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 Ga 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 A³⁶⁶S mutant reduces affinity of α_s 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%) 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-R²⁰¹C) 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.