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.     

Differential interaction of GRK2 with members of the G alpha q family

Regulators of G protein signaling (RGS) proteins bind to active G alpha subunits and accelerate the rate of GTP hydrolysis and/or block interaction with effector molecules, thereby decreasing signal duration and strength. RGS proteins are defined by the presence of a conserved 120-residue region termed the RGS domain. Recently, it was shown that the G protein-coupled receptor kinase 2 (GRK2) contains an RGS domain that binds to the active form of G alpha(q). Here, the ability of GRK2 to interact with other members of the G alpha(q) family, G alpha(11), G alpha(14), and G alpha(16), was tested. The signaling of all members of the G alpha(q) family, with the exception of G alpha(16), was inhibited by GRK2. Immunoprecipitation of full-length GRK2 or pull down of GST-GRK2-(45-178) resulted in the detection of G alpha(q), but not G alpha(16), in an activation-dependent manner. Moreover, activated G alpha(16) failed to promote plasma membrane (PM) recruitment of a GRK2-(45-178)-GFP fusion protein. Assays with chimeric G alpha(q)(-)(16) subunits indicated that the C-terminus of G alpha(q) mediates binding to GRK2. Despite showing no interaction with GRK2, G alpha(16) does interact with RGS2, in both inositol phosphate and PM recruitment assays. Thus, GRK2 is the first identified RGS protein that discriminates between members of the G alpha(q) family, while another RGS protein, RGS2, binds to both G alpha(q) and G alpha(16).     

RGS11 interacts preferentially with R7BP over Gαoa - Characterization of Gβ5-free RGS11

Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Gγ-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gβ5-containing complexes that exhibit a number of unique functional properties. However, presence of Gβ5 reduced the affinity of R7-RGS7 for Gα subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gβ5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gβ5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Gα_oa. rRGS11 was capable of interacting with Gα_oa and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (K_D) of 904 (±208) nM, and 308 (±97) nM, respectively. It also induced several-fold increase in the GTPase activity of Gα_oa. The binding of rRGS11 was differential with a binding preference for R7BP over Gα_oa implying extended roles of R7BP. In addition, we identified a novel interaction between Gα_oa and R7BP with a K_D of 592 (±150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.     

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

Polarity exchange at the interface of regulators of G protein signaling with G protein alpha-subunits

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha(t) and Galpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha(i1), this lysine was changed to glutamate for compensation. Galpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(i)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha(i1) and Galpha(i1)K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. Galpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha(i1). Analogous mutants of Galpha(q) (Galpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha(i) and Galpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha-RGS specificities in living cells.     

Functional and structural diversity of the human Dickkopf gene family

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.     

Interaction of 14-3-3 protein with regulator of G protein signaling 7 is dynamically regulated by tumor necrosis factor-alpha

Regulators of G protein signaling (RGS) constitute a family of proteins with a conserved RGS domain of approximately 120 amino acids that accelerate the intrinsic GTP hydrolysis of activated Galpha(i) and Galpha(q) subunits. The phosphorylation-dependent interaction of 14-3-3 proteins with a subset of RGS proteins inhibits their GTPase-accelerating activity in vitro. The inhibitory interaction between 14-3-3 and RGS7 requires phosphorylation of serine 434 of RGS7. We now show that phosphorylation of serine 434 is dynamically regulated by TNF-alpha. Cellular stimulation by TNF-alpha transiently decreased the phosphorylation of serine 434 of RGS7, abrogating the inhibitory interaction with 14-3-3. We examined the effect of 14-3-3 on RGS-mediated deactivation kinetics of G protein-coupled inwardly rectifying K(+) channels (GIRKs) in Xenopus oocytes. 14-3-3 inhibited the function of wild-type RGS7, but not that of either RSG7(P436R) or RGS4, two proteins that do not bind 14-3-3. Our findings are the first evidence that extracellular signals can modulate the activity of RGS proteins by regulating their interaction with 14-3-3.     

Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1

Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cbeta (PLC-beta) signaling cascade which may allow for rapid deactivation of intracellular Ca(2+) release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-beta, G-betagamma, and both deactivated (GDP-bound) and activated (GTPgammaS-bound) Galpha(q). We find that RGS4 not only binds to activated Galpha(q), as predicted, but also to Gbetagamma and PLCbeta(1). These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCbeta(1) or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Galpha(q), Gbetagamma and phospholipase Cbeta(1) will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Galpha(q) to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.     

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.     

Spinophilin/neurabin reciprocally regulate signaling intensity by G protein-coupled receptors

Spinophilin (SPL) and neurabin (NRB) are structurally similar scaffolding proteins with several protein binding modules, including actin and PP1 binding motifs and PDZ and coiled-coil domains. SPL also binds regulators of G protein signaling (RGS) proteins and the third intracellular loop (3iL) of G protein-coupled receptors (GPCRs) to reduce the intensity of Ca(2+) signaling by GPCRs. The role of NRB in Ca(2+) signaling is not known. In the present work, we used biochemical and functional assays in model systems and in SPL(-/-) and NRB(-/-) mice to show that SPL and NRB reciprocally regulate Ca(2+) signaling by GPCRs. Thus, SPL and NRB bind all members of the R4 subfamily of RGS proteins tested (RGS1, RGS2, RGS4, RGS16) and GAIP. By contract, SPL, but not NRB, binds the 3iL of the GPCRs alpha(1B)-adrenergic (alpha(1B)AR), dopamine, CCKA, CCKB and the muscarinic M3 receptors. Coexpression of SPL or NRB with the alpha(1B)AR in Xenopus oocytes revealed that SPL reduces, whereas NRB increases, the intensity of Ca(2+) signaling by alpha(1B)AR. Accordingly, deletion of SPL in mice enhanced binding of RGS2 to NRB and Ca(2+) signaling by alphaAR, whereas deletion of NRB enhanced binding of RGS2 to SPL and reduced Ca(2+) signaling by alphaAR. This was due to reciprocal modulation by SPL and NRB of the potency of RGS2 to inhibit Ca(2+) signaling by alphaAR. These findings suggest a novel mechanism of regulation of GPCR-mediated Ca(2+) signaling in which SPL/NRB forms a functional pair of opposing regulators that modulates Ca(2+) signaling intensity by GPCRs by determining the extent of inhibition by the R4 family of RGS proteins.     

Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for alpha subunits of heterotrimeric G proteins (Galpha), thereby accelerating G protein inactivation. RGS proteins can also block Galpha-mediated signal production by competing with downstream effectors for Galpha binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on Galpha(q)-mediated phospholipase Cbeta (PLCbeta) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M3 receptors, which couple to endogenous Gq protein and mediate a stimulatory effect of carbachol on PLCbeta, or constitutively active Galphaq*, which is inert to GTP hydrolysis and activates PLCbeta independent of receptor activation. In M3-expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, Galphaq* -induced PLCbeta activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M3, Galphaq/Galphaq*, PLCbeta, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.     

Receptor-selective effects of endogenous RGS3 and RGS5 to regulate mitogen-activated protein kinase activation in rat vascular smooth muscle cells

Regulators of G protein signaling (RGS) proteins compose a highly diverse protein family best known for inhibition of G protein signaling by enhancing GTP hydrolysis by Galpha subunits. Little is known about the function of endogenous RGS proteins. In this study, we used synthetic ribozymes targeted to RGS2, RGS3, RGS5, and RGS7 to assess their function. After demonstrating the specificity of in vitro cleavage by the RGS ribozymes, rat aorta smooth muscle cells were used for transient transfection with the RGS-specific ribozymes. RGS3 and RGS5 ribozymes differentially enhanced carbachol- and angiotensin II-induced MAP kinase activity, respectively, whereas RGS2 and RGS7 ribozymes had no effect. This enhancement was pertussis toxin-insensitive. Thus RGS3 is a negative modulator of muscarinic m3 receptor signaling, and RGS5 is a negative modulator of angiotensin AT1a receptor signaling through G(q/11). Also, RGS5 ribozyme enhanced angiotensin-stimulated inositol phosphate release. These results indicate the feasibility of using the ribozyme technology to determine the functional role of endogenous RGS proteins in signaling pathways and to define novel receptor-selective roles of endogenous RGS3 and RGS5 in modulating MAP kinase responses to either carbachol or angiotensin.     

