Selective uncoupling of RGS action by a single point mutation in the G protein α-subunit

Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effecters that propagate a signal, G protein signaling is governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. RGS proteins (regulators of G protein signaling) were identified as potent negative regulators of G protein signaling pathways in simple eukaryotes and are now known to act as GTPase-activating proteins (GAPs) for G; protein alpha-subunits in vitro, It is not known, however, if G alpha GAP activity is responsible far the regulatory action of RGS proteins in vivo. We describe here a G alpha mutant in yeast (gpa1(sst)) that phenotypically mimics the loss of its cognate RGS protein (SST2), The gpa1(sst) mutant is resistant to an activated allele of SST2 in vivo and is unresponsive to RGS GAP activity in vivo. The analogous mutation in a mammalian G(q) alpha is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through G alpha and that RGS-GAP activity is responsible for their desensitizing activity in cells. The G alpha(sst) mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.