Galpha Research Articles

Noncanonical Wnt Signaling through G Protein-Linked PKCδ Activation Promotes Bone Formation

Wnt signaling regulates a variety of developmental processes in animals. Although the beta-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here, we delineate a pathway in which Wnt3a signals through the Galpha(q/11) subunits of G proteins to activate phosphatidylinositol signaling and PKCdelta in the murine ST2 cells. Galpha(q/11)-PKCdelta signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCdelta homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCdelta-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation.

A novel type of G protein inhibitor, YM‐254890, acts as a Gq‐specific guanine nucleotide dissociation inhibitor via the switch I region of Galpha subunit

The cyclic depsipeptide YM-254890 was isolated from the culture broth of Chromobacterum QS3666 as an inhibitor of ADP-induced platelet aggregation. YM-254890 specifically inhibits the Gq-mediated cell response in intact cells, but the details of the inhibitory mechanism of YM-254890 remain to be clarified. In this presentation, we demonstrate that YM-254890 functions as a Gαq-specific guanine nucleotide dissociation inhibitor (GDI). GTPγS binding and the GDP dissociation assay using a purified G protein α subunit indicated that YM-254890 directly inhibits the spontaneous GDP/GTP exchange on Gαq and stabilizes the GDP-bound conformation of Gαq. In contrast, YM-254890 had no effect on the GTPγS binding to Gαs, Gαi, Gαo, and Gα13. YM-254890 did not compete with Gβ γ for binding to Gαq and inhibited the activation of heterotrimeric Gαqβ γ by AlF4−. Moreover, chimera and site-directed mutagenesis studies showed that the switch I region including Ile 190 of Gαq is important for YM-254890 sensitivity. Our results indicate that YM-254890 acts as a novel type of Gq-specific GDI via the switch I region of Galpha subunit.

The KlSTE2 and KlSTE3 genes encode MATα‐ and MATa‐specific G‐protein‐coupled receptors, respectively, which are required for mating of Kluyveromyces lactis haploid cells

Mating in yeast is initiated by binding of pheromone to G-protein-coupled receptors expressed in haploid cells. We analysed the role of KlSte2p and KlSte3p receptors in the Kluyveromyces lactis mating pathway. By sequence analysis, KlSte2p and KlSte3p are the homologues of the Saccharomyces cerevisiae alpha-pheromone and a-pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MATalpha and therefore is the receptor for the K. lactis a-pheromone and KlSTE3 gene is expressed in the MATa cells and binds the alpha-pheromone. The KlSTE2 gene is silent in MATa cells, while it is highly expressed in MATalpha cells, and conversely the KlSTE3 gene is expressed in MATa cells and repressed in MATalpha cells. Disruption mutants of both genes were found to be sterile, and this defect is reversed by plasmidic copies of each gene. The cytoplasmic C-terminus of KlSte3p interacts strongly with the KlGpa1p (Galpha) subunit, which is involved in the transduction of the pheromone stimulus to induce mating. Remarkably, this same domain does not interact with a constitutive active allele of the Galpha subunit, indicating that the C-terminus is able to discriminate between the active (GTP-bound) and inactive (GDP-bound) forms of the Galpha subunit.

Structural and dynamical changes in an alpha-subunit of a heterotrimeric G protein along the activation pathway.

The Galpha subunits of heterotrimeric G proteins (Galphabetagamma) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in "switch" sequences of Galpha, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gbetagamma subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Galphai1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix alpha4, a putative effector binding region. In the inactive Galphai1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gbetagamma, motions of R1 are highly constrained, reflecting direct contact interactions at the Galphai1-Gbeta interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding alpha2/beta4 loop and smaller allosteric changes at the Galphai1-Gbetagamma interface distant from the receptor binding surface. Upon addition of GTPgammaS, the activated Galphai1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in alpha4, distant from the interaction surface, supports a role for this helix in effector binding.

Signal Transfer from GPCRs to G Proteins: ROLE OF THE Gα N-TERMINAL REGION IN RHODOPSIN-TRANSDUCIN COUPLING

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).

