Second site suppressor mutations of a GTPase-deficient G-protein α-subunit -: Selective inhibition of Gβγ-mediated signaling

G proteins transmit signals from cell surface receptors to intracellular effectors, The intensity of the signal is governed by the rates of GTP binding (leading to subunit dissociation) and hydrolysis. Mutants that cannot hydrolyze GTP (e.g. G(s)alpha(Q227L), G(i2)alpha(Q205L)) are constitutively activated and can lead to cell transformation and cancer. Here we have used a genetic screen to identify intragenic suppressors of a GTPase-deficient form of the G alpha in yeast, Gpal(Q323L). Sequencing revealed second-site mutations in three conserved residues, K54E, R327S, and L353 Delta (codon deletion). Each mutation alone results in a complete loss of the beta gamma-mediated mating response in yeast, indicating a dominant-negative mode of inhibition. Likewise, the corresponding mutations in a mammalian G(i2)alpha (K46E, R209S, L235 Delta) lead to inhibition of G beta gamma-mediated mitogen-activated protein (MAP) kinase phosphorylation in cultured cells. The most potent of these beta gamma inhibitors (R209S) has no effect on G(i2)alpha-mediated regulation of adenylyl cyclase, Despite its impaired ability to release py, purified recombinant Gpal(R327S) is, fully competent to bind and hydrolyze GTP. These mutants will be useful for uncoupling G beta gamma- and G alpha-mediated signaling events in whole cells and animals. In addition, they serve as a model for drugs that could directly inhibit G protein activity and cell transformation.