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 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 beta 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.     

Membrane Anchor R9AP Potentiates GTPase-accelerating Protein Activity of RGS11��G��5 Complex and Accelerates Inactivation of the mGluR6-Go 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 β subunit, Gβ₅, 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·Gβ₅ complex at Gα_o. Single turnover GTPase assays reveal that R9AP co-localizes RGS11·Gβ₅ and Gα_o on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11·Gβ₅. Reconstitution of mGluR6-Gα_o signaling in Xenopus oocytes indicates that RGS11·Gβ₅-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Gα_o signaling by the RGS11·Gβ₅·R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes.     

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.     

Regulator of G protein signaling 6 (RGS6) protein ensures coordination of motor movement by modulating GABAB receptor signaling

γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.     

Regulator of G protein signaling 2 deficiency causes endothelial dysfunction and impaired endothelium-derived hyperpolarizing factor-mediated relaxation by dysregulating Gi/o signaling

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium- or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K(+) channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)α activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.     

Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4

RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/G_q signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/G_q signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/G_q signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function.     

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.     

Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

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.     

Regulators of G protein signaling 6 and 7. Purification of complexes with gbeta5 and assessment of their effects on g protein-mediated signaling pathways.

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o).     

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 G_i/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from G_i/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 G_i/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.     

Beta-agonist-associated reduction in RGS5 expression promotes airway smooth muscle hyper-responsiveness

Although short-acting and long-acting inhaled β(2)-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent β(2)-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to β(2)-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cβ and myosin light chain). In contrast, β-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5(-/-) mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive β(2)-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.     

Exercise enhancement by RGS14 disruption is mediated by brown adipose tissue

Enhanced exercise capacity is not only a feature of healthful aging, but also a therapy for aging patients and patients with cardiovascular disease. Disruption of the Regulator of G Protein Signaling 14 (RGS14) in mice extends healthful lifespan, mediated by increased brown adipose tissue (BAT). Accordingly, we determined whether RGS14 knockout (KO) mice exhibit enhanced exercise capacity and the role of BAT in mediating exercise capacity. Exercise was performed on a treadmill and exercise capacity was assessed by maximal running distance and work to exhaustion. Exercise capacity was measured in RGS14 KO mice and their wild types (WT), and also in WT mice with BAT transplantation from RGS14 KO mice or from other WT mice. RGS14 KO mice demonstrated 160 ± 9% increased maximal running distance and 154 ± 6% increased work to exhaustion, compared to WT mice. RGS14 KO BAT transplantation to WT mice, resulted in a reversal of phenotype, with the WT mice receiving the BAT transplant from RGS14 KO mice demonstrating 151 ± 5% increased maximal running distance and 158 ± 7% increased work to exhaustion, at three days after BAT transplantation, compared to RGS14 KO donors. BAT transplantation from WT to WT mice also resulted in increased exercise performance, but not at 3 days, but only at 8 weeks after transplantation. The BAT induced enhanced exercise capacity was mediated by (1) mitochondrial biogenesis and SIRT3; (2) antioxidant defense and the MEK/ERK pathway, and increased hindlimb perfusion. Thus, BAT mediates enhanced exercise capacity, a mechanism more powerful with RGS14 disruption.     

Structural basis for the 14-3-3 protein-dependent inhibition of the regulator of G protein signaling 3 (RGS3) function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.     

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 I_KACh. 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-I_KACh-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-I_KACh signaling pathway in SAN cells to carbachol and a significant slowing of M₂R-I_KACh 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-I_KACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M₂R-I_KACh signaling in mice.     

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.     

Regulator of G protein signaling 6 (RGS6) induces apoptosis via a mitochondrial-dependent pathway not involving its GTPase-activating protein activity

Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨ(m)) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨ(m) was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.     

G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction

A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.