Turning G Proteins On and Off Using Peptide Ligands

Intracellular Galpha subunits represent potential therapeutic targets for a number of diseases. Here we describe three classes of new molecules that modulate G protein signaling by direct targeting of Galpha. Using messenger RNA display, we have identified unique peptide sequences that bind Galpha i1 . Functionally, individual peptides were found that either enhance or repress basal levels of G protein-activated inwardly rectifying potassium (GIRK) channel signaling, a downstream effector of G protein activation, indicating that the peptides directly turn G proteins on or off in vivo . A third functional class acts as a signaling attenuator; basal GIRK channel activity is unaffected but responses to repeated G protein activation are reduced. These data demonstrate that G protein-directed ligands can achieve physiological effects similar to those resulting from classical receptor targeting and may serve as leads for developing new classes of therapeutics.

DEP-Domain-Mediated Regulation of GPCR Signaling Responses

G protein-coupled receptors (GPCRs) mediate cellular responses to a variety of stimuli, but how specific responses are regulated has been elusive, as the types of GPCRs vastly outnumber the classes of G protein heterotrimers available to initiate downstream signaling. In our analysis of signaling proteins containing DEP domains ( approximately 90 residue sequence motifs first recognized in fly Dishevelled, worm EGL-10, and mammalian Pleckstrin), we find that DEP domains are responsible for specific recognition of GPCRs. We examined the yeast regulator of G protein signaling (RGS) protein Sst2 and demonstrate that the DEP domains in Sst2 mediate binding to its cognate GPCR (Ste2). DEP-domain-mediated tethering promotes downregulation by placing the RGS protein in proximity to its substrate (receptor-activated Galpha subunit). Sst2 docks to the Ste2 cytosolic tail, but only its unphosphorylated state, allowing for release and recycling of this regulator upon receptor desensitization and internalization. DEP-domain-mediated targeting of effectors and regulators to specific GPCRs provides a means to dictate the nature, duration, and specificity of the response.

The Apelin Receptor Is Coupled to Gi1 or Gi2 Protein and Is Differentially Desensitized by Apelin Fragments

The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles.

Activation of the Phosphatidylinositol 3-Kinase Vps34 by a G Protein α Subunit at the Endosome

In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.

Activation state-dependent interaction between Galphai and p67phox.

The phagocyte NADPH oxidase consists of multiple protein subunits that interact with each other to form a functional superoxide-generating complex. Although the essential components for superoxide production have been well characterized, other proteins potentially involved in the regulation of NADPH oxidase activation remain to be identified. We report here that the Galphai subunit of heterotrimeric G proteins is a novel binding partner for p67phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67phox preferably interacted with inactive Galphai. Expression of p67phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Galphai. We identified a fragment of p67phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Galphai. Because these domains are involved in the interaction with p47phox and p40phox, the relationship between the respective binding events was investigated. Wild-type Galphai, but not its QL mutant, could promote the interaction between p67phox and p47phox. However, the interaction between p67phox and p40phox was not affected by either Galphai form. These results provide the first evidence for an interaction between p67phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Galphai in the regulation or activation of NADPH oxidase.

The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein alpha subunit confers functional specificity to G-proteins. Vertebrate and insect Galpha-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Galpha genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Galpha subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Galpha gene classes, C. elegans and other nematodes also possess two lineage-specific Galpha gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Galpha genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution.

Constitutive Activation of Gαs within Forebrain Neurons Causes Deficits in Sensorimotor Gating Because of PKA-Dependent Decreases in cAMP

Sensorimotor gating, the ability to automatically filter sensory information, is deficient in a number of psychiatric disorders, yet little is known of the biochemical mechanisms underlying this critical neural process. Previously, we reported that mice expressing a constitutively active isoform of the G-protein subunit Galphas (Galphas(*)) within forebrain neurons exhibit decreased gating, as measured by prepulse inhibition of acoustic startle (PPI). Here, to elucidate the biochemistry regulating sensorimotor gating and to identify novel therapeutic targets, we test the hypothesis that Galphas(*) causes PPI deficits via brain region-specific changes in cyclic AMP (cAMP) signaling. As predicted from its ability to stimulate adenylyl cyclase, we find here that Galphas(*) increases cAMP levels in the striatum. Suprisingly, however, Galphas(*) mice exhibit reduced cAMP levels in the cortex and hippocampus because of increased cAMP phosphodiesterase (cPDE) activity. It is this decrease in cAMP that appears to mediate the effect of Galphas(*) on PPI because Rp-cAMPS decreases PPI in C57BL/6J mice. Furthermore, the antipsychotic haloperidol increases both PPI and cAMP levels specifically in Galphas(*) mice and the cPDE inhibitor rolipram also rescues PPI deficits of Galphas(*) mice. Finally, to block potentially the pathway that leads to cPDE upregulation in Galphas(*) mice, we coexpressed the R(AB) transgene (a dominant-negative regulatory subunit of protein kinase A (PKA)), which fully rescues the reductions in PPI and cAMP caused by Galphas(*). We conclude that expression of Galphas(*) within forebrain neurons causes PPI deficits because of a PKA-dependent decrease in cAMP and suggest that cAMP PDE inhibitors may exhibit antipsychotic-like therapeutic effects.

