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.     

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.     

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.     

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.     

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.     

Engineering a V2 Vasopressin Receptor Agonist- and Regulator of G-Protein-Signaling-Sensitive G Protein

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to G(s)alpha. Furthermore, G(s)alpha is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V(2) receptor with a series of chimeric G protein alpha subunits in which the C-terminal 6-12 amino acids of G(i1)alpha were replaced with the equivalent sequence of G(s)alpha allowed robust vasopressin-stimulated [(35)S]GTPgammaS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V(2) receptor and either G(s)alpha or G(i1)alpha. However, it produced a concentration-dependent stimulation of V(max) for a V(2) receptor-G(i1)alpha/Gs6alpha fusion protein. This construct bound [(3)H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V(2) receptor. RGS1 enhanced vasopressin stimulation of V(2) receptor-G(i1)alpha/G(s)6alpha in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both V(max) of the GTPase activity and the observed K(m) for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V(2) receptor agonist ligands and may be amenable to many other G(s)alpha-coupled receptors.     

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.     

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.     

Characterization of RGS5 in regulation of G protein-coupled receptor signaling

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.     

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.     

eat-11 encodes GPB-2, a Gbeta(5) ortholog that interacts with G(o)alpha and G(q)alpha to regulate C. elegans behavior

In C. elegans, a G(o)/G(q) signaling network regulates locomotion and egg laying [1-8]. Genetic analysis shows that activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is suppressed by perturbations of this network, which include loss of the GOA-1 G(o)alpha, DGK-1 diacylglycerol kinase, EAT-16 G protein gamma subunit-like (GGL)-containing RGS protein, or an unidentified protein encoded by the gene eat-11 [9]. We cloned eat-11 and report that it encodes the Gbeta(5) ortholog GPB-2. Gbeta(5) binds specifically to GGL-containing RGS proteins, and the Gbeta(5)/RGS complex can promote the GTP-hydrolyzing activity of Galpha subunits [10, 11]. However, little is known about how this interaction affects G protein signaling in vivo. In addition to EAT-16, the GGL-containing RGS protein EGL-10 participates in G(o)/G(q) signaling; EGL-10 appears to act as an RGS for the GOA-1 G(o)alpha, while EAT-16 appears to act as an RGS for the EGL-30 G(q)alpha [4, 5]. We have combined behavioral, electrophysiological, and pharmacological approaches to show that GPB-2 is a central member of the G(o)/G(q) network and that GPB-2 may interact with both the EGL-10 and EAT-16 RGS proteins to mediate the opposing activities of G(o)alpha and G(q)alpha. These interactions provide a mechanism for the modulation of behavior by antagonistic G protein networks.     

Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.     

The human delta opioid receptor activates G(i1)alpha more efficiently than G(o1)alpha

To assess the relative capacity of the human delta opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the alpha-subunits of either G(i1) or G(o1), containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [(3)H]naltrindole with high affinity. D-ala(2),D-leu(5) Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by D-ala(2),D-leu(5) enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to D-ala(2),D-leu(5) enkephalin was more than three times greater for G(i1)alpha than for G(o1)alpha. As the effect of agonist in both cases was to increase V:(max) without increasing the observed K:(m) for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G(i1)alpha compared with G(o1)alpha. An equivalent fusion protein between the human mu opioid receptor-1 and G(i1)alpha produced a similar D-ala(2),D-leu(5) enkephalin-induced GTP turnover number as the delta opioid receptor-G(i1)alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G(i1)alpha.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

RGS2 is upregulated by and attenuates the hypertrophic effect of alpha1-adrenergic activation in cultured ventricular myocytes

Regulator of G protein signaling (RGS) proteins counter the effects of G protein-coupled receptors (GPCRs) by limiting the abilities of G proteins to propagate signals, although little is known concerning their role in cardiac pathophysiology. We investigated the potential role of RGS proteins on alpha1-adrenergic receptor signals associated with hypertrophy in primary cultures of neonatal rat cardiomyocytes. Levels of mRNA encoding RGS proteins 1-5 were examined, and the alpha1-adrenergic agonist phenylephrine (PE) significantly increased RGS2 gene expression but had little or no effect on the others. The greatest changes in RGS2 mRNA occurred within the first hour of agonist addition. We next investigated the effects of RGS2 overexpression produced by infecting cells with an adenovirus encoding RGS2-cDNA on cardiomyocyte responses to PE. As expected, PE increased cardiomyocyte size and also significantly upregulated alpha-skeletal actin and ANP expression, the markers of hypertrophy, as well as the Na-H exchanger 1 isoform. These effects were blocked in cells infected with the adenovirus expressing RGS2. We also examined hypertrophy-associated MAP kinase pathways, and RGS2 overexpression completely prevented the activation of ERK by PE. In contrast, the activation of both JNK and p38 unexpectedly were increased by RGS2, although the ability of PE to further activate the p38 pathway was reduced. These results indicate that RGS2 is an important negative-regulatory factor in cardiac hypertrophy produced by alpha1-adrenergic receptor stimulation through complex mechanisms involving the modulation of mitogen-activated protein kinase signaling pathways.     

Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.     

RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity

Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma-like (GGL) domain that mediates dimeric interaction with Gbeta(5). Gbeta(5)/R7 dimers stimulated steady state GTPase activity of Galpha-subunits of the G(i) family, but not of Galpha(q) or Galpha(11), when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta(5)/R7 proteins revealed differences in potencies and efficacies toward Galpha-subunits of the G(i) family. Although all four Gbeta(5)/R7 proteins exhibited similar potencies toward Galpha(o), Gbeta(5)/RGS9 and Gbeta(5)/RGS11 were more potent GAPs of Galpha(i1), Galpha(i2), and Galpha(i3) than were Gbeta(5)/RGS6 and Gbeta(5)/RGS7. The maximal GAP activity exhibited by Gbeta(5)/RGS11 was 2- to 4-fold higher than that of Gbeta(5)/RGS7 and Gbeta(5)/RGS9, with Gbeta(5)/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta(5)/RGS7 and Gbeta(5)/RGS9 inhibited Gbeta(5)/RGS11-stimulated GTPase activity of Galpha(o). Therefore, R7 family RGS proteins are G(i) family-selective GAPs with potentially important differences in activities.     

Modulation of the affinity and selectivity of RGS protein interaction with G alpha subunits by a conserved asparagine/serine residue.

The crystal structure of the complex between a G protein alpha subunit (Gi alpha 1) and its GTPase-activating protein (RGS4) demonstrated that RGS4 acts predominantly by stabilization of the transition state for GTP hydrolysis [Tesmer, J. J., et al. (1997) Cell 89, 251]. However, attention was called to a conserved Asn residue (Asn128) that could play a catalytic role by interacting, directly or indirectly, with the hydrolytic water molecule. We have analyzed the effects of several disparate substitutions for Asn128 on the GAP activity of RGS4 toward four G alpha substrates (Go, Gi, Gq, and Gz) using two assay formats. The results substantiate the importance of this residue but indicate that it is largely involved in substrate binding and that its function may vary with different G alpha targets. Various mutations decreased the apparent affinity of RGS4 for substrate G alpha proteins by several orders of magnitude, but had variable and modest effects on maximal rates of GTP hydrolysis when tested with different G alpha subunits. One mutation, N128F, that differentially decreased the GAP activity toward G alpha i compared with that toward G alpha q could be partially suppressed by mutation of the nearby residue in G alpha i to that found in G alpha q (K180P). Detection of GAP activities of the mutants was enhanced in sensitivity up to 100-fold by assay at steady state in proteoliposomes that contain heterotrimeric G protein and receptor.