Regulators of G protein signaling (RGS) and RGS-like proteins are a family (>30 members) of highly diverse, multifunctional signaling proteins that bind directly to activated G alpha subunits. Family members are defined by a shared RGS domain, which is responsible for G alpha binding and markedly stimulates the GTPase activity of G alpha subunits leading to their deactivation and termination of downstream signals. Although much has been learned in recent years about the biochemistry of RGS/G alpha interactions, considerably less is known about the broader cellular roles and regulation of RGS proteins. Recent findings indicate that cellular mechanisms such as covalent modification, alternative gene splicing, and protein processing can dictate the activity and subcellular localization of RGS proteins. Many family members also directly link G proteins to a growing list of signaling proteins with diverse cellular roles. New findings indicate that RGS proteins act not as dedicated inhibitors but, rather, as tightly regulated modulators and integrators of G protein signaling. In some cases, RGS proteins modulate the lifetime and kinetics of both slow-acting (e.g., Ca(2+) oscillations) and fast-acting (e.g., ion conductances, phototransduction) signaling responses. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. These and other recent studies with animal model systems indicate that RGS proteins play important roles in both physiology and disease. Recognition of the central functions these proteins play in vital cellular processes has focused our attention on RGS proteins as exciting new candidates for therapeutic intervention and drug development.

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.

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.

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)-CA3-CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2-7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG-CA3-CA1 circuit.long-term potentiation | hippocampus | G protein signaling | RGS proteins | ERK

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 Gi␣ family, but not Gs␣ or G12␣. RGS4 regulates Gi␣ family members and Gq␣. 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 Gq␣ function. RGS2 selectively binds Gq␣, but not other G␣ proteins (Gi, Go, Gs, G12͞13) in brain membranes; RGS4 binds Gq␣ and Gi␣ family members. RGS2 binds purified recombinant Gq␣, but not Go␣, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gs␣ or Gi␣ family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Gi␣ 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 Gq␣-directed activation of phospholipase C␤1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gq␣ function.

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 G i␣ subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for G q␣ , the G ␣ protein responsible for activation of phospholipase C␤. Furthermore, these RGS proteins block activation of phospholipase C␤ by guanosine 5-(3-O-thio)triphosphate-G q␣ . GAP activity does not explain this effect, which apparently results from occlusion of the binding site on G ␣ for effector. Inhibitory effects of RGS proteins on G proteinmediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.The ␣ subunits of signal-transducing, heterotrimeric G proteins are molecular switches and clocks, and these functions are imparted to the proteins by conformational changes that result from the binding and hydrolysis of GTP. G ␣ proteins are inactive as GDP-bound species because of reduced affinity (compared with the GTP-bound protein) for downstream effectors and, in addition, enhanced affinity for the G protein ␤␥ subunit complex. Binding of G ␣ -GDP to ␤␥ occludes sites for interaction with downstream effectors on both ␣ and ␤␥. G proteins are activated by appropriate plasma membranebound, heptahelical receptors, which catalyze exchange of GDP for GTP, and they are deactivated as a result of their intrinsic GTPase activity.The last few years have brought heightened appreciation of regulation of G ␣ -catalyzed GTP hydrolysis by proteins known as GTPase-activating proteins (GAPs). Such GAP activities were first demonstrated with certain effectors for G protein action-notably phospholipase C␤ and the ␥ subunit of retinal cyclic GMP phosphodiesterase (1-7). Genetic studies (particularly in yeast and worms) have now resulted in discovery of a large family (at least 20 members in mammals) of negative regulators of G protein signaling (RGS proteins), and biochemical characterization of a few of the family members has demonstrated that they, too, act as GAPs, especially toward members of the G i subfamily of G ␣ proteins (8, 9).Mammalian RGS proteins are presumed to play an important role in the down-regulation or desensitization of G protein-mediated signaling pathways, as one such protein (Sst2p) does in yeast (10-16). However, much remains to be learned, including such obvious and crucial questions as mechanisms of acceleration of GTPase activity, cellular distribution of the family members, the specificity of their interactions with G ␣ subunits, mechanisms of regulation of RGS protein activity, and the importance of RGS protein-mediated inhibition of G protein signaling compared with other mechanisms (particularly receptor-directed kinases) that have been documented over the past two decades.In the relatively few reports that have appeared to date, RGS proteins have been shown to interact preferentially with members o...

is not myristoylated (3). However, when synthesized in Escherichia coli, a, has reduced affinities for f8y, adenylyl cyclase, and Ca2+ channels (4-6). Hypothetically, the differences between native and recombinant a, are due to the lack of unknown posttranslational modifications of the recombinant protein (4). Furthermore, the structural features of a, necessary for association with membranes have not been fully characterized (7-9). Other a subunits, including members of the Gq family (activators of phospholipase C-*), lack the requisite glycine residue at position 2 (10) and are also presumably not myristoylated. Some membrane-associated proteins, including certain forms of p2lms and receptors such as rhodopsin, are palmitoylated (11). Palmitate is almost always linked to cysteine residues through a thioester bond. The function of proteinbound palmitate is poorly defined. In an attempt to identify posttranslational modifications of as and aq, we examined a subunits for incorporation of radioactive palmitate. We report here that tritiated palmitate is incorporated into a, and aq and, in addition, into a subunits that are also myristoylated (ao, ai, and az). tHowever, simple interpretation of these data is hampered by the fact that myristoylation is a stable modification, whereas palmitoylation is dynamic. The specific activity of protein-bound myristate would reflect that of the precursor pool over the time course of the experiment. The specific activity of protein-bound palmitate might reflect that of the precursor pool only at the end of the experiment if tumover were sufficiently rapid. MATERIALS AND METHODS 3675The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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