A Global Map of G Protein Signaling Regulation by RGS Proteins.

The control over the extent and timing of G protein signaling is provided by the regulator of G protein signaling (RGS) proteins that deactivate G protein α subunits (Gα). Mammalian genomes encode 20 canonical RGS and 16 Gα genes with key roles in physiology and disease. To understand the principles governing the selectivity of Gα regulation by RGS, we examine the catalytic activity of all canonical human RGS proteins and their selectivity for a complete set of Gα substrates using real-time kinetic measurements in living cells. The data reveal rules governing RGS-Gα recognition, the structural basis of its selectivity, and provide principles for engineering RGS proteins with defined selectivity. The study also explores the evolution of RGS-Gα selectivity through ancestral reconstruction and demonstrates how naturally occurring non-synonymous variants in RGS alter signaling. These results provide a blueprint for decoding signaling selectivity and advance our understanding of molecular recognition principles.

Pharmacogenomics of GPCR Drug Targets.

Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (∼34%) Food and Drug Administration (FDA)-approved drugs and account for a global sales volume of over 180 billion US dollars annually, the prevalence of genetic variation among GPCRs targeted by drugs is unknown. By analyzing data from 68,496 individuals, we find that GPCRs targeted by drugs show genetic variation within functional regions such as drug- and effector-binding sites in the human population. We experimentally show that certain variants of µ-opioid and Cholecystokinin-A receptors could lead to altered or adverse drug response. By analyzing UK National Health Service drug prescription and sales data, we suggest that characterizing GPCR variants could increase prescription precision, improving patients' quality of life, and relieve the economic and societal burden due to variable drug responsiveness. VIDEO ABSTRACT.

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells.

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Effects of Small Molecule Ligands on ACKR3 Receptors.

Chemokines such as stromal derived factor 1 and their G protein coupled receptors are well-known regulators of the development and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C chemokine motif receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3). ACKR3 has been described as an atypical "biased" receptor because it does not appear to signal through G proteins and, instead, signals solely through the ß-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mammalian G α isoforms and have generated a comprehensive map of the G α activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective agonists for ACKR3 as assessed by their ability to recruit ß-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the transmembrane domains of ACKR3. The development of more selective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signaling axis and better understand the structural determinants for ACKR3 activation. SIGNIFICANCE STATEMENT: We are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions between these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.

The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects.

The interplay between G protein-coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain.

PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2.

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

Novel GNB1 mutations disrupt assembly and function of G protein heterotrimers and cause global developmental delay in humans.

Global developmental delay (GDD), often accompanied by intellectual disability, seizures and other features is a severe, clinically and genetically highly heterogeneous childhood-onset disorder. In cases where genetic causes have been identified, de novo mutations in neuronally expressed genes are a common scenario. These mutations can be best identified by exome sequencing of parent-offspring trios. De novo mutations in the guanine nucleotide-binding protein, beta 1 (GNB1) gene, encoding the Gß1 subunit of heterotrimeric G proteins, have recently been identified as a novel genetic cause of GDD. Using exome sequencing, we identified 14 different novel variants (2 splice site, 2 frameshift and 10 missense changes) in GNB1 in 16 pediatric patients. One mutation (R96L) was recurrently found in three ethnically diverse families with an autosomal dominant mode of inheritance. Ten variants occurred de novo in the patients. Missense changes were functionally tested for their pathogenicity by assaying the impact on complex formation with Gγ and resultant mutant Gßγ with Gα. Signaling properties of G protein complexes carrying mutant Gß1 subunits were further analyzed by their ability to couple to dopamine D1R receptors by real-time bioluminescence resonance energy transfer (BRET) assays. These studies revealed altered functionality of the missense mutations R52G, G64V, A92T, P94S, P96L, A106T and D118G but not for L30F, H91R and K337Q. In conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in GNB1 as a cause of GDD and provide insights how perturbation in heterotrimeric G protein function contributes to the disease.

LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/ß-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/ß-catenin signaling or cell proliferation in vivo Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell-cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell-cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell-cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1-Rac1 pathway to strengthen cell-cell adhesion in normal adult crypt stem cells and colon cancer cells.

Orphan Receptor GPR158 Is an Allosteric Modulator of RGS7 Catalytic Activity with an Essential Role in Dictating Its Expression and Localization in the Brain.

Regulators of G protein signaling control the duration and extent of signaling via G protein-coupled receptor (GPCR) pathways by accelerating the GTP hydrolysis on G protein α subunits thereby promoting termination of GPCR signaling. A member of this family, RGS7, plays a critical role in the nervous system where it regulates multiple neurotransmitter GPCRs that mediate vision, memory, and the action of addictive drugs. Previous studies have established that in vivo RGS7 forms mutually exclusive complexes with the membrane protein RGS7-binding protein or the orphan receptor GPR158. In this study, we examine the impact of GPR158 on RGS7 in the brain. We report that knock-out of GPR158 in mice results in marked post-transcriptional destabilization of RGS7 and substantial loss of its association with membranes in several brain regions. We further identified the RGS7-binding site in the C terminus of GPR158 and found that it shares significant homology with the RGS7-binding protein. The proximal portion of the GPR158 C terminus additionally contained a conserved sequence that was capable of enhancing RGS7 GTPase-activating protein activity in solution by an allosteric mechanism acting in conjunction with the regulators of the G protein signaling-binding domain. The distal portion of the GPR158 C terminus contained several phosphodiesterase E γ-like motifs and selectively recruited G proteins in their activated state. The results of this study establish GPR158 as an essential regulator of RGS7 in the native nervous system with a critical role in controlling its expression, membrane localization, and catalytic activity.

Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9-2 protein complexes in living cells.

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by µ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein ß subunit Gß5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gß5 regulated both Gi and Go with a bias toward Go, but RGS7/Gß5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.

Rules and mechanisms governing G protein coupling selectivity of GPCRs.

G protein-coupled receptors (GPCRs) convert extracellular stimuli into intracellular signaling by coupling to heterotrimeric G proteins of four classes: Gi/o, Gq, Gs, and G12/13. However, our understanding of the G protein selectivity of GPCRs is incomplete. Here, we quantitatively measure the enzymatic activity of GPCRs in living cells and reveal the G protein selectivity of 124 GPCRs with the exact rank order of their G protein preference. Using this information, we establish a classification of GPCRs by functional selectivity, discover the existence of a G12/13-coupled receptor, G15-coupled receptors, and a variety of subclasses for Gi/o-, Gq-, and Gs-coupled receptors, culminating in development of the predictive algorithm of G protein selectivity. We further identify the structural determinants of G protein selectivity, allowing us to synthesize non-existent GPCRs with de novo G protein selectivity and efficiently identify putative pathogenic variants.

Type 5 G protein beta subunit (Gbeta5) controls the interaction of regulator of G protein signaling 9 (RGS9) with membrane anchors.

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein ß subunit, Gß5, and membrane anchor R7BP or R9AP. Association with Gß5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gß5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gß5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gß5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gß5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gß5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.

Interaction of transducin with uncoordinated 119 protein (UNC119): implications for the model of transducin trafficking in rod photoreceptors.

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gα(t1)). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gα(t1). The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gα(t1) with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gα(t1)GDP as well as with heterotrimeric transducin (G(t)). This interaction of UNC119 with G(t) led to displacement of Gß(1)γ(1) from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G(t) from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G(t), but had no effect on the GTP-hydrolysis by Gα(t1). A model for the role of UNC119 in the IS→OS translocation of G(t) is proposed based on the UNC119 ability to dissociate G(t) subunits from each other and the membrane. We also found that UNC119 inhibited activation of G(o) by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.

Ligand-directed bias of G protein signaling at the dopamine D2 receptor.

