Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.     

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.     

RGS proteins: more than just GAPs for heterotrimeric G proteins

Members of the newly described RGS family of proteins have a common RGS domain that contains GTPase-activating activity for many Galpha subunits of heterotrimeric G proteins. Their ability to dampen signalling via Galphai-, Galphaq- and Galpha12/13-coupled pathways makes them crucial players in mediating the multitude of cellular processes controlled by heterotrimeric G proteins. Some RGS proteins also contain additional motifs that link them to other signalling networks, where they constitute effector-type molecules. This review summarizes recent findings on RGS proteins, especially those that implicate RGS proteins in more than just enhancing the GTPase activity of their Galpha subunit targets.     

An N-terminal region of Caenorhabditis elegans RGS proteins EGL-10 and EAT-16 directs inhibition of G(alpha)o versus G(alpha)q signaling

Regulator of G protein signaling (RGS) proteins contain an RGS domain that inhibits G(alpha) signaling by activating G(alpha) GTPase activity. Certain RGS proteins also contain a Ggamma-like (GGL) domain and a poorly characterized but conserved N-terminal region. We assessed the functions of these subregions in the Caenorhabditis elegans RGS proteins EGL-10 and EAT-16, which selectively inhibit GOA-1 (G(alpha)(o)) and EGL-30 (G(alpha)(q)), respectively. Using transgenes in C. elegans, we expressed EGL-10, EAT-16, their subregions, or EGL-10/EAT-16 chimeras. The chimeras showed that the GGL/RGS region of either protein can act on either GOA-1 or EGL-30 and that a key factor determining G(alpha) target selectivity is the manner in which the N-terminal and GGL/RGS regions are linked. We also found that coexpressing N-terminal and GGL/RGS fragments of EGL-10 gave full EGL-10 activity, whereas either fragment alone gave little activity. Biochemical analysis showed that coexpressing the two fragments caused both to increase in abundance and also caused the GGL/RGS fragment to move to the membrane, where the N-terminal fragment is localized. By coimmunoprecipitation, we found that the N-terminal fragment complexes with the C-terminal fragment and its associated Gbeta subunit, GPB-2. We conclude that the N-terminal region directs inhibition of G(alpha) signaling by forming a complex with the GGL/RGS region and affecting its stability, membrane localization, and G(alpha) target specificity.     

RGS3 and RGS4 differentially associate with G protein-coupled receptor-Kir3 channel signaling complexes revealing two modes of RGS modulation. Precoupling and collision coupling

RGS3 and RGS4 are GTPase-activating proteins expressed in the brain and heart that accelerate the termination of G(i/o)- and G(q)-mediated signaling. We report here the determinants mediating selective association of RGS4 with several G protein-coupled receptors (GPCRs) that form macromolecular complexes with neuronal G protein-gated inwardly rectifying potassium (Kir3 or GIRK) channels. Kir3 channels are instrumental in regulating neuronal firing in the central and peripheral nervous system and pacemaker activity in the heart. By using an epitope-tagged degradation-resistant RGS4 mutant, RGS4(C2V), immunoprecipitation of several hemagglutinin-tagged G(i/o)-coupled and G(q)-coupled receptors expressed in Chinese hamster ovary (CHO-K1) cells readily co-precipitated both Kir3.1/Kir3.2a channels and RGS4(C2V). In contrast to RGS4(C2V), the closely related and functionally active RGS3 "short" isoform (RGS3s) did not interact with any of the GPCR-Kir3 channel complexes examined. Deletion and chimeric RGS constructs indicate both the N-terminal domain and the RGS domain of RGS4(C2V) are necessary for association with m2 receptor-Kir3.1/Kir3.2a channel complexes, where the GPCR was found to be the major target for RGS4(C2V) interaction. The functional impact of RGS4(C2V) "precoupling" to the GPCR-Kir3 channel complex versus RGS3s "collision coupling" was a 100-fold greater potency in the acceleration of G protein-dependent Kir3 channel-gating kinetics with no attenuation in current amplitude. These findings demonstrate that RGS4, a highly regulated modulator and susceptibility gene for schizophrenia, can directly associate with multiple GPCR-Kir3 channel complexes and may affect a wide range of neurotransmitter-mediated inhibitory and excitatory events in the nervous and cardiovascular systems.     

Assembly and trafficking of heterotrimeric G proteins

To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This current topic will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.     

RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.     

Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.     

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.     

Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K(+) channel and control regulation of RGS7 by G beta 5

Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K(+) channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit G beta 5, is regulated by G beta 5 by two distinct mechanisms. First, G beta 5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase-activating protein activity.     

Non-canonical functions of RGS proteins

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.     

