Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gα q reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gα q , causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] to the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol in an essential step for the physiological action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli (1-3). These cascades use

Synergistic activation by heterotrimeric guanine nucleotide–binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β-dependent functions, we identified the site in p110β that binds to the Gβγ subunit of G proteins. Mutation of this site eliminated Gβγ-dependent activation of PI3Kβ (a dimer of p110β and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide–mediated activation. Disrupting the p110β-Gβγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kβ in fibroblasts, and reduced proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kβ could provide a therapeutic approach for tumors that depend on p110β for growth and metastasis.

Regulator of G-protein signaling (RGS) proteins areGTPase activating proteins (GAPs) of heterotrimeric Gproteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein ␣-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a G␥-like (GGL) domain that mediates dimeric interaction with G␤ 5 . G␤ 5 /R7 dimers stimulated steady state GTPase activity of G␣-subunits of the G i family, but not of G␣ q or G␣ 11 , when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the G␤ 5 /R7 proteins revealed differences in potencies and efficacies toward G␣-subunits of the G i family. Although all four G␤ 5 /R7 proteins exhibited similar potencies toward G␣ o , G␤ 5 /RGS9 and G␤ 5 /RGS11 were more potent GAPs of G␣ i1 , G␣ i2 , and G␣ i3 than were G␤ 5 /RGS6 and G␤ 5 /RGS7. The maximal GAP activity exhibited by G␤ 5 /RGS11 was 2-to 4-fold higher than that of G␤ 5 /RGS7 and G␤ 5 /RGS9, with G␤ 5 /RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious G␤ 5 /RGS7 and G␤ 5 /RGS9 inhibited G␤ 5 / RGS11-stimulated GTPase activity of G␣ o . Therefore, R7 family RGS proteins are G i family-selective GAPs with potentially important differences in activities.Heterotrimeric guanine nucleotide-binding proteins (G-proteins) act as molecular switches in multiple GPCR 1 signaling pathways via regulation of specific effector molecules such as phospholipase C and adenylyl cyclase. The biological activity of G-protein ␣-subunits is determined by the identity of the bound guanine nucleotide (GTP or GDP), which in turn is governed by the relative rates of guanine nucleotide exchange and hydrolysis of GTP by the intrinsic GTPase activity of G␣-subunits.These opposing reactions are stimulated by agonist-occupied GPCR and GTPase-activating proteins (GAPs).Although some effector proteins exhibit GAP activity (1-3), the primary regulators of GTPase activity of G␣-subunits are a diverse family of regulator of G-protein signaling (RGS) proteins that act as GAPs for heterotrimeric G-protein ␣-subunits (4 -7). This family is defined by a conserved RGS domain, which markedly increases the rate of GTP hydrolysis by G␣-subunits and terminates effector activation by both G␣-and G␤␥-subunits. More than 30 RGS proteins have been identified and organized into subfamilies based on sequence similarity and domain structure. These families vary in size and complexity, from the R4 family whose structure is largely limited to the RGS domain to the R12 and RhoGEF families whose members are large multifunctional proteins containing several domains (for reviews see Refs. 8 -10).The R7 RGS family is a unique multidomain family, which consists of RGS proteins containing a novel G-␥-like (GGL) domain homologous to the G␥-subunit of heterotrimeric Gproteins (11). This domain, found in the mammalian proteins RGS6, -7, -9, and -11 and the Caenorhabdi...

-methyl-(N )-methanocarba-2Ј-deoxyadenosine-3Ј,5Ј-bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y 1 receptor (P2Y1-R) antagonist

GTP-bound subunits of the Gq family of G alpha subunits directly activate phospholipase C-beta (PLC-beta) isozymes to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-betas are GTPase activating proteins (GAPs) that also promote the formation of GDP-bound, inactive G beta subunits. Both phospholipase activation by G alpha-GTP subunits and GAP activity require a C-terminal region unique to PLC-beta isozymes. The crystal structure of the C-terminal region from an avian PLC-beta, determined at 2.4 A resolution, reveals a novel fold composed almost entirely of three long helices forming a coiled-coil that dimerizes along its long axis in an antiparallel orientation. The dimer interface is extensive ( approximately 3,200 A(2)), and, based on gel exclusion chromatography, full length PLC-betas are dimeric, indicating that PLC-betas likely function as dimers. Sequence conservation, mutational data and molecular modeling show that an electrostatically positive surface of the dimer contains the major determinants for binding G beta q. Effector dimerization, as highlighted by PLC-betas, provides a viable mechanism for regulating signaling cascades linked to heterotrimeric G proteins.

