Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-ß isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-ß3 by Gß1γ2 In contrast, the peptide robustly prevented activation of PLC-ß3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-ß3 at least as effectively as a dominant-negative form of full-length PLC-ß3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells.

Membrane-induced allosteric control of phospholipase C-ß isozymes.

All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-ß (PLC-ß) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-ß isozymes are autoinhibited, and several proteins, including Gαq, Gßγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gß1γ2 did not activate purified PLC-ß3 under these conditions despite their robust capacity to activate PLC-ß3 at membranes. In addition, mutants of PLC-ß3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-ß isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-ß isozymes.

G protein-coupled receptor-mediated activation of p110ß by Gßγ is required for cellular transformation and invasiveness.

Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110ß from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110ß 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 p110ß-dependent functions, we identified the site in p110ß that binds to the Gßγ subunit of G proteins. Mutation of this site eliminated Gßγ-dependent activation of PI3Kß (a dimer of p110ß and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide-mediated activation. Disrupting the p110ß-Gßγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kß in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kß could provide a therapeutic approach for tumors that depend on p110ß for growth and metastasis.

Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex.

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-ßs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-ß3 bound to activated Gα(q) reveals a conserved module found within PLC-ßs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-ß3 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-ß3 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.

Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor.

The human P2Y1 receptor (P2Y1-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 P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-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) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) >> ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Galpha(q)beta(1)gamma(2) was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki 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-beta1 were potent and efficacious GTPase-activating proteins for Galphaq and Galpha11 in P2Y1-R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.

Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins.

P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y(1) receptor and the human P2Y(12) receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 mug/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.

A unique fold of phospholipase C-beta mediates dimerization and interaction with G alpha q.

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.

RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity.

Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma-like (GGL) domain that mediates dimeric interaction with Gbeta(5). Gbeta(5)/R7 dimers stimulated steady state GTPase activity of Galpha-subunits of the G(i) family, but not of Galpha(q) or Galpha(11), when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta(5)/R7 proteins revealed differences in potencies and efficacies toward Galpha-subunits of the G(i) family. Although all four Gbeta(5)/R7 proteins exhibited similar potencies toward Galpha(o), Gbeta(5)/RGS9 and Gbeta(5)/RGS11 were more potent GAPs of Galpha(i1), Galpha(i2), and Galpha(i3) than were Gbeta(5)/RGS6 and Gbeta(5)/RGS7. The maximal GAP activity exhibited by Gbeta(5)/RGS11 was 2- to 4-fold higher than that of Gbeta(5)/RGS7 and Gbeta(5)/RGS9, with Gbeta(5)/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta(5)/RGS7 and Gbeta(5)/RGS9 inhibited Gbeta(5)/RGS11-stimulated GTPase activity of Galpha(o). Therefore, R7 family RGS proteins are G(i) family-selective GAPs with potentially important differences in activities.

Purification and functional reconstitution of the human P2Y12 receptor.

The human P2Y12 receptor (P2Y12-R) is a member of the G protein coupled P2Y receptor family, which is intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y12-R, Gbeta1gamma2, and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y12-R and Galphai2beta1gamma2 was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was markedly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 = 80 nM) examined, whereas ADP, the cognate agonist of the P2Y12-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action of 2MeSADP; therefore, ATP is a relatively low-affinity antagonist of the P2Y12-R. The G protein selectivity of the P2Y12-R was examined by reconstitution with various G protein alpha-subunits in heterotrimeric form with Gbeta1gamma2. The most robust coupling of the P2Y12-R was to Galphai2, but effective coupling also occurred to Galphai1 and Galphai3. In contrast, little or no coupling occurred to Galphao or Galphaq. These results illustrate that the signaling properties of the P2Y12-R can be studied as a purified protein under conditions that circumvent the complications that occur in vivo because of nucleotide metabolism and interconversion as well as nucleotide release.

Quantitation of the P2Y(1) receptor with a high affinity radiolabeled antagonist.

2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.