R7 Regulators Of G Protein Signaling Research Articles

Association of Mouse Vulnerability to Alzheimer’s Disease Pathology with Mutations of Gnb5: A Modulator of G‐protein Coupled Receptor Signaling

The prevalence and devastating effects of Alzheimer’s Disease (AD) and lack of effective treatments underscore the need to determine genes and molecular pathways with pathophysiologic relevance to develop targeted therapies. Prior unpublished bioinformatic analysis by our laboratory of genomic and brain transcript expression data from subjects with and without AD has identified hundreds of potential AD causative genes, one being GNB5, which encodes the protein Gβ5. Gβ5 is a divergent member of the G‐protein β subunit family and is primarily expressed in neuronal tissues. It is known to stabilize members of the R7‐Regulator of G‐protein Signaling (RGS) subfamily that exert GTPase Accelerating Protein (GAP) activities on Gαi/o proteins during G‐protein coupled receptor (GPCR) signaling. Homozygous lack of Gβ5 causes significant neuronal and behavioral impairments in both mice and humans; however, the impact of GNB5 heterozygosity requires further examination. For this project, we demonstrated through fear conditioning behavioral tasks involving hippocampus and amygdala function that learning and memory deficits existed in mice heterozygous for Gnb5. In addition, the potential relevance of GNB5 in AD pathogenesis was confirmed in an APP/PSEN1 double transgenic AD mouse model in which missing even one copy of the Gnb5 gene enhanced amyloid plaque and neurofibrillary tangle formations in multiple brain regions of mice including, the entorhinal cortex, hippocampus, and frontal cortex. These results were confirmed by traditional immunohistochemistry methods as well as immunostaining of transparent mouse brains cleared using the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) method. We further demonstrated that the exacerbated AD pathology may be caused, at least in part, by the increased vulnerability of heterozygous GNB5 mouse neurons to the toxicity of Aβ42 peptide oligomers which have been linked to beta‐amyloid plaque formation. Our data suggest the involvement of GNB5 regulated GPCR signaling in the progression of AD pathology and illustrates a potential new therapeutic target for attenuation of cellular pathology associated with AD.

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein

RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.

Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily

Regulators of G protein Signaling (RGS) promote deactivation of heterotrimeric G proteins thus controlling the magnitude and kinetics of responses mediated by G protein-coupled receptors (GPCR). In the nervous system, RGS7 and RGS9-2 play essential role in vision, reward processing, and movement control. Both RGS7 and RGS9-2 belong to the R7 subfamily of RGS proteins that form macromolecular complexes with R7-binding protein (R7BP). R7BP targets RGS proteins to the plasma membrane and augments their GTPase-accelerating protein (GAP) activity, ultimately accelerating deactivation of G protein signaling. However, it remains unclear if R7BP serves exclusively as a membrane anchoring subunit or further modulates RGS proteins to increase their GAP activity. To directly answer this question, we utilized a rapidly reversible chemically induced protein dimerization system that enabled us to control RGS localization independent from R7BP in living cells. To monitor kinetics of Gα deactivation, we coupled this strategy with measuring changes in the GAP activity by bioluminescence resonance energy transfer-based assay in a cellular system containing μ-opioid receptor. This approach was used to correlate changes in RGS localization and activity in the presence or absence of R7BP. Strikingly, we observed that RGS activity is augmented by membrane recruitment, in an orientation independent manner with no additional contributions provided by R7BP. These findings argue that the association of R7 RGS proteins with the membrane environment provides a major direct contribution to modulation of their GAP activity.

G protein beta 5 is targeted to D2-dopamine receptor-containing biochemical compartments and blocks dopamine-dependent receptor internalization.

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins.

A Mechanism Regulating G Protein-coupled Receptor Signaling That Requires Cycles of Protein Palmitoylation and Depalmitoylation

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K(+) channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4(-/-), Rgs6(-/-), and Rgs4(-/-):Rgs6(-/-) mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6(-/-) mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6(-/-) and Gβ5(-/-) mice, suggest that the partial rescue of phenotypes in Rgs4(-/-):Rgs6(-/-) mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

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 β subunit Gβ5 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/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 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.

