GTPase-activating proteins (GAPs) regulate heterotrimeric G proteins by increasing the rates at which their subunits hydrolyze bound GTP and thus return to the inactive state. G protein GAPs act allosterically on G subunits, in contrast to GAPs for the Ras-like monomeric GTP-binding proteins. Although they do not contribute directly to the chemistry of GTP hydrolysis, G protein GAPs can accelerate hydrolysis >2000-fold. G protein GAPs include both effector proteins (phospholipase C-¿, p115RhoGEF) and a growing family of regulators of G protein signaling (RGS proteins) that are found throughout the animal and fungal kingdoms. GAP activity can sharpen the termination of a signal upon removal of stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network. GAPs are regulated by various controls of their cellular concentrations, by complex interactions with G¿ or with G¿5 through an endogenous G-like domain, and by interaction with multiple other proteins.

A novel class of regulators of G protein signaling (RGS) proteins has been identified recently. Genetic evidence suggests that RGS proteins inhibit G protein-mediated signaling at the level of the receptor-G protein interaction or the G protein alpha subunit itself. We have found that two RGS family members, GAIP and RGS4, are GTPase-activating proteins (GAPs), accelerating the rate of GTP hydrolysis by Gi alpha 1 at least 40-fold. All Gi subfamily members assayed were substrates for these GAPs; Gs alpha was not. RGS4 activates the GTPase activity of certain Gi alpha 1 mutants (e.g., R178C), but not others (e.g., Q204L). The GAP activity of RGS proteins is consistent with their proposed role as negative regulators of G protein-mediated signaling.

Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site. G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function.

The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G 12 and G 13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G 12 or G 13 in stress fiber formation induced through a variety of G q /G 11 -coupled receptors. Using fibroblast cell lines derived from wildtype and G␣ q /G␣ 11 -deficient mice, we show that agonistdependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B 2 , vasopressin V 1A , endothelin ET A , and serotonin 5-HT 2C receptors induced stress fiber formation in either the presence or absence of G␣ q /G␣ 11 . Stress fiber assembly induced through the muscarinic M 1 and the metabotropic glutamate subtype 1␣ receptors was dependent on G q /G 11 proteins. The activation of the G q /G 11 -coupled endothelin ET B and angiotensin AT 1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B 2 , and 5-HT 2C receptor-mediated stress fiber formation was dependent on G␣ 13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ET A , and V 1A receptors induced stress fiber accumulation via G␣ 12 in an EGF receptorindependent manner. Our data demonstrate that many G q /G 11 -coupled receptors induce stress fiber assembly in the absence of G␣ q and G␣ 11 and that this involves either a G␣ 12 or a G␣ 13 /EGF receptor-mediated pathway.Heterotrimeric guanine nucleotide-binding proteins (G-proteins) 1 act as molecular switches that couple receptors for hormones, neurotransmitters, and other extracellular stimuli to effector systems such as enzymes or ion channels (1-4). Gproteins are composed of an ␣-, ␤-, and ␥-subunit and are characterized by the identity of the ␣-subunit that binds and hydrolyzes GTP. On the basis of sequence and functional homologies, G-protein ␣-subunits can be classified into four families: (a) G␣ s , (b) G␣ i/o , (c) G␣ q , and (d) G␣ 12 (5).The G␣ 12 subfamily consists of the ubiquitously expressed members G␣ 12 and G␣ 13 , whose functions are still incompletely understood. They share a 67% sequence identity and are less than 45% homologous to other G-protein ␣-subunits (6). G 12 and G 13 are activated through various receptors, including receptors for thrombin, thromboxane A 2 , and lysophosphatidic acid (LPA) (7-9). Because no receptors selectively coupling to G␣ 12 or G␣ 13 have been found thus far, G 12 /G 13 -mediated signaling pathways have been studied using constitutively active mutants of G␣ 12 and G␣ 13 . Both G␣ 12 and G␣ 13 can regulate the Na ϩ /H ϩ antiporter (10 -12), the Jun kinase/stress-activated protein kinase pathway (13), and the Rho-dependent formation of actin stress fibers (14). Whereas there are clear similarities in the effects induced by constitutively active forms of G␣ 12 and G␣ 13 , differences in the involved signal transduction mechanisms have been reported (9,11...

Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via G q . Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10 4 -fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates G q class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by G␣ q and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled G q signaling.Heterotrimeric G proteins of the G q class are mediators of Ca 2ϩ responses in animal cells. Signaling is initiated by agonist binding to heptahelical transmembrane receptors complexed with G q ␣␤␥ and phospholipase C-␤ (PLC␤) 1 (1), which generates IP 3 to trigger Ca 2ϩ release from internal stores (2).Many cells express several G q -coupled receptors that regulate the location, intensity, and propagation of intracellular Ca 2ϩ waves. For example, pancreatic acini respond to acetylcholine, bombesin, and cholecystokinin by activating the same set of G q class proteins and mobilizing the same Ca 2ϩ pool, but each receptor evokes distinct patterns of Ca 2ϩ waves (3). Ca 2ϩ release may be regulated by intracellular proteins that interact with guanine nucleotide binding proteins, such as regulators of G protein signaling (RGS) proteins.2 RGS proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling (5-8). Mammals express over 20 different RGS proteins, of which RGS4 has received the most extensive biochemical characterization (5, 7-12). RGS4 is composed of a central domain of 120 amino acids that is homologous to other RGS proteins, termed the RGS box, flanked by less well conserved N-and C-terminal sequences (13). In rat pancreatic acinar cells, RGS4 preferentially inhibited G q/11 -mediated signaling evoked by carbachol relative to bombesin and cholecystokinin regardless of the identity of the G q class ␣ subunit. 2Regulatory specificity was apparently conferred by direct or indirect interaction between RGS4 and the receptor.In the present study, we used deletion mutations to identify two domains in RGS4 that regulate agonist-dependent Ca 2ϩ signaling. The RGS box accelerates GTP hydrolysis by G␣ q whereas the N terminus conveys high affinity and receptorselective inhibition. These combined activities are required for receptor selectivity and high potency i...