RGS14 limits seizure-induced mitochondrial oxidative stress and pathology in hippocampus.

RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. In these neurons, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies showed that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity, possibly affording protection to CA2 PCs. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show that the loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Our proteomics data show that the loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro. As a readout of oxidative stress, we found that RGS14 KO dramatically increased 3- nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we unexpectedly found no differences in neuronal injury in CA2 PCs. However, we observed a striking and surprising lack of microgliosis in CA1 and CA2 of RGS14 KO compared to WT. Together, our data demonstrate a newly appreciated role for RGS14 in limiting intense seizure activity and pathology in hippocampus. Our findings are consistent with a model where RGS14 limits seizure onset and mortality and, after seizure, is upregulated to support mitochondrial function, prevent oxidative stress in CA2 PCs, and promote microglial activation in hippocampus.

RGS14 is a bifunctional regulator of Galphai/o activity that exists in multiple populations in brain.

Members of the regulators of G protein signaling (RGS) family modulate Galpha-directed signals as a result of the GTPase-activating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To define the cellular and biochemical properties of RGS14 we utilized two different affinity purified antisera that specifically recognize recombinant and native RGS14. In brain, we observed two RGS14-like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent-extractable and the other detergent-insensitive. Recombinant RGS14 binds specifically to activated Galphai/o, but not Galphaq/11, Galpha12/13, or Galphas in brain membranes. In reconstitution studies, we found that RGS14 is a non-selective GAP for Galphai1 and Galphao and that full-length RGS14 is an approximately 10-fold more potent stimulator of Galpha GTPase activity than the RGS domain alone. In contrast, neither full-length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Galphai but not Galphao, and this activity is restricted to the C-terminus containing the GoLoco domain. These findings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the first examples of an RGS protein that is a bifunctional regulator of Galpha actions.

Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling.

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 Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), 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 GTPgammaS-stimulated Galpha(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-betat activity than of GTPgammaS-promoted PLC-betat 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 4beta-phorbol 12beta-myristate 13alpha-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 GTPgammaS-stimulated PLC-betat 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 Galpha(q)betagamma. 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.

RGS7 is palmitoylated and exists as biochemically distinct forms.

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Galpha and Gbeta5 are enriched in brain, but biochemical mechanisms governing RGS7/Galpha/Gbeta5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and Gbeta5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gbeta5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with Gbeta5, are equally effective stimulators of Galpha(o) GTPase activity, suggesting that palmitoylation does not prevent RGS7/Galpha(o) interactions. The isolated core RGS domain of RGS7 selectively binds activated Galpha(i/o) in brain extracts and is an effective stimulator of both Galpha(o) and Galpha(i1) GTPase activities in vitro. In contrast, the RGS7/Gbeta5 complex selectively interacts with Galpha(o) only, suggesting that features outside the RGS domain and/or Gbeta5 association dictate RGS7-Galpha interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.

G protein selectivity is a determinant of RGS2 function.

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Galpha subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward G(q) versus G(i) family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of G(q)-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of G(i)-mediated signaling. RGS2 mutants were identified that display increased potency toward G(i) family members without affecting potency toward G(q). These mutations and the structure of RGS4-G(i)alpha(1) complexes suggest that RGS2-G(i)alpha interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the alpha8-alpha9 loop of RGS2 and alphaA of G(i) class alpha subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Galpha subunits.

Emerging roles for RGS proteins in cell signalling.

Regulators of G-protein signalling (RGS proteins) are a family of highly diverse, multifunctional signalling proteins that share a conserved 120 amino acid domain (RGS domain). RGS domains bind directly to activated Galpha subunits and act as GTPase-activating proteins (GAPs) to attenuate and/or modulate hormone and neurotransmitter receptor-initiated signalling by both Galpha-GTP and Gbetagamma. Apart from this structural domain, which is shared by all known RGS proteins, these proteins differ widely in their overall size and amino acid identity and possess a remarkable variety of structural domains and motifs. These biochemical features impart signalling functions and/or enable RGS proteins to interact with a growing list of unexpected protein-binding partners with diverse cellular roles. New appreciation for the broader cellular functions of RGS proteins challenges established models of G-protein signalling and serves to identify these proteins as central participants in receptor signalling and cell physiology.

