RGS Homology Domain Research Articles

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling.

G protein‐coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2‐mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates’ ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation‐independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

Gα s Directly Drives PDZ‐RhoGEF Signaling to Cdc42

G proteins stimulate RhoGEFs that control cell morphology through actin-cytoskeleton remodeling. Gα12 and Gα13 interact with RH-RhoGEFs (RGS-homology domain RhoGEFs), driving RhoA activation. However, whether additional Gα proteins directly regulate RH-RhoGEFs was not known. To respond this question, we first evaluated the morphological effects of constitutively active DH/PH constructs from p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, consistent with their known specificity. Intriguingly, PRG-DH/PH also induced filopodia-like cell protrusions and directly activated Cdc42. Constitutively active Gαs (GαsQ227L) increased the ability of PRG-DH/PH or endogenous PRG to interact with and activate Cdc42. Using a chemogenetic approach, with DREADD receptors, we demonstrated that Gs-coupled receptors, but not those coupled to Gi or Gq, drive PRG activity towards Cdc42. Given that Gαs interacted with PRG DH/PH region, we looked for potential inhibitory effects of the minimal fragment from PRG (linker) that binds GαsQ227L. This construct, the PRG-linker, interfered with GαsQ227L:PRG interaction and PRG:Cdc42 affinity. Besides, it attenuated Gαs signaling by Gs-DREADDs and Gs-coupled prostaglandin receptors (assessed by phosphorylation of CREB and PKA substrates). Altogether, our results demonstrate that Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor.

G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although β3-adrenergic receptor (β3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce β3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of β3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human β3AR presented a short-term desensitization of cAMP response stimulated by the β3AR agonist, BRL37344, and not by forskolin. We found that β3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased β3AR desensitization. Consistently, stimulation of β3AR increased interaction between GRK2 and Gαs subunit. Furthermore, in rat cardiomyocytes endogenously expressing β3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the β3AR in heart function and disease.

Regulator of G-Protein Signaling (RGS) Protein Modulation of Opioid Receptor Signaling as a Potential Target for Pain Management.

Opioid drugs are the gold standard for the management of pain, but their use is severely limited by dangerous and unpleasant side effects. All clinically available opioid analgesics bind to and activate the mu-opioid receptor (MOR), a heterotrimeric G-protein-coupled receptor, to produce analgesia. The activity of these receptors is modulated by a family of intracellular RGS proteins or regulators of G-protein signaling proteins, characterized by the presence of a conserved RGS Homology (RH) domain. These proteins act as negative regulators of G-protein signaling by serving as GTPase accelerating proteins or GAPS to switch off signaling by both the Gα and βγ subunits of heterotrimeric G-proteins. Consequently, knockdown or knockout of RGS protein activity enhances signaling downstream of MOR. In this review we discuss current knowledge of how this activity, across the different families of RGS proteins, modulates MOR activity, as well as activity of other members of the opioid receptor family, and so pain and analgesia in animal models, with particular emphasis on RGS4 and RGS9 families. We discuss inhibition of RGS proteins with small molecule inhibitors that bind to sensitive cysteine moieties in the RH domain and the potential for targeting this family of intracellular proteins as adjuncts to provide an opioid sparing effect or as standalone analgesics by promoting the activity of endogenous opioid peptides. Overall, we conclude that RGS proteins may be a novel drug target to provide analgesia with reduced opioid-like side effects, but that much basic work is needed to define the roles for specific RGS proteins, particularly in chronic pain, as well as a need to develop newer inhibitors.

Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening

AimsG protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. Materials and methodsWe performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549 cells. GRK2(45–178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. Key findingsWe identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SignificanceWe obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.

Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins

BackgroundRegulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.HypothesisDifferences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.ApproachThe rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.ResultsDeuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.ConclusionThese results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.Support or Funding InformationNIH T32 GM092715NSF 1507588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Gα12-specific Binding Domain in AKAP-Lbc and p114RhoGEF.

