title: A Gsalpha mutant designed to inhibit receptor signaling through Gs.
abstract: Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein alpha subunit (Galpha), thereby generating two potential signaling molecules, Galpha-GTP and free Gbetagamma. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Galpha mutation. Here we describe a mutant alpha subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the alpha subunit (alphas) of Gs three separate mutations chosen because they impair alphas function in complementary ways: the A366S mutant reduces affinity of alphas for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active alphas mutant (alphas-R201C) or to agonists for receptors that activate Gq or Gi. This alphas triple mutant and cognate Galpha mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.
authors.affiliation: Departments of Cellular and Molecular Pharmacology and Medicine and Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
authors.forename: T
authors.initials: T
authors.lastname: Iiri
authors.forename: S M
authors.initials: SM
authors.lastname: Bell
authors.forename: T J
authors.initials: TJ
authors.lastname: Baranski
authors.forename: T
authors.initials: T
authors.lastname: Fujita
authors.forename: H R
authors.initials: HR
authors.lastname: Bourne
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors; D011974:Receptors, LH / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection / Q000235:genetics
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins