A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.     

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.     

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.     

RGS10 Negatively Regulates Platelet Activation and Thrombogenesis

Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10^-/- mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.     

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.     

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.     

RGS13 regulates germinal center B lymphocytes responsiveness to CXC chemokine ligand (CXCL)12 and CXCL13

Normal lymphoid tissue development and function depend upon directed cell migration. Providing guideposts for cell movement and positioning within lymphoid tissues, chemokines signal through cell surface receptors that couple to heterotrimeric G proteins, which are in turn subject to regulation by regulator of G protein signaling (RGS) proteins. In this study, we report that germinal center B lymphocytes and thymic epithelial cells strongly express one of the RGS family members, RGS13. Located between Rgs1 and Rgs2, Rgs13 spans 42 kb on mouse chromosome 1. Rgs13 encodes a 157-aa protein that shares 82% amino acid identity with its 159-aa human counterpart. In situ hybridization with sense and antisense probes localized Rgs13 expression to the germinal center regions of mouse spleens and Peyer's patches and to the thymus medulla. Affinity-purified RGS13 Abs detected RGS13-expressing cells in the light zone of the germinal center. RGS13 interacted with both Gialpha and Gqalpha and strongly impaired signaling through G(i)-linked signaling pathways, including signaling through the chemokine receptors CXCR4 and CXCR5. Prolonged CD40 signaling up-regulated RGS13 expression in human tonsil B lymphocytes. These results plus previous studies of RGS1 indicate the germinal center B cells use two RGS proteins, RGS1 and RGS13, to regulate their responsiveness to chemokines.     

Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl₄)-induced acute and chronic liver injury in Rgs5^LacZ/LacZ reporter mice. Furthermore, CCl₄ treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl₄ and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.     

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.     

RGS16 attenuates pulmonary Th2/Th17 inflammatory responses

The regulators of G protein signaling (RGS) protein superfamily negatively controls G protein-coupled receptor signal transduction pathways. RGS16 is enriched in activated/effector T lymphocytes. In this paper, we show that RGS16 constrains pulmonary inflammation by regulating chemokine-induced T cell trafficking in response to challenge with Schistosoma mansoni. Naive Rgs16(-/-) mice were "primed" for inflammation by accumulation of CCR10(+) T cells in the lung. Upon pathogen exposure, these mice developed more robust granulomatous lung fibrosis than wild-type counterparts. Distinct Th2 or putative Th17 subsets expressing CCR4 or CCR10 accumulated more rapidly in Rgs16(-/-) lungs following challenge and produced proinflammatory cytokines IL-13 and IL-17B. CCR4(+)Rgs16(-/-) Th2 cells migrated excessively to CCL17 and localized aberrantly in challenged lungs. T lymphocytes were partially excluded from lung granulomas in Rgs16(-/-) mice, instead forming peribronchial/perivascular aggregates. Thus, RGS16-mediated confinement of T cells to Schistosome granulomas mitigates widespread cytokine-mediated pulmonary inflammation.     

Rgs5 targeting leads to chronic low blood pressure and a lean body habitus

RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.     

Thioesterase Superfamily Member 2 (Them2) and Phosphatidylcholine Transfer Protein (PC-TP) Interact To Promote Fatty Acid Oxidation and Control Glucose Utilization

Thioesterase superfamily member 2 (Them2) is a mitochondrion-associated long-chain fatty acyl coenzyme A (CoA) thioesterase that is highly expressed in the liver and oxidative tissues. Them2 activity in vitro is increased when it interacts with phosphatidylcholine transfer protein (PC-TP), a cytosolic lipid binding protein. Them2^−/− and Pctp^−/− mice exhibit enhanced hepatic insulin sensitivity and increased adaptive thermogenesis, and Them2^−/− mice are also resistant to diet-induced hepatic steatosis. Although we showed previously that a Them2–PC-TP complex suppresses insulin signaling, the enzymatic activity of Them2 suggests additional direct involvement in regulating hepatic nutrient homeostasis. Here we used cultured primary hepatocytes to elucidate biochemical and cellular mechanisms by which Them2 and PC-TP regulate lipid and glucose metabolism. Under conditions simulating fasting, Them2^−/− and Pctp^−/− hepatocytes each exhibited decreased rates of fatty acid oxidation and gluconeogenesis. In results indicative of Them2-dependent regulation by PC-TP, chemical inhibition of PC-TP failed to reproduce these changes in Them2^−/− hepatocytes. In contrast, rates of glucose oxidation and lipogenesis in the presence of high glucose concentrations were decreased only in Them2^−/− hepatocytes. These findings reveal a primary role for Them2 in promoting mitochondrial oxidation of fatty acids and glucose in the liver.     

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.     

CTRP1 protein enhances fatty acid oxidation via AMP-activated protein kinase (AMPK) activation and acetyl-CoA carboxylase (ACC) inhibition

We previously described the adipokine CTRP1, which has up-regulated expression following exposure to the anti-diabetic drug rosiglitazone and increased circulating levels in adiponectin-null mice (Wong, G. W., Krawczyk, S. A., Kitidis-Mitrokostas, C., Revett, T., Gimeno, R., and Lodish, H. F. (2008) Biochem. J. 416, 161-177). Although recombinant CTRP1 lowers blood glucose in mice, its physiological function, mechanisms of action, and roles in metabolic stress remain unknown. Here, we show that circulating levels of CTRP1 are strikingly reduced in diet-induced obese mice. Overexpressing CTRP1 in transgenic mice improved insulin sensitivity and decreased high-fat diet-induced weight gain. Reduced adiposity resulted from enhanced fatty acid oxidation and energy expenditure, effects mediated by AMP-activated protein kinase (AMPK). In skeletal muscle of transgenic mice, AMPKα and its downstream target, acetyl-CoA carboxylase (ACC), were hyperphosphorylated, indicative of AMPK activation and ACC inhibition. Inactivation of ACC promotes mitochondrial fat oxidation. Consistent with the direct effect of CTRP1 on AMPK signaling, recombinant CTRP1 administration acutely stimulated muscle AMPKα and ACC phosphorylation in vivo. In isolated soleus muscle, recombinant CTRP1 activated AMPK signaling to increase fatty acid oxidation ex vivo, an effect abrogated by an AMPK inhibitor. These results provide the first in vivo evidence that CTRP1 is a novel regulator of fatty acid metabolism.     

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.     

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.