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

Defective Retinal Depolarizing Bipolar Cells in Regulators of G Protein Signaling (RGS) 7 and 11 Double Null Mice

Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gβ5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gβ5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S is undetectable, whereas levels of the photoreceptor-specific Gβ5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in retina, the DBC-related defects in Gβ5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.

R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal

The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.

Membrane Attachment Is Key to Protecting Transducin GTPase-Activating Complex from Intracellular Proteolysis in Photoreceptors

The members of the R7 regulator of G-protein signaling (RGS) protein subfamily are versatile regulators of G-protein signaling throughout the nervous system. Recent studies indicate that they are often found in complexes with membrane anchor proteins that serve as versatile modulators of their activity, intracellular targeting, and stability. One striking example is the interplay between the membrane anchor R9AP and the RGS9-1 · Gβ5 GTPase-activating complex responsible for the rapid inactivation of the G-protein transducin in vertebrate photoreceptor cells during their recovery from light excitation. The amount of this complex in photoreceptors sets their temporal resolution and is precisely regulated by the expression level of R9AP, which serves to protect the RGS9-1 and Gβ5 subunits from intracellular proteolysis. In this study, we investigated the mechanism by which R9AP performs its protective function in mouse rods and found that it is entirely confined to recruiting RGS9-1 · Gβ5 to cellular membranes. Furthermore, membrane attachment of RGS9-1 · Gβ5 is sufficient for its stable expression in rods even in the absence of R9AP. Our second finding is that RGS9-1 · Gβ5 possesses targeting information that specifies its exclusion from the outer segment and that this information is neutralized by association with R9AP to allow outer segment targeting. Finally, we demonstrate that the ability of R9AP · RGS9-1 · Gβ5 to accelerate GTP hydrolysis on transducin is independent of its means of membrane attachment, since replacing the transmembrane domain of R9AP with a site for lipid modification did not impair the catalytic activity of this complex.

Type 5 G Protein β Subunit (Gβ5) 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 β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 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 Gβ5 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 Gβ5. 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 Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 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 Gβ5 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.

A Conserved Protein Interaction Interface on the Type 5 G Protein β Subunit Controls Proteolytic Stability and Activity of R7 Family Regulator of G Protein Signaling Proteins

Regulators of G protein signaling (RGS) proteins of the R7 subfamily limit signaling by neurotransmitters in the brain and by light in the retina. They form obligate complexes with the Gβ5 protein that are subject to proteolysis to control their abundance and alter signaling. The mechanisms that regulate this proteolysis, however, remain unclear. We used genetic screens to find mutations in Gβ5 that selectively destabilize one of the R7 RGS proteins in Caenorhabditis elegans. These mutations cluster at the binding interface between Gβ5 and the N terminus of R7 RGS proteins. Equivalent mutations within mammalian Gβ5 allowed the interface to still bind the N-terminal DEP domain of R7 RGS proteins, and mutant Gβ5-R7 RGS complexes initially formed in cells but were then rapidly degraded by proteolysis. Molecular dynamics simulations suggest the mutations weaken the Gβ5-DEP interface, thus promoting dynamic opening of the complex to expose determinants of proteolysis known to exist on the DEP domain. We propose that conformational rearrangements at the Gβ5-DEP interface are key to controlling the stability of R7 RGS protein complexes.

RSBP-1 is a membrane-targeting subunit required by the Galpha(q)-specific but not the Galpha(o)-specific R7 regulator of G protein signaling in Caenorhabditis elegans.

