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