Phosphorylation of RGS14 by protein kinase A potentiates its activity toward G alpha i

Regulators of G protein signaling (RGS proteins) modulate Galpha-directed signals because of the GTPase activating protein (GAP) activity of their conserved RGS domain. RGS14 and RGS12 are unique among RGS proteins in that they also regulate Galpha(i) signals because of the guanine nucleotide dissociation inhibitor (GDI) activity of a GoLoco motif near their carboxy-termini. Little is known about cellular regulation of RGS proteins, although several are phosphorylated in response to G-protein directed signals. Here we show for the first time the phosphorylation of native and recombinant RGS14 in host cells. Direct stimulation of adenylyl cyclase or introduction of dibutyryl-cAMP induces phosphorylation of RGS14 in cells. This phosphorylation occurs through activation of cAMP-dependent protein kinase (PKA) since phosphate incorporation is completely blocked by a selective inhibitor of PKA but only partially or not at all blocked by inhibitors of other G-protein regulated kinases. We show that purified PKA phosphorylates two specific sites on recombinant RGS14, one of which, threonine 494 (Thr494), is immediately adjacent to the GoLoco motif. Because of this proximity, we focused on the possible effects of PKA phosphorylation on the GDI activity of RGS14. We found that mimicking phosphorylation on Thr494 enhanced the GDI activity of RGS14 toward Galpha(i) nearly 3-fold, with no associated effect on the GAP activity toward either Galpha(i) or Galpha(o). These findings implicate cAMP-induced phosphorylation as an important modulator of RGS14 function since phosphorylation could enhance RGS14 binding to Galpha(i)-GDP, thereby limiting Galpha(i) interactions with downstream effector(s) and/or enhancing Gbetagamma-dependent signals.     

Two G12 family G proteins, G alpha12 and G alpha13, show different subcellular localization

The G alpha subunits of the G12 family of heterotrimeric G proteins, G alpha12 and G alpha13, are closely related in sequences and some effectors, but they often act through different pathways or bind to different proteins. We have examined subcellular distribution of these two G proteins and found that endogenous G alpha12 and G alpha13 localize in membrane and cytoplasmic fractions, respectively. Exogenously expressed G alpha12 and G alpha13 also localize in membrane and cytoplasmic fractions, respectively, in COS-7 cells. Stimulation of lysophosphatidic acid receptor coupled to G alpha13 markedly promotes the translocation of G alpha13 from cytoplasm to membrane. This different localization of G alpha12 and G alpha13 may explain some of the nonoverlapping actions of G alpha12 and G alpha13.     

Purification and characterization of subforms of the guanine-nucleotide-binding proteins G alpha i and G alpha o

Five different pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G proteins) were purified from bovine brain. Immunochemical characterization of alpha subunits identified two G alpha o proteins (G alpha o-I and G alpha o-II), two 41-kDa G alpha i proteins (G alpha i-I and G alpha i-II) and the 40-kDa G alpha i2 protein. Site-directed antisera specific for G alpha o proteins did not differentiate between G alpha o-I and G alpha o-II. However, in situ peptide mapping using polyacrylamide gel electrophoresis revealed distinct cleavage products with different proteases for each of these proteins. Additionally comparison of Rf values demonstrated a slightly faster migration for G alpha o-II than for G alpha o-I, which is the only type of G alpha o protein present in cell membranes of the neuroblastoma/glioma cell line NG 108-15. The importance of these structural differences and possible functional implications are discussed.     

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.     

The Ras‐binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins

RGS14 is a 60 kDa protein that contains a regulator of G protein signaling (RGS) domain near its N‐terminus, a central region containing a pair of tandem Ras‐binding domains (RBD), and a GPSM (G protein signaling modulator) domain (a.k.a. Gi/o‐Loco binding [GoLoco] motif) near its C‐terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full‐length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter‐domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR‐activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation. J. Cell. Biochem. 114: 1414–1423, 2013. © 2012 Wiley Periodicals, Inc.     

RGS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.     

The regulator of G protein signaling RGS4 selectively enhances alpha 2A-adreoreceptor stimulation of the GTPase activity of Go1alpha and Gi2alpha

Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Galpha(i1) and Galpha(i3) but markedly enhanced agonist-stimulation of the proteins containing Galpha(i2) and Galpha(o1.) The effect of RGS4 on the alpha(2A)-adrenoreceptor-Galpha(o1) fusion protein was concentration-dependent with EC(50) of 30 +/- 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-Galpha(o1) or alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-Galpha(o1) and alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V(max) and significantly increased K(m) of the fusion proteins for GTP. The increase in K(m) for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for alpha(2A)-adrenoreceptor-Galpha(o1) than for alpha(2A)-adrenoreceptor-Galpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.