Regulator of G protein signaling (RGS) proteins inhibit G protein signaling by activating Galpha GTPase activity, but the mechanisms that regulate RGS activity are not well understood. The mammalian R7 binding protein (R7BP) can interact with all members of the R7 family of RGS proteins, and palmitoylation of R7BP can target R7 RGS proteins to the plasma membrane in cultured cells. However, whether endogenous R7 RGS proteins in neurons require R7BP or membrane localization for function remains unclear. We have identified and knocked out the only apparent R7BP homolog in Caenorhabditis elegans, RSBP-1. Genetic studies show that loss of RSBP-1 phenocopies loss of the R7 RGS protein EAT-16, but does not disrupt function of the related R7 RGS protein EGL-10. Biochemical analyses find that EAT-16 coimmunoprecipitates with RSBP-1 and is predominantly plasma membrane-associated, whereas EGL-10 does not coimmunoprecipitate with RSBP-1 and is not predominantly membrane-associated. Mutating the conserved membrane-targeting sequence in RSBP-1 disrupts both the membrane association and function of EAT-16, demonstrating that membrane targeting by RSBP-1 is essential for EAT-16 activity. Our analysis of endogenous R7 RGS proteins in C. elegans neurons reveals key differences in the functional requirements for membrane targeting between members of this protein family.

RGS7 and -11 Complexes Accelerate the ON-Bipolar Cell Light Response

The retinal ON-bipolar cell (ON-BPC) light response is initiated upon deactivation of the metabotropic glutamate receptor mGluR6 and the G protein Go. G protein-based signaling cascades are typically accelerated by interaction of the G protein alpha subunit with a member of the regulator of G protein signaling (RGS) protein family. The goal of this study was to determine whether RGS7 and/or -11 serve this function in retinal ON-BPCs. Retinas from mice lacking RGS11 (RGS11(-/-)), or with a deletion mutation in RGS7 (RGS7(Delta/Delta)), or both, were compared to wild-type (WT) by immunofluorescence confocal microscopy. The retinal light response was measured with the electroretinogram (ERG). The kinetics of simulated light responses from individual rod bipolar cells were recorded by whole-cell patch-clamp electrophysiology. Levels of the R7 RGS interaction partners, Gbeta5 and R9AP, were reduced in the outer plexiform layer of the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. ERG recordings demonstrated a delay in the rising phase of the ERG b-wave, larger photopic b-wave amplitudes, and increased scotopic threshold response sensitivity in the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. The ERG measured from the RGS7(Delta/Delta) retina was normal. Patch-clamp recordings of chemically simulated light responses of rod BPCs revealed a 25-ms delay in the onset of the ON-BPC response in the RGS7(Delta/Delta)/RGS11(-/-) mouse compared with the WT. RGS11 plays a role in the deactivation of Galphao, which precedes activation of the depolarizing current in ON-BPCs. RGS7 must also serve a role as changes in RGS7(Delta/Delta)/RGS11(-/-) mice were greater than those in RGS11(-/-) mice.

The R7 RGS Protein Family: Multi-Subunit Regulators of Neuronal G Protein Signaling

G protein-coupled receptor signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. Four proteins in this group, RGS6, RGS7, RGS9, and RGS11, share a common molecular organization of three modules: (i) the catalytic RGS domain, (ii) a GGL domain that recruits G beta(5), an outlying member of the G protein beta subunit family, and (iii) a DEP/DHEX domain that mediates interactions with the membrane anchor proteins R7BP and R9AP. As heterotrimeric complexes, R7 RGS proteins not only associate with and regulate a number of G protein signaling pathway components, but have also been found to form complexes with proteins that are not traditionally associated with G protein signaling. This review summarizes our current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein-protein interactions, and physiological roles.

