Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.     

Activation of RGS9-1GTPase acceleration by its membrane anchor,R9AP

The GTPase-accelerating protein (GAP) complex RGS9-1.G beta(5) plays an important role in the kinetics of light responses by accelerating the GTP hydrolysis of G alpha(t) in vertebrate photoreceptors. Much, but not all, of this complex is tethered to disk membranes by the transmembrane protein R9AP. To determine the effect of the R9AP membrane complex on GAP activity, we purified recombinant R9AP and reconstituted it into lipid vesicles along with the photon receptor rhodopsin. Full-length RGS9-1.G beta(5) bound to R9AP-containing vesicles with high affinity (K(d) < 10 nm), but constructs lacking the DEP (dishevelled/EGL-10/pleckstrin) domain bound with much lower affinity, and binding of those lacking the entire N-terminal domain (i.e. the dishevelled/EGL-10/pleckstrin domain plus intervening domain) was not detectable. Formation of the membrane-bound complex with R9AP increased RGS9-1 GAP activity by a factor of 4. Vesicle titrations revealed that on the time scale of phototransduction, the entire reaction sequence from GTP uptake to GAP-catalyzed hydrolysis is a membrane-delimited process, and exchange of G alpha(t) between membrane surfaces is much slower than hydrolysis. Because in rod cells different pools exist of RGS9-1.G beta(5) that are either associated with R9AP or not, regulation of the association between R9AP and RGS9-1.G beta(5) represents a potential mechanism for the regulation of recovery kinetics.     

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.     

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.     

Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity

Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G(i) and/or G(q) class G protein alpha subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP(3) but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP(3). Calmodulin binds all RGS proteins we tested in a Ca(2+)-dependent manner but does not directly affect GAP activity. Indeed, Ca(2+)/calmodulin binds a complex of RGS4 and a transition state analog of Galpha(i1)-GDP-AlF(4)(-). Ca(2+)/calmodulin reverses PIP(3)-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca(2+)/calmodulin competition with PIP(3) may provide an intracellular mechanism for feedback regulation of Ca(2+) signaling evoked by G protein-coupled agonists.     

The Mutation in the N-Terminal Domain of RGS4 Disrupts PA-Conferred Inhibitory Effect on GAP Activity

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for G protein alpha-subunits and are thought to be responsible for rapid deactivation of G protein mediated signaling pathway. In this present study, we demonstrate that PA is the most efficient candidate to inhibit GAP activity of RGS4. The functional significance of N-terminus of RGS4 in respose to PA-granted inhibition on GAP activity has been studied with the site mutation in the N-terminus of RGS4. These site-directed mutations in the N-terminal domain do not severely disrupt its association with liposomes of PA. However, RGS4L23E diminishes the inhibition of GAP activity by PA compared with the wild type RGS4, whereas RGSR22E abrogates the inhibitory effect by PA on GAP activity. The correspondent conformational discrepancy in the RGS domain of these mutants in the presence of PA vesicles was detected from fluorescence experiments. It is suggested that the functional pertinence between the N-terminus and RGS domain may be important to modulate PA-conferred inhibitory effect on its GAP activity.     

Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10

RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys(95) in RGS4, Cys(66) in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys(2) or Cys(12). The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [(3)H]palmitate labeling of Cys(95). Membrane-bound RGS4 is palmitoylated both at Cys(95) and Cys(2/12), but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G(i) and G(q) families of G proteins. Palmitoylation of Cys(95) on RGS4 or Cys(66) on RGS10 inhibits GAP activity 80-100% toward either Galpha(i) or Galpha(z) in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity >/=20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Galpha(z) and increased G(z) GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.     

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.     

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.     

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.     

Modulation of transducin GTPase activity by chimeric RGS16 and RGS9 regulators of G protein signaling and the effector molecule

RGS9, a member of the family of regulators of G protein signaling (RGS), serves as a GTPase-activating protein (GAP) for the transducin alpha-subunit (Gtalpha) in the vertebrate visual transduction cascade. The GAP activity of RGS9 is uniquely potentiated by the gamma-subunit of the effector enzyme, cGMP-phosphodiesterase (Pgamma). In contrast, Pgamma attenuates the GAP effects of several other RGS proteins, including RGS16. We demonstrate here that the Pgamma subunit exerts its effects on the GTPase activity of the Gtalpha-RGS complex via the C-terminal domain, Pgamma-63-87. The structural determinants that control the direction of Pgamma effects on the RGS-Gtalpha system are localized within the RGS domains. The addition of Pgamma caused an increase in the maximal stimulation of Gtalpha GTPase activity by RGS9d without affecting the EC50 value. Modulation of Gtalpha GTPase activity by chimeric RGS16 and RGS9 proteins and Pgamma has been investigated. This analysis suggests that in addition to the differences in primary structures, the overall conformations of the RGS fold in RGS9 and RGS16 are likely to be responsible for the opposite effects of Pgamma on the RGS9 and RGS16 GAP activity. The RGS9 alpha3-alpha5 region constituted the minimal insertion of the RGS9 domain into RGS16 that reversed the inhibitory effect of Pgamma. A model of the RGS9 complex with Gtalpha shows the alpha3-alpha5 helices in RGS9 facing the proximate Pgamma binding site on Gtalpha. Our results and this model demonstrate that the mechanism of potentiation of RGS9 GAP activity by Pgamma involves a more rigid stabilization of the Gtalpha switch regions when Gtalpha is bound to both RGS9 and Pgamma.     

