Selective Uncoupling of RGS Action by a Single Point Mutation in the G Protein α-Subunit

Abstract

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 ␣-subunits in vitro. It is not known, however, if G␣ GAP activity is responsible for the regulatory action of RGS proteins in vivo. We describe here a G␣ 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 G q ␣ is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through G␣ and that RGS-GAP activity is responsible for their desensitizing activity in cells. The G␣ sst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.A wide variety of cellular signals (hormones, neurotransmitters, light, odors) act through a three component system composed of cell surface receptors, heterotrimeric G proteins, and effector proteins (1). The mating pheromones in yeast Saccharomyces cerevisiae act through receptors (STE2, STE3 gene products), a G protein ␣␤␥ heterotrimer (GPA1, STE4, STE18), and a mitogen-activated protein kinase signaling cascade that promotes cell division arrest and fusion (2). If mating is unsuccessful, however, the cells become refractory to pheromone stimulation and will eventually resume normal growth. RGS1 proteins have recently been identified as a fourth component of the G protein signaling pathway (2, 3). The founding member of the RGS family, called SST2, was identified in a genetic screen for negative regulators of the pheromone response pathway in yeast (4). Loss of function sst2 mutants render cells supersensitive to a pheromone stimulus and unable to recover from pheromone-induced growth arrest. Dominant gain-of-function alleles of SST2 have the opposite effect, rendering cells insensitive to pheromone stimulation (5). Further genetic and biochemical experiments revealed that Sst2 interacts directly with the G protein ␣-subunit, Gpa1 (6).Behavioral genetic analyses in C. elegans uncovered a homologue of Sst2, called EGL-10 (7). egl-10 was shown to negatively regulate goa-1, which encodes the G␣ that mediates serotonindependent egg laying behavior. Two mammalian homologues, GAIP and RGS10, were identified by their interaction with G␣-subunits in a two-hybrid screen (8, 9). An additional 15 mammalian members of the family were found by expression cloning, degenerate polymerase chain reaction, low stringency hybridization, and as expressed sequence tags (7-11). All of the RGS proteins share a conserved "...

Regulation of Stress Response Signaling by the N-terminal Dishevelled/EGL-10/Pleckstrin Domain of Sst2, a Regulator of G Protein Signaling in Saccharomyces cerevisiae

“…P20L grew better than those with full-length Sst2, as previously reported (51). Cells expressing C-Sst2 grew poorly in the presence or absence of high salt, and even more poorly in the presence of salt plus pheromone (Fig.…”

G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction

“…The binding defect is because of a Gly 3 Ser substitution in switch I of the G␣ subunit. Gly 3 Ser substitutions in mammalian G␣ subunits G␣ i , G␣ o , and G␣ q also result in resistance to RGS proteins of the R4 and R7 subfamilies (63,64).…”

“…To test this hypothesis, we evaluated the ability of the RGS-resistant mutant, G␣ q -G188S, to interact with GRK2. An RGS-resistant G␣ mutant was first described for the G␣ subunit of the Saccharomyces cerevisiae trimeric G protein, Gpa1p (63). gpa1 sst mutants are supersensitive to the mating pheromone, ␣-factor, because of a failure to bind the RGS protein, Sst2p.…”

Regulators of G Protein Signaling and Transient Activation of Signaling

“…We have shown previously that Sst2 interacts genetically (10,11) and physically (12) with Gpa1 and can accelerate its GTPase activity more than 40-fold (13). Mutants lacking SST2 are ϳ100-fold more sensitive to pheromone (12,14,15).…”