Direct Modulation of Phospholipase D Activity by Gβγ

Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.

Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling

Regulators of G protein signaling (RGS proteins) bind directly to activated Galpha subunits to inhibit their signaling. However, recent findings show that RGS proteins selectively regulate signaling by certain G protein-coupled receptors (GPCRs) in cells, irrespective of the coupled G protein. New studies support an emerging model that suggests RGS proteins utilize both direct and indirect mechanisms to form stable functional pairs with preferred GPCRs to selectively modulate the signaling functions of those receptors and linked G proteins. Here, we discuss these findings and their implications for established models of GPCR signaling.

Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl cyclase activation requiring neurofibromin and Ras

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in humans, and animal models. We identify a novel growth factor stimulated adenylyl cyclase (AC) pathway in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galpha(s). This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galpha(s), can activate AC. We also show that Galpha(s) is the major Galpha isoform in fly brains, and define a second AC pathway stimulated by serotonin and histamine requiring NF1 and Galpha(s), as well as a third, classical Galpha(s)-dependent AC pathway, which is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, we show that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, we demonstrate that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galpha(s)-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies.

Calcium Mobilization in Human Platelets is Differentially Modulated by PAR‐1 and PAR‐4 through Gi/o and PI3K

Protease activated receptors (PARs) on the platelet surface play an essential role in mediating hemostasis. Human platelets express PAR-1 and PAR-4, and these receptor subtypes are activated by thrombin. Knockout mice for the various G-alpha subunits of the heterotrimeric G-protein family have provided good tools for the study of PAR-4 signaling in platelets. However, differences in G-protein coupling between human and mouse PAR-4 could exist, and mouse platelets do not express PAR-1. Therefore, determining which G-alpha subunits PAR-1 and PAR-4 activate in human platelets is paramount in understanding the physiological effects of thrombin and its inhibitors. Both receptors have been shown to couple to Gq and G12/13 pathways. However, neither receptor has been shown to have a Gi/o signaling component. The main objective of our research was to determine the role of Gi/o in human platelet PAR-mediated calcium mobilization and integrin activation. We have found that PAR-1, but not PAR-4, directly couples to Gi/o family members to activate PI3K in human platelets using flow cytometry. We have also shown that this PI3K potentiates intraplatelet calcium mobilization in a conditional manner using Fura-2 loaded platelets, and also causes integrin glycoprotein IIbIIIa activation using flow cytometry. PAR-4 seems to lack this Gi/o pathway, and we are currently developing a cell permeable peptide based strategy to explore this possibility. Demonstration of a Gi/o signaling component downstream of PAR-1 in human platelets is a novel finding with potential therapeutic implications. This work was supported by the Training Grant in the Pharmacological Sciences (Vanderbilt-NIH).

Regulator of G‐protein signaling (RGS) proteins differentially control chondrocyte differentiation

Control of chondrocyte differentiation is attained, in part, through G-protein signaling, but the functions of the RGS family of genes, well known to control G-protein signaling at the Galpha subunit, have not been studied extensively in chondrogenesis. Recently, we have identified the Rgs2 gene as a regulator of chondrocyte differentiation. Here we extend these studies to additional Rgs genes. We demonstrate that the Rgs4, Rgs5, Rgs7, and Rgs10 genes are differentially regulated during chondrogenic differentiation in vitro and in vivo. To investigate the roles of RGS proteins during cartilage development, we overexpressed RGS4, RGS5, RGS7, and RGS10 in the chondrogenic cell line ATDC5. We found unique and overlapping effects of individual Rgs genes on numerous parameters of chondrocyte differentiation. In particular, RGS5, RGS7, and RGS10 promote and RGS4 inhibits chondrogenic differentiation. The identification of Rgs genes as novel regulators of chondrogenesis will contribute to a better understanding of both normal cartilage development and the etiology of chondrodysplasias and osteoarthritis.

