The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with G␣ o and G␣ s as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of G␣ proteins including G␣ q , G␣ i1 , and G␣ o , but not G␣ s . The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound G␣ proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the G␣ protein; this complex dissociates upon binding of GTP to G␣.Heterotrimeric guanine nucleotide-binding regulatory proteins mediate signal transduction between many membranebound receptors and intracellular effectors (1). Traditionally, activation of heterotrimeric G proteins 1 is accomplished exclusively by the action of GPCRs, seven transmembrane-spanning proteins that typically reside in the plasma membrane. These receptors act as guanine nucleotide exchange factors (GEFs), binding the inactive GDP-bound conformation of G proteins and stimulating release of GDP from G␣. To ensure directionality of exchange, GEFs stabilize a nucleotide-free transition state of G␣ that is disrupted by binding of GTP (2, 3). This facilitates dissociation of G␣⅐GTP from the G␤␥ dimer and release of these proteins from the receptor. Dissociated G protein subunits then participate in interactions with a variety of effectors.G protein signaling is attenuated when G␣ hydrolyzes the ␥ phosphate of its bound GTP and G␣⅐GDP reassociates with ␤␥. GTPase-activating proteins (GAPs) facilitate the inactivation of many G proteins. Most of these GAPs contain a regulator of G protein signaling (RGS) domain that binds preferentially to the G␣⅐GTP transition state and accelerates GTPase activity (4, 5). More than 20 unique RGS domain-containing proteins have been discovered, and the nature of their G protein specificity and their mode of action in cells are subjects of intense interest (6, 7).Nonreceptor activators of G proteins may operate in lieu of or in conjunction with GPCRs to enhance signaling, but their physiological role is not well understood (8 -11). Activators of G protein signaling AGS1 and AGS3 were identified in a genetic screen in yeast designed to isolate expressed mammalian cDNAs that encode proteins that bypass the need for a receptor (12). AGS3 possesses G␣ guanine nucleotide dissociation inhibitor activity but may activate G proteins by liberating G␤␥ (10, 13). AGS1 encodes a Ras-like small GTPase that, when bound to GTP, possesses in vitro guanine nucleotide exchange activity for members of th...

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.

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the ␣ subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein ␥ subunit-like (GGL) domain between its Dishevelled͞Egl-10͞Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different G ␤ subunits reveals specific interaction between RGS11 and G ␤5 . The expression of mRNA for RGS11 and G ␤5 in human tissues overlaps. The G ␤5 ͞RGS11 heterodimer acts as a GAP on G ␣o , apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G ␣ proteins and form complexes with specific G ␤ subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.Proteins belonging to the RGS (regulators of G protein signaling) family constitute a newly appreciated group of at least 20 mammalian gene products that act as GTPaseactivating proteins (GAPs) on the ␣ subunits of heterotrimeric, signal-transducing G proteins (1-3). As such, RGS proteins can serve as negative regulators of G proteinmediated signaling pathways by speeding the inactivation of GTP-bound G ␣ subunits. Although several members of the RGS family are relatively simple Ϸ25 kDa proteins that contain little more than a characteristic RGS domain, others include modules that impart additional functions. For example, RGS12 can associate in vitro with certain G protein-coupled receptors by virtue of an alternatively spliced PDZ (PSD-95͞ Dlg͞Z0-1) domain (4), and p115, a guanine nucleotide exchange factor for the low-molecular-weight GTPase rho, contains an RGS domain that imparts sensitivity to regulation by G protein ␣ subunits (5, 6).We describe here a novel G protein ␥ subunit-like domain (GGL; pronounced giggle) that is found in several mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11) and in EGL-10, an RGS protein of Caenorhabditis elegans. The GGL domains of RGS11 and RGS7 interact preferentially with the G protein ␤ 5 subunit, and the complex of RGS11 and ␤ 5 has GAP activity toward the G protein ␣ o subunit. MATERIALS AND METHODSGeneration of Expression Constructs. cDNAs for RGS11 and various G protein subunits were cloned from human brain or retinal mRNA, from mouse retinal mRNA, or were obtained as described (7,8); all amplified cDNAs were verified by sequencing. Human RGS7 cDNA was a kind gift of Paul F. Worley (Johns Hopkins University). cDNAs encoding G protein subunits were subcloned into the mammalian expression vector pcDNA3.1-Zeo (Invitrogen), and G ␥ and RGS protein cDNAs were subcloned in-frame with an N-terminal tandem hemagglutinin (HA)-epitope tag into a modified pcDNA3.1 vector. Recombinant baculoviruses expressing native or hexahistidine-tagged RGS11 or G ␤5 subunits were generated by using the Bac-To-Bac system by following the manufacturer's protocols (Life Technologies, Gaithersburg, MD).In Vitro Transcription and Translation. Reactions were performed using the T...

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein ␣ subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/ Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3 end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3 end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPaseactivating protein for G i class ␣ subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3 exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.The mammalian "regulators of G-protein signaling" (RGS) 1 gene family was first identified by sequence and functional similarity to fungal and nematode genes captured in genetic screens for negative regulators of specific G-protein-coupled receptor (GPCR) signals (1-3). In vitro biochemical analyses soon established that this gene family encodes potent accelerators ("GAPs") of the intrinsic GTP hydrolysis activity of Gprotein ␣ subunits, revealing a molecular mechanism by which RGS proteins drive G-proteins into their inactive GDP-bound form and hence down-regulate GPCR signal transduction in vivo (reviewed in Refs. 4 and 5). However, the mechanisms by which individual RGS proteins desensitize pathways activated by particular GPCRs remain to be elucidated. Tightly regulated transcription has been described for RGS1 (3), RGS2 (6), and RGS3-RGS11 (7), and palmitoylation of the cysteine-rich N terminus of G␣-interacting protein (GAIP) has also been observed (8); however, transcriptional regulation and post-translational modifications of particular RGS family members can each only be expected to afford a gross level of intracellular control over the temporal and spatial expression of G␣-directed GAP activity.We and others have hypothesized that regions outside the RGS fold contribute to regulation of G␣ GAP activity and/or targeting of individual RGS proteins to particular receptor signaling pathways (4, 5, 9, 10). Here, we report the GAP activity of RGS12 and identify a PDZ-like N-terminal sequence within two splice forms. PDZ domains...