receptor signaling through Gs

Hormonal signals activate trimeric G pro- teins by substituting GTP for GDP bound to the G protein a subunit (Ga), thereby generating two potential signaling molecules, Ga-GTP and free Gp3y. The usefulness of domi- nant 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 a subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of ad- enylyl cyclase. To construct this mutant, we introduced into the a subunit (as) of Gs three separate mutations chosen because they impair as function in complementary ways: the A366S mutant reduces affinity of as 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 hor-