G-protein-coupled receptors (GPCRs) represent the largest family of drug targets. Upon activation, GPCRs signal primarily via a diverse set of heterotrimeric G proteins. Most GPCRs can couple to several different G protein subtypes. However, how drugs act at GPCRs contributing to the selectivity of G protein recognition is poorly understood. Here, we examined the G protein selectivity profile of the dopamine D2 receptor (D2), a GPCR targeted by antipsychotic drugs. We show that D2 discriminates between six individual members of the Gi/o family, and its profile of functional selectivity is remarkably different across its ligands, which all engaged D2 with a distinct G protein coupling pattern. Using structural modeling, receptor mutagenesis, and pharmacological evaluation, we identified residues in the D2 binding pocket that shape these ligand-directed biases. We further provide pharmacogenomic evidence that natural variants in D2 differentially affect its G protein biases in response to different ligands.

Membrane anchoring subunits specify selective regulation of RGS9·Gbeta5 GAP complex in photoreceptor neurons.

The RGS9·Gß5 complex is the key regulator of neuronal G-protein signaling and shows remarkable selectivity of subunit composition. In retinal photoreceptors, RGS9·Gß5 is bound to the membrane anchor R9AP and the complex regulates visual signaling. In the basal ganglia neurons, RGS9·Gß5 is instead associated with a homologous protein, R7BP, and regulates reward circuit. Switching this selective subunit composition of the complex in rod photoreceptors allowed us to study the molecular underpinning of signaling specificity in diverse G-protein pathways. We have found that both membrane anchoring subunits play a conserved role in regulating protein levels of RGS9·Gß5 and enhancing the ability of RGS·Gß5 complexes to stimulate GTPase activity of G proteins. However, notable differences exist in the subcellular targeting of alternatively configured complexes. Unlike R9AP, which relies on passive targeting mechanisms for the delivery to the outer segments of the photoreceptors, R7BP is excluded from this location and is instead specifically targeted to the plasma membrane. R7BP-containing complexes could be rerouted to the outer segments, where they are capable of regulating the phototransduction cascade by the active targeting signals derived from rhodopsin. These findings illustrate the diversity of the G-protein signaling regulation by RGS·Gß5 complexes achieved by differential recruitment of the membrane anchors.

Membrane anchor R9AP potentiates GTPase-accelerating protein activity of RGS11 x Gbeta5 complex and accelerates inactivation of the mGluR6-G(o) signaling.

The R7 subfamily of RGS proteins critically regulates neuronal G protein-signaling pathways that are essential for vision, nociception, motor coordination, and reward processing. A member of the R7 RGS family, RGS11, is a GTPase-accelerating protein specifically expressed in retinal ON-bipolar cells where it forms complexes with the atypical G protein beta subunit, Gbeta(5), and transmembrane protein R9AP. Association with R9AP has been shown to be critical for the proteolytic stability of the complex in the retina. In this study we report that R9AP can in addition stimulate the GTPase-accelerating protein activity of the RGS11 x Gbeta(5) complex at Galpha(o). Single turnover GTPase assays reveal that R9AP co-localizes RGS11 x Gbeta(5) and Galpha(o) on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11 x Gbeta(5). Reconstitution of mGluR6-Galpha(o) signaling in Xenopus oocytes indicates that RGS11 x Gbeta(5)-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Galpha(o) signaling by the RGS11 x Gbeta(5) x R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes.

Diversity of the Gßγ complexes defines spatial and temporal bias of GPCR signaling.

The signal transduction by G-protein-coupled receptors (GPCRs) is mediated by heterotrimeric G proteins composed from one of the 16 Gα subunits and the inseparable Gßγ complex assembled from a repertoire of 5 Gß and 12 Gγ subunits. However, the functional role of compositional diversity in Gßγ complexes has been elusive. Using optical biosensors, we examined the function of all Gßγ combinations in living cells and uncovered two major roles of Gßγ diversity. First, we demonstrate that the identity of Gßγ subunits greatly influences the kinetics and efficacy of GPCR responses at the plasma membrane. Second, we show that different Gßγ combinations are selectively dispatched from the plasma membrane to various cellular organelles on a timescale from milliseconds to minutes. We describe the mechanisms regulating these processes and document their implications for GPCR signaling via various Gα subunits, thereby illustrating a role for the compositional diversity of G protein heterotrimers.

