Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize G i -family ␣ subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast G␣ subunit Gpa1 Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins initiates signals elicited by numerous hormone, neurotransmitter, and sensory stimuli (1). Receptors activate G proteins by stimulating the release of GDP from the ␣ subunit, allowing GTP to bind and to induce dissociation of the G protein ␣ and ␤␥ subunits, which interact with effector proteins to modulate cellular responses (2-5).The duration and strength of receptor-generated physiological responses are regulated by the rate at which GTP is hydrolyzed by ␣ subunit (6, 7). It has been known for some time that the physiological turn-off of some G protein-mediated signals is faster than would be predicted from the in vitro GTPase activity of isolated G protein subunits (8, 9). The solution to this paradox appears to reside in the newly recognized family of regulator of G protein signaling (RGS) 1 proteins, first identified genetically in the yeast Saccharomyces cerevisiae and in the nematode, Caenorhabditis elegans (10 -13). At least 19 RGS protein cDNAs have been identified in mammalian tissues, all sharing a homologous carboxyl-terminal region of ϳ120 amino acid residues termed the RGS domain (13-15). Biochemical studies with ␣ i and ␣ q family of G proteins demonstrated that RGS4 and G␣-interacting protein (GAIP) act as GTPase accelerating proteins (GAPs) (16, 17), which could account for inhibition of G protein-mediated responses (15). GAP activity of Sst2 for Gpa1 has also been recently demonstrated (18). The mechanism by which GTPase activity is enhanced by RGS appears to be the stabilization of the transition state conformation of G␣ for nucleotide hydrolysis (19,20). RGS4 also directly inhibits the interaction of the GTP␥S-bound ␣ q subunit with phospholipase C␤, presumably by binding to the effector region of activated ␣ q (16).A mutant yeast G␣ subunit, Gpa1 sst , was recently identified in a screen for novel strains showing the "supersensitive to pheromone" (sst) phenotype. It has a single glycine to serine mutation and escapes from negative regulation by the RGS protein, Sst2 (21). Since many RGS proteins affect G i -family G proteins, and the crystal structure of the RGS4⅐␣ i1 complex was recently reported, we wanted to see if the corresponding G 3 S mutation in ␣ i1 and ␣ o would produce insensitivity to RGS. A major objective was to obtain a detailed biochemical and mechanistic analysis of this newly identified class of mutations.We report that G ␣ i1 and ␣ o subunit G 3 S mutants are insensitive to GTPase activation by two different RGS proteins. Quantitative flow cytometry studies demonstrated that a Ͼ30 -100-fold reduction in affinity of RGS for the ␣ su...

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 "...

Mammalian phosducins are known to bind G protein ␤␥ subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to G␤␥. Binding was enhanced by pheromone stimulation and by the addition of GTP␥S, conditions that favor dissociation of G␤␥ from G␣. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2⌬ mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1⌬ mutant were viable and exhibited a 6 -7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70 -80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling.

The Broad-Complex (BR-C) is essential for metamorphosis in Drosophila melanogaster. This locus is coextensive with the 2B5 ecdysone-responsive early puff and is necessary for puffing and transcription of many subsequently activated late genes in the developing salivary gland. Mapping of 31 cDNA clones indicates that approximately 100 kb of the genome is devoted to the synthesis of many BR-C RNAs. Sequence analyses of these cDNA clones show that the BR-C encodes a family of related proteins characterized by a common core amino-terminal domain fused to alternate carboxy domains each containing a pair of zinc fingers. Most proteins also contain domains rich in distinctive amino acids located between the common core and zinc finger regions. BR-C mutant alleles resulting from chromosomal rearrangements at 2B5 are associated with deletions of 5'-untranslated sequences, separation of the core coding domain from the downstream zinc finger domains, or a P element insertional disruption of a zinc finger coding sequence. We infer that the BR-C directly regulates late gene expression by specifying the synthesis of a family of proteins with DNA binding potential.

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