Selective uncoupling of RGS action by a single point mutation in the G protein alpha-subunit.
Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effectors 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 Galpha GAP activity is responsible for the regulatory action of RGS proteins in vivo. We describe here a Galpha 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 vitro. The analogous mutation in a mammalian Gqalpha is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through Galpha and that RGS-GAP activity is responsible for their desensitizing activity in cells. The Galphasst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.
DiBello
P R
PR
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Garrison
T R
TR
Apanovitch
D M
DM
Hoffman
G
G
Shuey
D J
DJ
Mason
K
K
Cockett
M I
MI
Dohlman
H G
HG
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D005656:Fungal Proteins / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000032:analysis; D019204:GTP-Binding Proteins / Q000235:genetics*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D010675:Pheromones / Q000032:analysis; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012701:Serotonin / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection / Q000235:genetics; D020710:RGS Proteins
D002118:Calcium; D005656:Fungal Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D007295:Inositol Phosphates; D010675:Pheromones; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012701:Serotonin; C094301:SST2 protein, S cerevisiae; D020710:RGS Proteins