A G(sα) mutant designed to inhibit receptor signaling through G(s)

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein α subunit (Gα), thereby generating two potential signaling molecules, Gα–GTP and free Gβγ. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Gα mutation. Here we describe a mutant α subunit designed to inhibit receptor-mediated hormonal activation of G(s), the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the α subunit (α(s)) of G(s) three separate mutations chosen because they impair α(s) function in complementary ways: the A(366)S mutant reduces affinity of α(s) for binding GDP, whereas the G(226)A and E(268)A mutations impair the protein’s ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) G(s)-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 α(s) mutant (α(s)–R(201)C) or to agonists for receptors that activate G(q) or G(i). This α(s) triple mutant and cognate Gα mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.