Immunohistochemical localization of G protein βγ subunits in the lateral wall of the rat cochlea

The role of G protein-mediated signal transduction in the production of endolymph, an extracellular fluid of unusual ionic composition, is beginning to be understood. The identity of Galpha subunits in the stria vascularis and the spiral ligament of the lateral wall of the cochlear duct is well established. However, little is known about the presence of betagamma subunits. This study used immunohistochemistry to investigate the distribution of G protein betagamma subunits in the lateral wall of the cochlea. Temporal bones of 6- to 8-week-old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit-specific polyclonal antibodies. The results show that the pattern of immunoreactivity varies for the G protein beta1-4 and gamma1-3, 5 and 7 subunits in the stria vascularis and spiral ligament. In the stria vascularis, immunoreactivity was detected for beta2, beta3, beta4, gamma1, gamma2 and gamma7 subunits. All five types of fibrocytes in the spiral ligament exhibited positive staining for gamma2 and gamma7. However, immunoreactivity for beta1-4 subunits was variable. Immunoreactivity for gamma3 and gamma5 subunits was not detected in the lateral cochlear wall. The expression pattern of G protein betagamma subunits in lateral wall provides a basis for interpreting the functions of G protein-coupled receptors in cochlear fluid homeostasis.

Bring Your Own G Protein: TABLE 1

G protein-coupled receptor (GPCR)-Galpha fusion proteins were first characterized more than 10 years ago as a strategy for studying receptor-G protein signaling. A large number of studies have used this approach to characterize receptor coupling to members of the Gs, Gi, and Gq families of Galpha subunits, but this strategy has not been widely used to study Galpha12 and Galpha13. As described in the article by Zhang et al. in this issue of Molecular Pharmacology (p. 1433) characterization of the signaling properties of thromboxane A2 receptor (TPalpha) -Galpha12 and -Galpha13 fusion constructs demonstrates the applicability of this strategy to members of this unique family of Galpha subunits, and how this strategy can be used to resolve otherwise difficult problems of receptor pharmacology associated with these proteins. The general strategy of making receptor-Galpha fusion constructs has wide applicability to a number of research problems, but there are perhaps also "hidden messages" in how different receptor-Galpha subunit fusion pairs behave.

Endogenous Regulator of G-Protein Signaling Proteins Regulate the Kinetics of Gαq/11-Mediated Modulation of Ion Channels in Central Nervous System Neurons

Slow synaptic potentials are generated when metabotropic G-protein-coupled receptors activate heterotrimeric G-proteins, which in turn modulate ion channels. Many neurons generate excitatory postsynaptic potentials mediated by G-proteins of the Galphaq/11 family, which in turn activate phospholipase C-beta. Accessory GTPase-activating proteins (GAPs) are thought to be required to accelerate GTP hydrolysis and rapidly turn off G-proteins, but the involvement of GAPs in neuronal Galphaq/11 signaling has not been examined. Here, we show that regulator of G-protein signaling (RGS) proteins provide necessary GAP activity at neuronal Galphaq/11 subunits. We reconstituted inhibition of native 2-pore domain potassium channels in cerebellar granule neurons by expressing chimeric Galpha subunits that are activated by Galphai/o-coupled receptors, thus bypassing endogenous Galphaq/11 subunits. RGS-insensitive variants of these chimeras mediated inhibition of potassium channels that developed and recovered more slowly than inhibition mediated by RGS-sensitive (wild-type) chimeras or native Galphaq/11 subunits. These changes were not accompanied by a change in agonist sensitivity, as might be expected if RGS proteins acted primarily as effector antagonists. The slowed recovery from potassium channel inhibition was largely reversed by an additional mutation that mimics the RGS-bound state. These results suggest that endogenous RGS proteins regulate the kinetics of rapid Galphaq/11-mediated signals in central nervous system neurons by providing GAP activity.