The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.     

The mechanism of membrane-translocation of regulator of G-protein signaling (RGS) 8 induced by Galpha expression

RGS (regulators of G-protein signaling) proteins comprise a large family that modulates heterotrimeric G-protein signaling. This protein family has a common RGS domain and functions as GTPase-activating proteins for the alpha-subunits of heterotrimeric G-proteins located at the plasma membrane. RGS8 was identified as a neuron-specific RGS protein, which belongs to the B/R4 subfamily. We previously showed that RGS8 protein was translocated to the plasma membrane from the nucleus on coexpression of GTPase-deficient Galphao (GalphaoQL). Here, we first examined which subtypes of Galpha can induce the translocation of RGS8. When the Galphai family was expressed, the translocation of RGS8 did occur. To investigate the mechanism of this translocation, we generated a mutant RGS8 with reduced affinity to Galphao and an RGS-insensitive (RGS-i) mutant of GalphaoQL. Co-expression experiments with both mutants revealed that disruption of the Galpha-RGS8 interaction abolished the membrane-translocation of RGS8 despite the apparent membrane localization of RGS-i GalphaoQL. These results demonstrated that RGS8 is recruited to the plasma membrane where G-proteins are activated mainly by direct association with Galpha.     

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

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.     

Mammalian RGS proteins: multifunctional regulators of cellular signalling

Regulators of G-protein signalling (RGS) proteins are a large and diverse family initially identified as GTPase activating proteins (GAPs) of heterotrimeric G-protein Galpha-subunits. At least some can also influence Galpha activity through either effector antagonism or by acting as guanine nucleotide dissociation inhibitors (GDIs). As our understanding of RGS protein structure and function has developed, so has the realisation that they play roles beyond G-protein regulation. Such diversity of function is enabled by the variety of RGS protein structure and their ability to interact with other cellular molecules including phospholipids, receptors, effectors and scaffolds. The activity, sub-cellular distribution and expression levels of RGS proteins are dynamically regulated, providing a layer of complexity that has yet to be fully elucidated.     

Structure of the p115RhoGEF rgRGS domain-Galpha13/i1 chimera complex suggests convergent evolution of a GTPase activator

p115RhoGEF, a guanine nucleotide exchange factor (GEF) for Rho GTPase, is also a GTPase-activating protein (GAP) for G12 and G13 heterotrimeric Galpha subunits. The GAP function of p115RhoGEF resides within the N-terminal region of p115RhoGEF (the rgRGS domain), which includes a module that is structurally similar to RGS (regulators of G-protein signaling) domains. We present here the crystal structure of the rgRGS domain of p115RhoGEF in complex with a chimera of Galpha13 and Galphai1. Two distinct surfaces of rgRGS interact with Galpha. The N-terminal betaN-alphaN hairpin of rgRGS, rather than its RGS module, forms intimate contacts with the catalytic site of Galpha. The interface between the RGS module of rgRGS and Galpha is similar to that of a Galpha-effector complex, suggesting a role for the rgRGS domain in the stimulation of the GEF activity of p115RhoGEF by Galpha13.     

The RGS14 GoLoco domain discriminates among Galphai isoforms

Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its RGS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galphai1, but not Galphao. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galphai1 helical domain and the GoLoco C terminus (Kimple, R. J., Kimple, M. E., Betts, L., Sondek, J., and Siderovski, D. P. (2002) Nature 416, 878-881). Three isoforms of Galphai exist in mammalian cells. In this study, we tested whether all three isoforms were subject to RGS14 GDI activity. We found that RGS14 inhibits guanine nucleotide exchange on Galphai1 and Galphai3 could, but not Galphai2. Galphai2 be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, we found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galphai selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galphai1 and Galphai3 as the likely targets of RGS14-GoLoco regulation in vivo.     

RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain

Members of the regulators of G protein signaling (RGS) family modulate Galpha-directed signals as a result of the GTPase-activating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To define the cellular and biochemical properties of RGS14 we utilized two different affinity purified antisera that specifically recognize recombinant and native RGS14. In brain, we observed two RGS14-like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent-extractable and the other detergent-insensitive. Recombinant RGS14 binds specifically to activated Galphai/o, but not Galphaq/11, Galpha12/13, or Galphas in brain membranes. In reconstitution studies, we found that RGS14 is a non-selective GAP for Galphai1 and Galphao and that full-length RGS14 is an approximately 10-fold more potent stimulator of Galpha GTPase activity than the RGS domain alone. In contrast, neither full-length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Galphai but not Galphao, and this activity is restricted to the C-terminus containing the GoLoco domain. These findings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the first examples of an RGS protein that is a bifunctional regulator of Galpha actions.     

Novel activity of RGS14 on Goalpha and Gialpha nucleotide binding and hydrolysis distinct from its RGS domain and GDI activity

The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for Gialpha and Goalphaand a guanine nucleotide dissociation inhibitor (GDI) for Gialpha. This GDI activity is isolated to a region of the protein distinct from the RGS domain that contains an additional G protein-binding domain (RBD/GL). Here, we report that RGS14 missing its RGS domain (R14-RBD/GL) binds directly to Go and Gi to modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. In brain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14) bind Goalpha-GDP, Gialpha-GDP, and also Gbetagamma. When reconstituted with M2 muscarinic receptors (M2) plus either Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulates the steady-state GTPase activities of both G proteins, whereas R14-RBD/GL has little or no effect. R14-RBD/GL potentiates RGS4 GAP activity in membrane-based assays by increasing the apparent affinity of RGS4 for Gialpha and Goalpha, suggesting a cooperative interaction between the RBD/GL domain, RGS4, and Galpha. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assays with purified Gialpha or Goalpha, suggesting that membranes and/or receptors are required for this activity. When these findings are taken together, they indicate that regions of RGS14 outside of the RGS domain can bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Galphanucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDI activity.     

Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling

To define receptor subdomains important for protein interaction and identify components of novel signal transduction complexes for the mu- and delta-opioid receptors (mu-OR, delta-OR), we generated glutathione S-transferase fusion proteins of the carboxyl-termini of the mu-opioid receptor (mu-CT), the delta- (delta-CT), and the third intracellular loop of the delta-opioid receptor (delta-i3L) to search for interactive proteins. Results from pull down experiments have demonstrated for the first time that Gbetagamma complexes, derived from the heterotrimeric Galphatbeta1gamma1, purified Gbeta1gamma1, or Gbeta endogenously present in cell lysates and rat striatal extracts, interact with all mu- and delta-opioid receptor subdomains. On the other hand, the C-terminal peptides of the delta- and the mu-ORs exhibit differential profiles for Galpha subunit binding. Indeed, while mu-CT was unable to bind any form of Galpha, both the delta-CT and the delta-i3L displayed interactive regions for heterotrimeric Galphatbeta1gamma1, inactive Galpha(GDP) and active Galpha(GTPgammaS). Regulators of G protein signaling (RGS) proteins are another class of proteins that can modulate G protein signaling events. We demonstrate for the first time that RGS4 directly interacts with the mu-CT, the delta-CT as well as delta-i3L in a dose dependent manner. Moreover, RGS4 modulates mu-OR signaling and can form stable heterotrimeric complexes with the activated Galpha. Collectively, our data demonstrate that the C-termini of the mu- and delta-ORs provide direct physical scaffolding in which G protein subunits and RGS4 protein can be bound.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction

A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.     

Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.     

New roles for Galpha and RGS proteins: communication continues despite pulling sisters apart

Large G protein alpha subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Galpha subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein-protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Galpha subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

Regulators of G protein signaling 6 and 7. Purification of complexes with gbeta5 and assessment of their effects on g protein-mediated signaling pathways.

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o).     

RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans

Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. We found that mutating a regulator of G protein signaling, RGS-7, resulted in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galphao-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, we found that RIC-8 stimulates GTP binding by Galphao and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galphao, demonstrating that Galphao passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector.     

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。     

Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of Gi- and Gq-mediated signaling.

RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein alpha subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Gialpha. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Galpha subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Galpha subunits on cellular membranes.