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 inhibits Gαq-dependent uveal melanoma cell growth

Activating mutations in Gα_q/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gα_q/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gα_q-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation Gα_q^Q209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gα_q. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cβ, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gα_q, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with Gα_q^Q209L as a partial effector antagonist.     

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.     

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.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

Identification of critical residues in G(alpha)13 for stimulation of p115RhoGEF activity and the structure of the G(alpha)13-p115RhoGEF regulator of G protein signaling homology (RH) domain complex

RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).     

Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine,MCP-1 and activation of TLR4 and Gi/o proteins signaling

The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.     

HBXIP,a binding protein of HBx,regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity

To maintain the integrity of the genome, cells need to detect and repair DNA damage before they complete cell division. Hepatitis B x-interacting protein (HBXIP), a binding protein of HBx (Hepatitis B virus × protein), is aberrantly overexpressed in human cancer cells and show to promote cell proliferation and inhibit apoptosis. The present study is designed to investigate the role of HBXIP on the DNA damage response. Our results show that HBXIP acts as an important regulator of G2/M checkpoint in response to DNA damage. HBXIP knockdown increases phospho-histone H2AX expression and foci formation after treatment with ionizing radiation (IR). HBXIP regulates the ATM-Chk2 pathway following DNA damage. Depletion of HBXIP abrogates IR-induced G2/M cell cycle checkpoints, accompanying decrease the expression of phospho-Cdc25C, phospho-Cdc2 (Tyr15) and p27. We also show that downregulation of HBXIP expression sensitizes cancer cells to chemotherapy, as evidenced by an increase in apoptosis and cleavage of caspase-3 and caspase-9. Our data suggest that HBXIP can function as a mediator protein for DNA damage response signals to activate the G2/M checkpoint to maintain genome integrity and prevent cell death.     

Inhibition of EGF-induced ERK/MAP kinase-mediated astrocyte proliferation by μ opioids: integration of G protein and β-arrestin 2-dependent pathways

Although μ, κ, and δ opioids activate extracellular signal‐regulated kinase (ERK)/mitogen‐activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which μ opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)‐induced ERK‐mediated astrocyte proliferation. The μ‐opioid agonists ([d‐ ala², mephe⁴, gly‐ol⁵] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via G_i/o protein, calmodulin (CaM), and β‐arrestin2‐dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the μ‐opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W‐7, and siRNA silencing of β‐arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation‐mediated mechanism. This was disclosed by abolishment of μ‐opioid‐induced ERK phosphorylation with the EGF receptor‐specific tyrosine phosphorylation inhibitor, AG1478, and μ‐opioid‐induced reduction of EGF receptor tyrosine phosphorylation by PTX, and β‐arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long‐term (h) treatment of primary astrocytes with [d ‐ala²,mephe⁴,gly‐ol⁵] enkephalin or morphine, attenuated EGF‐induced ERK phosphorylation and proliferation (as measured by 5′‐bromo‐2′‐deoxy‐uridine labeling). PTX and β‐arrestin2 siRNA but not W‐7 reversed the μ‐opioid inhibition. Unexpectedly, β‐arrestin‐2 siRNA diminished both EGF‐induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different μ‐opioid signaling pathways to ERK and their cellular responses.