Changes in Striatal Signaling Induce Remodeling of RGS Complexes Containing Gβ5 and R7BP Subunits

Neurotransmitter signaling via G protein coupled receptors is crucially controlled by regulators of G protein signaling (RGS) proteins that shape the duration and extent of the cellular response. In the striatum, members of the R7 family of RGS proteins modulate signaling via D2 dopamine and mu-opioid receptors controlling reward processing and locomotor coordination. Recent findings have established that R7 RGS proteins function as macromolecular complexes with two subunits: type 5 G protein beta (Gbeta5) and R7 binding protein (R7BP). In this study, we report that the subunit compositions of these complexes in striatum undergo remodeling upon changes in neuronal activity. We found that under normal conditions two equally abundant striatal R7 RGS proteins, RGS9-2 and RGS7, are unequally coupled to the R7BP subunit, which is present in complex predominantly with RGS9-2 rather than with RGS7. Changes in the neuronal excitability or oxygenation status resulting in extracellular calcium entry, uncouples RGS9-2 from R7BP, triggering its selective degradation. Concurrently, released R7BP binds to mainly intracellular RGS7 and recruits it to the plasma membrane and the postsynaptic density. These observations introduce activity-dependent remodeling of R7 RGS complexes as a new molecular plasticity mechanism in striatal neurons and suggest a general model for achieving rapid posttranslational subunit rearrangement in multisubunit complexes.

Localization and differential interaction of R7 RGS proteins with their membrane anchors R7BP and R9AP in neurons of vertebrate retina

G protein signaling in the retina is crucially regulated by the R7 family of regulators of G protein signaling (RGS) proteins, which act to stimulate the rate of G protein inactivation. Recent findings indicate that R7 RGS proteins form complexes with two newly identified membrane anchors: RGS9 Anchor Protein (R9AP) and R7 Binding Protein (R7BP), which play essential roles in modulating the expression and localization of R7 RGS proteins. Here we demonstrate that the four R7 RGS proteins: RGS6, RGS7, RGS9 and RGS11 differentially associate with two membrane anchors. R9AP was found to form complexes with RGS9 and RGS11 which were substantially enriched in the photoreceptors. In contrast, complexes of R7BP with R7 RGS proteins were predominantly localized to the synaptic projections of retina neurons, suggesting their involvement in regulation of synaptic transmission between retina neurons. Furthermore, studies of knockout mice revealed that R9AP is necessary for the expression of only RGS9 but not for RGS6, 7 or 11. Together these data suggest that R7 RGS proteins in the retina are present as macromolecular complexes with their membrane anchors that could differentially regulate their function in various retina neurons.

The effect of the RGS7‐Gβ5‐R7BP complex on GPCR mediated calcium mobilization

G protein coupled receptors (GPCRs) are major targets for therapeutic drugs, hence understanding the mechanism of signal transduction through these receptors is crucial for the development of novel therapies. Regulators of G protein signaling (RGS) proteins accelerate the deactivation of G proteins by acting as GTPase activating proteins (GAPs) towards Gα subunits. The R7 subfamily of RGS proteins contains a Dishevelled, Egl-10, Pleckstrin (DEP) and Gγ like (GGL) domain along with the characteristic RGS domain. It is via the GGL domain that R7 RGS proteins form a stable complex with the Gβ subunit, Gβ5 in vivo. Recently, an additional member of this complex R7 binding protein (R7BP) has been identified. R7BP binds to all R7 RGS proteins via the DEP domain. This interaction facilitates the recruitment of the RGS7-Gβ5 complex to the plasma membrane. Previously we demonstrated that RGS7-Gβ5 attenuates Ca2+ mobilization from M3 receptor in transfected cells. In this study, using Fura 2, we show that RGS7-Gβ5 acts in a receptor-specific manner, as it does not have an effect on M1, another Gq-coupled GPCR. We also investigated the effect of R7BP on the activity of RGS7-Gβ5 and found that it diminishes the negative regulation of M3 receptor signaling. Additional insights into this molecular mechanism were obtained via microscopy and GST pulldown assays. This research is supported by NIH grant GM 060019 (V.Z.S.).

Subcellular Targeting of RGS9-2 Is Controlled by Multiple Molecular Determinants on Its Membrane Anchor, R7BP

RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner Gbeta5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2.Gbeta5.R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.