Giα. The C1b domain of the type V enzyme contributed to affinity for Giα, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein α subunits.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM34497 (to A. G. G.) and DK46371 (to S. R. S.), Welch Foundation Grants I-1271 (to A. G. G.) and I-1229 (to S. R. S.), and the Raymond and Ellen Willie Distinguished Chair of Molecular Neuropharmacology (to A. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed. Tel.: 214-648-2370; Fax: 214-648-8812.
- Abbreviations:
- GTPγS
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guanosine 5′-3-O-(thio)triphosphate
- GDPβS
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guanosine 5′-2-O-(thio)diphosphate.
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- Received May 28, 1998.
- Revision received July 7, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