Extended Phenotyping and Functional Validation Facilitate Diagnosis of a Complex Patient Harboring Genetic Variants in MCCC1 and GNB5 Causing Overlapping Phenotypes.

Identifying multiple ultra-rare genetic syndromes with overlapping phenotypes is a diagnostic conundrum in clinical genetics. This study investigated the pathogenicity of a homozygous missense variant in GNB5 (GNB5L; NM_016194.4: c.920T > G (p. Leu307Arg); GNB5S; NM_006578.4: c.794T > G (p. Leu265Arg)) identified through exome sequencing in a female child who also had 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency (newborn screening positive) and hemoglobin E trait. The proband presented with early-onset intellectual disability, the severity of which was more in keeping with GNB5-related disorder than 3-MCC deficiency. She later developed bradycardia and cardiac arrest, and upon re-phenotyping showed cone photo-transduction recovery deficit, all known only to GNB5-related disorders. Patient-derived fibroblast assays showed preserved GNB5S expression, but bioluminescence resonance energy transfer assay showed abolished function of the variant reconstituted Gß5S containing RGS complexes for deactivation of D2 dopamine receptor activity, confirming variant pathogenicity. This study highlights the need for precise phenotyping and functional assays to facilitate variant classification and clinical diagnosis in patients with complex medical conditions.

Gαo is a major determinant of cAMP signaling in the pathophysiology of movement disorders.

The G protein alpha subunit o (Gαo) is one of the most abundant proteins in the nervous system, and pathogenic mutations in its gene (GNAO1) cause movement disorder. However, the function of Gαo is ill defined mechanistically. Here, we show that Gαo dictates neuromodulatory responsiveness of striatal neurons and is required for movement control. Using in vivo optical sensors and enzymatic assays, we determine that Gαo provides a separate transduction channel that modulates coupling of both inhibitory and stimulatory dopamine receptors to the cyclic AMP (cAMP)-generating enzyme adenylyl cyclase. Through a combination of cell-based assays and rodent models, we demonstrate that GNAO1-associated mutations alter Gαo function in a neuron-type-specific fashion via a combination of a dominant-negative and loss-of-function mechanisms. Overall, our findings suggest that Gαo and its pathological variants function in specific circuits to regulate neuromodulatory signals essential for executing motor programs.

Retina-specific GTPase accelerator RGS11/G beta 5S/R9AP is a constitutive heterotrimer selectively targeted to mGluR6 in ON-bipolar neurons.

Members of the R7 family of the regulators of G-protein signaling (R7 RGS) proteins form multi-subunit complexes that play crucial roles in processing the light responses of retinal neurons. The disruption of these complexes has been shown to lead to the loss of temporal resolution in retinal photoreceptors and deficient synaptic transmission to downstream neurons. Despite the well established role of one member of this family, RGS9-1, in controlling vertebrate phototransduction, the roles and organizational principles of other members in the retina are poorly understood. Here we investigate the composition, localization, and function of complexes containing RGS11, the closest homolog of RGS9-1. We find that RGS11 forms a novel obligatory trimeric complex with the short splice isoform of the type 5 G-protein beta subunit (G beta 5) and the RGS9 anchor protein (R9AP). The complex is expressed exclusively in the dendritic tips of ON-bipolar cells in which its localization is accomplished through a direct association with mGluR6, the glutamate receptor essential for the ON-bipolar light response. Although association with both R9AP and mGluR6 contributed to the proteolytic stabilization of the complex, postsynaptic targeting of RGS11 was not determined by its membrane anchor R9AP. Electrophysiological recordings of the light response in mouse rod ON-bipolar cells reveal that the genetic elimination of RGS11 has little effect on the deactivation of G alpha(o) in dark-adapted cells or during adaptation to background light. These results suggest that the deactivation of mGluR6 cascade during the light response may require the contribution of multiple GTPase activating proteins.