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.

Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale.

Regulation of intracellular cyclic adenosine 3,5-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G s -dependent receptors for isoproterenol and prostaglandin E 2 . Whereas two ligands, uridine 5-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G q /calcium and G i , the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G s -coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P 2 receptor and the heterotrimeric G 13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.Cyclic adenosine 3Ј,5Ј-monophosphate (cAMP), a ubiquitous second messenger, mediates a wide range of cellular functions including cell metabolism (1), cell proliferation and differentiation (1), immune responses (2, 3), memory formation (4), and cardiac contractility (5). Canonically, the concentration of intracellular cAMP is regulated by two distinct families of enzymes. The transmembrane adenylyl cyclases (ACs) 3 synthesize cAMP from adenosine triphosphate (6, 7), whereas the cAMP-specific phosphodiesterases metabolize cAMP to biologically inactive adenosine 5Ј-monophosphate (8, 9). ACs are primarily activated by G␣ s but their activities can also be differentially regulated by G␣ i , G␤␥, or Ca 2ϩ (10, 11). The activities of various phosphodiesterases can be regulated by protein kinase A (PKA), extracellular-regulated kinase (ERK), phosphoinositide 3-kinase, and the concentration of cAMP itself (12-16). Thus integration of signaling by stimuli that can regulate the intracellular concentration of cAMP will depend strongly on the various pathways and the subtypes of ACs and phosphodiesterases expressed in individual cells at any given time.Assessment of the regulation of intracellular cAMP in vivo has only become possible recently. Zaccolo et al. (17) first described a FRET sensor for cAMP based on the cAMP binding domain of PKA. Subsequently, several reports have described FRET sensors for cAMP based on binding of the nucleotide to the Epac proteins (18 -21). While these FRET sensors have been effective for measuring changes and localization of cAMP in single cells, measurements are tedious. Furthermore, the requirement for excitation of donor molecules pro...

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.

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Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.

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...

The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.