The human P2Y 1 receptor (P2Y 1 -R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y 1 -R antagonist [ binding assays for antagonists with the purified P2Y 1 -R were in good agreement with the K i and K B values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y 1 -R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) Ͼ ADP ϭ 2-methylthio-ATP ϭ adenosine-5Ј-O-(3-thio)triphosphate ϭ adenosine-5Ј-O-(2-thiodiphosphate) Ͼ Ͼ ATP was obtained. The signaling activity of the purified P2Y 1 -R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y 1 -R and G␣ q ␤ 1 ␥ 2 was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC 50 values of agonists for activation of the purified P2Y 1 -R were similar to their respective K i values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC 50 and K i values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-␤1 were potent and efficacious GTPase-activating proteins for G␣ q and G␣ 11 in P2Y 1 -R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y 1 -R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.Burnstock's hypothesis (Burnstock, 1972) of purinergic signaling largely has been confirmed over the past two decades by demonstration of regulated release of cellular nucleotides, by elucidation of the mechanisms of metabolism of extracellular nucleotides by a complex array of ecto-enzymes, and by delineation of myriad nucleotide-promoted physiological responses (Dubyak and El-Moatassim, 1993;Harden et al., 1995;Ralevic and Burnstock, 1998;Lazarowski et al., 2003). Moreover, the molecular cloning of two classes of ubiquitously distributed receptors for extracellular nucleotides, the ligand-gated P2X receptors (Khakh et al., 2000) and the G protein-coupled P2Y receptors , comprising at least 15 human genes, has amplified the importance of nucleotide-promoted signaling.Although a burgeoning interest in the biology and therapeutic potential of extracellular nucleotide signaling now exists, the complex regulatory pathways that underlie the action of extracellular nucleotides remain difficult to study. Few truly selective agonists and antagonists are available for the P2 receptors, and studies with intact tissues are compromised both by nucleotide release and by metabolism and interconversion of ...

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate G␣ q -mediated inositol lipid signaling. Although purified RGS2 blocks PLC-␤ activation by the nonhydrolyzable GTP analog guanosine 5-O-thiophosphate (GTP␥S), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTP␥S-stimulated G␣ 11 signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10 -20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-␤t activity than of GTP␥S-promoted PLC-␤t activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4␤-phorbol 12␤-myristate 13␣-acetate and, to a lesser extent, by the P2Y 2 receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTP␥S-stimulated PLC-␤t activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y 1 receptor and G␣ q ␤␥. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.A variety of hormone and neurotransmitter receptors transduce signals through heterotrimeric G proteins. In their inactive state, G proteins exist as heterotrimers consisting of ␣, ␤, and ␥ subunits with GDP bound to G␣. Upon agonist occupation, the receptor promotes GDP/GTP exchange, and the active GTP-bound G␣ subunit and G␤␥ dissociate to interact with target effector proteins. Signaling is terminated by the hydrolysis of GTP to GDP and the subsequent formation of the heterotrimer. Therefore, the magnitude and duration of signaling is determined by the length of time G␣ remains in the active GTP-bound conformation.A recently identified family of proteins termed RGS (regulators of G protein signaling) proteins interact directly with G␣ subunits to decrease the lifetime of the active GTP-bound complex (1-4). RGS proteins attenuate heterotrimeric G protein 1 signaling by functioning as both GTPase-activating proteins (GAPs) (5, 6) and inhibitors of G protein/effector interaction (6, 7). In vitro studies illustrate that RGS2 interacts with and fu...

Background: Phospholipase C-␤ (PLC-␤) isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate to propagate signals for several physiological responses. Results: Membranes are essential for the allosteric release of autoinhibition of PLC-␤ isozymes. Conclusion: Activators of PLC-␤ release autoinhibition by orientating the isozymes at the membrane. Significance: The model described provides a better understanding of PLC-␤ regulation and potential mechanisms to inhibit their activation.