Second Site Suppressor Mutations of a GTPase-deficient G-Protein α-Subunit

Donald M. Apanovitch, Taroh Iiri, Takatoshi Karasawa, Henry R. Bourne, Henrik G. Dohlman
1998 Journal of Biological Chemistry  
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 ␣ Q227L , G i2 ␣ 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␣ in yeast, Gpa1 Q323L . Sequencing revealed
more » ... cond-site mutations in three conserved residues, K54E, R327S, and L353⌬ (codon deletion). Each mutation alone results in a complete loss of the ␤␥-mediated mating response in yeast, indicating a dominant-negative mode of inhibition. Likewise, the corresponding mutations in a mammalian G i2 ␣ (K46E, R209S, L235⌬) lead to inhibition of G␤␥-mediated mitogen-activated protein (MAP) kinase phosphorylation in cultured cells. The most potent of these ␤␥ inhibitors (R209S) has no effect on G i2 ␣mediated regulation of adenylyl cyclase. Despite its impaired ability to release ␤␥, purified recombinant Gpa1 R327S is fully competent to bind and hydrolyze GTP. These mutants will be useful for uncoupling G␤␥-and G␣-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.
doi:10.1074/jbc.273.44.28597 pmid:9786851 fatcat:plarllj3tzeslollolzcafyi4a