The intrinsic GTPase activity of transducin (the Galpha subunit involved in visual transduction) controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. A human homolog of murine retinal specific RGS (hRGSr) was expressed in Escherichia coli and its role in the regulation of transducin GTPase activity was investigated. As with other RGS proteins, hRGSr interacts preferentially with a transitional conformation of the transducin alpha-subunit, while its binding to GtalphaGTPgammaS or GtalphaGDP is weak. hRGSr and Pgamma do not compete for the interaction with the transducin alpha-subunit. The affinity of the Pgamma interaction with transducin alpha-subunit is modestly enhanced by the addition of hRGSr. In a single turnover assay, hRGSr accelerates the GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold. Addition of Pgamma to the reconstituted system reduces the GTPase level accelerated by hRGSr. The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr are elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 microM rhodopsin these rates are elevated even further. These data suggest that the effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade (Natochin, 1997).

The rod outer segment phototransduction GAP (GTPase-accelerating protein) has been identified as RGS9, a member of the RGS family of G alpha GAPs. RGS9 mRNA expression is specific for photoreceptor cells, and RGS9 protein colocalizes with other phototransduction components to photoreceptor outer segment membranes. The RGS domain of RGS9 accelerates GTP hydrolysis by the visual G protein transducin, and this acceleration is enhanced by the gamma subunit of the phototransduction effector cGMP phosphodiesterase. These unique properties of RGS9 match those of the rod outer segment GAP and implicate it as a key element in the recovery phase of visual transduction (He, 1998).

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein alpha subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein beta subunit (Gbeta5L). RGS9 and Gbeta5L also form a complex when coexpressed in cell culture. These data are consistent with the recent observation that several RGS proteins, including RGS9, contain a G protein gamma-subunit like domain that can mediate RGS protein association with Gbeta5. An example of such a complex is reported whose cellular localization and function are clearly defined (Makino, 1999).

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Another unique property of RGS9 is reported: high specificity for Galphat. The core (RGS) domain of RGS9 stimulates Galphat GTPase activity 10-fold and Galphai1 GTPase activity only 2-fold at a concentration of 10 muM. Using chimeric Galphat/Galphai1 subunits it has been demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 are well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments are 185 nM and 2 muM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. This is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9 (Skiba, 1999).

Serpentine Galphai-linked receptors support rapid adhesion and directed migration of leukocytes and other cell types. The intracellular mechanisms mediating and regulating chemoattractant-directed adhesion and locomotion are only now beginning to be explored. Little is known about the GTPase activity of the Galphai proteins involved in adhesion and chemotaxis, or the significance of their regulation to these responses. Using transiently transfected lymphoid cells as a model system, it has been shown that expression of RGS1, RGS3, and RGS4 inhibits chemoattractant-induced migration. In contrast, RGS2, a regulator of Galphaq activity, has no effect on cell migration to any chemoattractant. RGS1, RGS3, and RGS4 also reduce rapid chemoattractant-triggered adhesion, although the proadhesive response appears quantitatively less sensitive to RGS action than chemotaxis. The results suggest that the duration of the Galphai signal may be a particularly important parameter in the chemotactic responses of leukocytes, and demonstrate the potential for RGS family members to regulate cellular adhesive and migratory behaviors (Bowman, 1998).

The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. An IL-2-induced gene was cloned from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Gialpha and Gqalpha proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation (Beadling, 1999).

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. The GTPase specificity of RGS12 is described and four alternatively spliced forms of human RGS12 mRNA are identified. 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 (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 GTPase-activating protein for Gi class alpha 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 (Snow, 1998a).

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. The cloning and characterization of another novel cDNA isolated from that screen is reported. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library has identified the full length D-AKAP2. It is composed of 372 amino acids, which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein, thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase (L. Huang, 1997).

Identification of a new family of proteins (RGS proteins) that function as negative regulators of G protein signaling has sparked new understanding of desensitization of this signaling process. Recent studies with several mammalian RGS proteins has delineated their ability to interact with and function as GTPase-activating proteins specifically for G proteins in the Gi family. The functional activity of RGS3 and a truncated form of RGS3 were investaged on G protein-coupled receptor-mediated activation of adenylyl cyclase, phosphoinositide phospholipase C, and mitogen-activated protein kinase in intact cells. Polymerase chain reaction and 5'-rapid amplification of cDNA ends analyses reveals the tissue-specific expression of a short form of the RGS3 transcript that encodes the approximate carboxyl-terminal half of RGS3. This truncated form of RGS3 (RGS3T) has been shown to function as a negative regulator of pheromone signaling in yeast. Baby hamster kidney cells transiently transfected with RGS3T cDNA exhibit a pronounced impairment in platelet-activating factor receptor-stimulated inositol phosphate production, a pertussis toxin-insensitive response. Similarly, calcitonin gene-related peptide receptor-stimulated increases in intracellular cAMP and pituitary adenylate-cyclase activating polypeptide receptor-stimulated increases in both cAMP and inositol phosphates are reduced significantly in RGS3T transfectants compared with vector-transfected control cells. In contrast, baby hamster kidney cells transfected with the full-length RGS3 cDNA show no impairment in cAMP and inositol phosphate production mediated by these G protein-coupled receptors. However, lysophosphatidic acid receptor-stimulated phosphorylation of endogenous ERK1 and ERK2 is impaired markedly in both RGS3 and RGS3T transfectants, demonstrating the functional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidence for regulatory effects of an RGS protein on Gs- and Gq-mediated signaling in intact cells and document that the carboxyl-terminal region of RGS3 comprises the structural domain for this activity (Chatterjee, 1997).

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. A novel p53 target gene (A28-RGS14) is reported that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways (Buckbinder, 1997).

RGS14 possesses an N-terminal RGS domain, two Raf-like Ras-binding domains, and a GoLoco motif, which has GDP dissociation inhibitor activity. This study shows that unique among the known mammalian RGS proteins, RGS14 localizes in centrosomes. Its first Ras-binding domain is sufficient to target RGS14 to centrosomes. RGS14 also shuttles between the cytoplasm and nucleus, and its nuclear export depends on the CRM-1 nuclear export receptor. Mutation of a nuclear export signal or treatment with leptomycin B causes nuclear accumulation of RGS14 and its association with promyelocytic leukemia protein nuclear bodies. Furthermore, a point mutant defective in nuclear export fails to target to centrosomes, suggesting that nuclear cytoplasmic shuttling is necessary for its proper localization. Mild heat stress, but not proteotoxic or transcription-linked stresses, re-localizes the RGS14 from the cytoplasm to promyelocytic leukemia nuclear bodies. Expression of RGS14, but not point mutants that disrupt the functional activity of its RGS domain or GoLoco motif, enhances the reporter gene activity. The multifunctional domains and the dynamic subcellular localization of RGS14 implicate it in a diverse set of cellular processes including centrosome and nuclear functions and stress-induced signaling pathways.