N-terminal half of the cGMP phosphodiesterase gamma-subunit contributes to stabilization of the GTPase-accelerating protein complex

In the visual signal terminating transition state, the cyclic GMP phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) stimulates GTPase activity of the α-subunit of transducin (αt) by enhancing the interaction between αt and its regulator of G protein signaling (RGS9), which is constitutively bound to the type 5 G protein β-subunit (β5). Although it is known from a crystal structure of partial molecules that the PDEγ C terminus contacts with both αt and RGS9, contributions from the intrinsically disordered PDEγ N-terminal half remain unclear. In this study, we were able to investigate this issue using a photolabel transfer strategy that allows for mapping the interface of full-length proteins. We observed label transfer from PDEγ N-terminal positions 50, 30, and 16 to RGS9·β5 in the GTPase-accelerating protein (GAP) complex composed of PDEγ·αt·RGS9·β5. In support of a direct PDEγ N-terminal interaction with RGS9·β5, the PDEγ N-terminal peptide PDEγ(1-61) abolished label transfer to RGS9·β5, and another N-terminal peptide, PDEγ(10-30), disassembled the GAP complex in label transfer and pulldown experiments. Furthermore, we determined that the PDEγ C-terminal interaction with αt was enhanced whereas the N-terminal interaction was weakened upon changing the αt conformation from the signaling state to the transition state. This "rearrangement" of PDEγ domain interactions with αt appears to facilitate the interaction of the PDEγ N-terminal half with RGS9·β5 and hence its contribution to optimal stabilization of the GAP complex.     

14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.     

Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.     

Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members

RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGS-box domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated Galpha(q). Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or Galpha(q). Together, these data indicate that novel features within the RGS2 amphipathic alpha helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.     

N-terminal tyrosine residues within the potassium channel Kir3 modulate GTPase activity of Galphai

trkB activation results in tyrosine phosphorylation of N-terminal Kir3 residues, decreasing channel activation. To determine the mechanism of this effect, we reconstituted Kir3, trkB, and the mu opioid receptor in Xenopus oocytes. Activation of trkB by BDNF (brain-derived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPase-activating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTPgammaS also prevented Kir3 deactivation, and the GTPgammaS effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for Gbetagamma but rather acted to accelerate GTPase activity, like RGS4. Tyrosine phosphatase inhibition by peroxyvanadate pretreatment reversibly mimicked the BDNF/trkB effect, indicating that tyrosine phosphorylation of Kir3 may have caused the GTPase acceleration. Tyrosine to phenylalanine substitution in the N-terminal domain of Kir3.4 blocked the BDNF effect, supporting the hypothesis that phosphorylation of these tyrosines was responsible. Like other GAPs, Kir3.4 contains a tyrosine-arginine-glutamine motif that is thought to function by interacting with G protein catalytic domains to facilitate GTP hydrolysis. These data suggest that the N-terminal tyrosine hydroxyls in Kir3 normally mask the GAP activity and that modification by phosphorylation or phenylalanine substitution reveals the GAP domain. Thus, BDNF activation of trkB could inhibit Kir3 by facilitating channel deactivation.     

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.     

RGS2: a multifunctional regulator of G-protein signaling

Regulators of G-protein signaling (RGS) proteins enhance the intrinsic rate at which certain heterotrimeric G-protein alpha-subunits hydrolyze GTP to GDP, thereby limiting the duration that alpha-subunits activate downstream effectors. This activity defines them as GTPase activating proteins (GAPs). As do other RGS proteins RGS2 possesses a 120 amino acid RGS domain, which mediates its GAP activity. In addition, RGS2 shares an N-terminal membrane targeting domain with RGS4 and RGS16. Found in many cell types, RGS2 expression is highly regulated. Functionally, RGS2 blocks Gq alpha-mediated signaling, a finding consistent with its potent Gq alpha GAP activity. Surprisingly, RGS2 inhibits Gs signaling to certain adenylyl cyclases. Like other RGS proteins, RGS2 lacks Gs alpha GAP activity, however it directly inhibits the activity of several adenylyl cyclase isoforms. Targeted mutation of RGS2 in mice impairs anti-viral immunity, increases anxiety levels, and alters synaptic development in hippocampal CA1 neurons. RGS2 has emerged as a multifunctional RGS protein that regulates multiple G-protein linked signaling pathways.