RGS4 inhibits signaling by group I metabotropic glutamate receptors.

Metabotropic glutamate receptors (mGluRs) couple to heterotrimeric G-proteins and regulate cell excitability and synaptic transmission in the CNS. Considerable effort has been focused on understanding the cellular and biochemical mechanisms that underlie regulation of signaling by G-proteins and their linked receptors, including the mGluRs. Recent findings demonstrate that regulators of G-protein signaling (RGS) proteins act as effector antagonists and GTPase-activating proteins for Galpha subunits to inhibit cellular responses by G-protein-coupled receptors. RGS4 blocks Gq activation of phospholipase Cbeta and is expressed broadly in rat brain. The group I mGluRs (mGluRs 1 and 5) couple to Gq pathways to regulate several effectors in the CNS. We examined the capacity of RGS4 to regulate group I mGluR responses. In Xenopus oocytes, purified RGS4 virtually abolishes the mGluR1a- and mGluR5a-mediated but not the inositol trisphospate-mediated activation of a calcium-dependent chloride current. Additionally, RGS4 markedly attenuates the mGluR5-mediated inhibition of potassium currents in hippocampal CA1 neurons. This inhibition is dose-dependent and occurs at concentrations that are virtually identical to those required for inhibition of phospholipase C activity in NG108-15 membranes and reconstituted systems using purified proteins. These findings demonstrate that RGS4 can modulate mGluR responses in neurons, and they highlight a previously unknown mechanism for regulation of G-protein-coupled receptor signaling in the CNS.

Organization of G proteins and adenylyl cyclase at the plasma membrane.

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein alpha and beta subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein alpha subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells.

Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein alpha subunits (the reaction responsible for their deactivation); they can also act as effector antagonists. We demonstrate herein that either of two such RGS proteins, RGS4 or GAIP, attenuated signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo was similar to that seen in vitro. RGS4 and GAIP both attenuated Gi-mediated inhibition of cAMP synthesis. RGS4 was more effective than GAIP in blocking Gq-mediated activation of phospholipase Cbeta.

RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha.

RGS proteins constitute a newly appreciated and large group of negative regulators of G protein signaling. Four members of the RGS family act as GTPase-activating proteins (GAPs) with apparent specificity for members of the Gi alpha subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for Gq alpha, the G alpha protein responsible for activation of phospholipase C beta. Furthermore, these RGS proteins block activation of phospholipase C beta by guanosine 5'-(3-O-thio) triphosphate-Gq alpha. GAP activity does not explain this effect, which apparently results from occlusion of the binding site on G alpha for effector. Inhibitory effects of RGS proteins on G protein-mediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.

RGS2/G0S8 is a selective inhibitor of Gqalpha function.

RGS (regulators of G protein signaling) proteins are GTPase activating proteins that inhibit signaling by heterotrimeric G proteins. All RGS proteins studied to date act on members of the Gialpha family, but not Gsalpha or G12alpha. RGS4 regulates Gialpha family members and Gqalpha. RGS2 (G0S8) is exceptional because the G proteins it regulates have not been identified. We report that RGS2 is a selective and potent inhibitor of Gqalpha function. RGS2 selectively binds Gqalpha, but not other Galpha proteins (Gi, Go, Gs, G12/13) in brain membranes; RGS4 binds Gqalpha and Gialpha family members. RGS2 binds purified recombinant Gqalpha, but not Goalpha, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gsalpha or Gialpha family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Gialpha family members. In contrast, RGS2 and RGS4 completely inhibit Gq-directed activation of phospholipase C in cell membranes. When reconstituted with phospholipid vesicles, RGS2 is 10-fold more potent than RGS4 in blocking Gqalpha-directed activation of phospholipase Cbeta1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gqalpha function.

Functional importance of the amino terminus of Gq alpha.

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.

Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases.