G蛋白信号调节因子的结构分类和功能

G蛋白信号调节因子是能够直接与激活的Gα亚基结合,显著刺激Gα亚基上的GTP酶活性,加速GTP水解,从而灭活或终止G蛋白信号的一组分子大小各异的多功能蛋白质家族。它们都共同拥有一个130个氨基酸的保守的RGS结构域,其功能是结合激活的Gα亚基,负调节G蛋白信号。许多RGS蛋白还拥有非RGS结构域,能够结合其它信号蛋白,从而整合和调节G蛋白信号之间以及G蛋白和其它信号系统之间的关系。     

Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.     

Pleiotropic phenotype of a genomic knock-in of an RGS-insensitive G184S Gnai2 allele

Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Galpha(i2) subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Galpha(i2)(G184S) knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Galpha(i2)(+/G184S) mice show some but not all of these abnormalities. Thus, loss of RGS actions at Galpha(i2) produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.     

Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling and adenylyl cyclase activity

Regulator of G protein signaling (RGS) proteins function as GTPase accelerating proteins (GAP) for Galpha subunits, attenuating G-protein-coupled receptor signal transduction. The present study tested the ability of members of different subfamilies of RGS proteins to modulate both G-protein-dependent and -independent signaling in mammalian cells. RGS4, RGS10, and RGSZ1 significantly attenuated Galphai-mediated signaling by 5-HT1A, but not by dopamine D2, receptor-expressing cells. Additionally, RGS4 and RGS10 significantly inhibited forskolin-stimulated cAMP production in both cell lines. In contrast, RGS2, RGS7, and RGSZ1 had no effect on forskolin-stimulated cAMP production in these cells. RGS2 and RGS7 significantly decreased Galphaq-mediated signaling by 5-HT2A receptors, confirming that the RGS4 and RGS10 effects on forskolin-stimulated cAMP production were specific, and not simply due to overexpression. Interestingly, similar expression levels of RGS4 protein resulted in greater inhibition of G-protein-independent cAMP production compared to G-protein-dependent GAP activity. Our results suggest specificity and selectivity of RGS proteins on G-protein-dependent and -independent signaling in mammalian cells.     

RGS7 and RGS8 differentially accelerate G protein-mediated modulation of K+ currents

The recently discovered family of RGS (regulators of G protein signaling) proteins acts as GTPase activating proteins which bind to alpha subunits of heterotrimeric G proteins. We previously showed that a brain-specific RGS, RGS8 speeds up the activation and deactivation kinetics of the G protein-coupled inward rectifier K+ channel (GIRK) upon receptor stimulation (Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529). Here we report the isolation of a full-length rat cDNA of another brain-specific RGS, RGS7. In situ hybridization study revealed that RGS7 mRNA is predominantly expressed in Golgi cells within granule cell layer of cerebellar cortex. We observed that RGS7 recombinant protein binds preferentially to Galphao, Galphai3, and Galphaz. When co-expressed with GIRK1/2 in Xenopus oocytes, RGS7 and RGS8 differentially accelerate G protein-mediated modulation of GIRK. RGS7 clearly accelerated activation of GIRK current similarly with RGS8 but the acceleration effect of deactivation was significantly weaker than that of RGS8. These acceleration properties of RGS proteins may play important roles in the rapid regulation of neuronal excitability and the cellular responses to short-lived stimulations.     

Whither Goest the RGS Proteins?

Studies of the desensitization of G protein-coupled signal transduction have led to the discovery of a family of guanosine triphosphatase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits — the “regulator of G protein signaling” or RGS proteins. In considering both documented and potential functions of several RGS protein family members with demonstrable multidomain compositions (p115RhoGEF, PDZRhoGEF, Axin, Axil/Conductin, D-AKAP2, the G protein-coupled receptor kinases [GRKs], the DEP/GGL/RGS subfamily [RGS6, RGS7, RGS9, RGS11], and RGS12), this review explores the shift in our appreciation of the RGS proteins from unidimensional desensitizing agents to multifocal signal transduction regulators.