Binding of GTP induces α subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the α subunit (αs) of Gs in genetically αs-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type αs to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-αs) quantitatively from cell extracts. We activated the epitope-tagged αs in intact cells by: (a) exposure of cells to cholera toxin, which activates αs by covalent modification; (b) mutational replacement of arginine201 in HA-αs by a cysteine residue, to create HA-αs-R201C; like the cholera toxin-catalyzed modification, this mutation activates αs by slowing its intrinsic GTPase activity; and (c) treatment of cells with the β-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to αs, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of αs (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-αs- R201C was found in the soluble fraction, as compared to 10% of wild type HA-αs. Isoproterenol rapidly (in <2 min) caused 20% of HA-αs to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-αs-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of αs in response to activation. Taken together, these findings show that activation of αs induces a conformational change that loosens its attachment to membranes and increases its degradation rate.
ASJC Scopus subject areas
- Cell Biology