The capacities of the alpha subunits of pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) to inhibit different isoforms of mammalian adenylyl cyclases were assessed. Membranes from Sf9 cells infected with recombinant baculoviruses encoding either type I, II, V, or VI adenylyl cyclase were reconstituted with purified G protein subunits. Types V and VI adenylyl cyclase are most sensitive to inhibition by Gi alpha 1, Gi alpha 2, and Gi alpha 3; type I adenylyl cyclase can be inhibited by these three Gi alpha proteins and by G(o) alpha as well. Type II adenylyl cyclase appears to be immune to inhibition by these proteins. Examination of the effects of native and mutant Gi alpha proteins, as well as analysis of competition for binding of Gs alpha to adenylyl cyclases, indicate that at least certain adenylyl cyclases have independent sites for interaction with Gs alpha (site 1, stimulatory) and Gi alpha (site 2, inhibitory). High concentrations of Gi alpha can interact with site 1 on types I and II adenylyl cyclase and activate the enzymes. Types I and II adenylyl cyclase also appear to have independent sites for interaction with G protein beta gamma subunits. The type I enzyme is strongly inhibited, while type II adenylyl cyclase is activated if Gs alpha is also present.

Activation of phospholipase C-beta 2 mutants by G protein alpha q and beta gamma subunits.

The beta- but not the gamma- and delta-type isozymes of inositol phospholipid-specific phospholipase C (PLC) are activated by G protein alpha q and beta gamma subunits. The beta-type PLC isozymes differ from other isozymes in that they contain a long carboxyl-terminal region downstream of the Y catalytic domain and a region rich in acidic amino acids between the two separated X and Y catalytic domains. To determine the sites on PLC-beta 2 that participate in the interaction of the enzyme with alpha q and beta gamma subunits, we introduced specific truncations and substitutions in the PLC-beta 2 cDNA at positions corresponding to the carboxyl-terminal and acidic amino acid-rich regions, respectively. After transient expression of these cDNA clones in CV-1 cells, the mutant enzymes were partially purified and their capacity to be activated by alpha q and beta gamma subunits determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC-beta 2 affected neither alpha q- nor beta gamma-dependent activation significantly. Carboxyl-terminal truncation to residue Gly-934 or to residue Ala-867 resulted in enzymes that were activated by beta gamma but not by alpha q. This result suggests that the carboxyl-terminal region of PLC-beta 2 is required for activation by alpha q, and that beta gamma subunits interact with a different region of the enzyme. Thus, alpha q and beta gamma subunits may independently modulate a single PLC-beta 2 molecule concurrently.

Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.

A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.

Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits.

Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC-gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC-beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC-beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway.

Lipid modifications of G proteins: alpha subunits are palmitoylated.

A small number of membrane-associated proteins are reversibly and covalently modified with palmitic acid. Palmitoylation of G-protein alpha and beta subunits was assessed by metabolic labeling of subunits expressed in simian COS cells or insect Sf9 cells. The fatty acid was incorporated into all of the alpha subunits examined (alpha s, alpha o, alpha i1, alpha i2, alpha i3, alpha z, and alpha q), including those that are also myristoylated (alpha o, alpha i, and alpha z). Palmitate was released by treatment with base, suggesting attachment to the protein through a thioester or ester bond. Limited tryptic digestion of activated alpha o and alpha s resulted in release of the amino-terminal portions of the proteins and radioactive palmitate. These data are consistent with palmitoylation of the proteins near their amino termini, most likely on Cys-3. Reversible acylation of G-protein alpha subunits may provide an additional mechanism for regulation of signal transduction.

G proteins.

The family of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serves an essential role in transducing receptor-generated signals across the plasma membrane. Recent findings reveal unexpected functional roles for individual G protein subunits. Thus, GTP-binding alpha-subunits and the beta gamma-subunit complex can influence the activity of effector molecules independently or simultaneously, either synergistically or in opposition, to elicit a complex constellation of cellular events.

Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C yields the second messengers inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol. This activity is regulated by a variety of hormones through G protein pathways. However, the specific G protein or proteins involved has not been identified. The alpha subunit of a newly discovered pertussis toxin-insensitive G protein (Gq) has recently been isolated and is now shown to stimulate the activity of polyphosphoinositide-specific phospholipase C (PI-PLC) from bovine brain. Both the maximal activity and the affinity of PI-PLC for calcium ion were affected. These results identify Gq as a G protein that regulates PI-PLC.