AKAP-Lbc is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in heart development and pro-fibrotic signaling in cardiomyocytes. Heterotrimeric G proteins of the G12/13 subfamily, comprising Gα12 and Gα13, are well characterized as stimulating a specialized group of RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking an RH domain, AKAP-Lbc is bound by Gα12 through an unknown mechanism to activate Rho signaling. We identified a Gα12-binding region near the C-terminus of AKAP-Lbc, closely homologous to a region of p114RhoGEF that we also discovered to interact with Gα12. This binding mechanism is distinct from the well-studied interface between RH-RhoGEFs and G12/13 α subunits, as demonstrated by Gα12 mutants selectively impaired in binding either this AKAP-Lbc/p114RhoGEF region or RH-RhoGEFs. AKAP-Lbc and p114RhoGEF showed high specificity for binding Gα12 in comparison to Gα13, and experiments using chimeric G12/13 α subunits mapped determinants of this selectivity to the N-terminal region of Gα12. In cultured cells expressing constitutively GDP-bound Gα12 or Gα13, the Gα12 construct was more potent in exerting a dominant-negative effect on serum-mediated signaling to p114RhoGEF, demonstrating coupling of these signaling proteins in a cellular pathway. In addition, charge-reversal of conserved residues in AKAP-Lbc and p114RhoGEF disrupted Gα12 binding for both proteins, suggesting they harbor a common structural mechanism for interaction with this α subunit. Our results provide the first evidence of p114RhoGEF as a Gα12 signaling effector, and define a novel region conserved between AKAP-Lbc and p114RhoGEF that allows Gα12 signaling input to these non-RH RhoGEFs.

Regulatory effects of GRK2 on GPCRs and non-GPCRs and possible use as a drug target (Review).

Gprotein-coupled receptor kinase2(GRK2) is a key member of the Gprotein-coupled receptor kinase(GRK) family. GRK2 activity is regulated by the C-terminus of GRK2 which contains a plekstrin homology domain and the N-terminus of GRK2 which contains the RGS homology domain with binding sites for several proteins and lipids such as Gprotein-coupled receptors(GPCRs), Gprotein, phospholipaseC, phosphatidylinositol 4,5-bisphosphate, extracellular signal‑regulated kinase, protein kinaseA and Gβγ. GRK2 phosphorylates the GPCR and enhances the affinity of binding to arrestins, which uncouple the receptors from G proteins, and target the receptors for desensitization and internalization. GRK2 also regulates non‑GPCR desensitization and internalization by phosphorylation, and is important in maintaining the balance between the receptors and signal transduction. Previous findings have indicated that the upregulation of GRK2 in heart failure enhances dysfunctional adrenergic signaling and myocyte death. Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast-like synoviocytes. In this review, we discussed the evidence for the association between altered GRK2 levels and various diseases, which suggests that GRK2 may be an effective drug target for preventing and treating heart failure, hypertension and inflammatory disease.

Using NMR‐Based Fragment Screening to Probe the Druggability of Regulator of G Protein Signaling 17

Regulator of G protein Signaling 17 (RGS17), as a GTPase accelerating protein (GAP), temporally controls G protein‐coupled receptor activity by accelerating the intrinsic rate of GTP hydrolysis of inhibitory Gα subunits, thus terminating signaling. Recent evidence has shown that RGS17 expression is drastically increased in malignancies in various tissues, including lung, breast, prostate, and liver, and inhibition of RGS17 via RNA interference is sufficient to slow cancer growth and progression. This makes RGS17 an attractive target for the development of small molecule inhibitors that could serve as chemotherapeutics with a novel mechanism of action. In order to identify novel druggable sites on the RGS homology domain of RGS17, we performed triple resonance NMR experiments using 15N, 13C doubly labeled protein samples to assign the protein. We then screened a library of 1000 fragments and assessed compound binding via chemical shift perturbation in protein‐detected 1H‐15N HSQC spectra. Seven hits were identified with affinities ranging from 530 μM to 5.2 mM and ligand efficiencies from 0.18 to 0.31. Interestingly, none of these identified hits bound directly to the region of RGS17 that forms the Gα interaction interface. Six fragments bound between α6 and α7 in the helical bundle domain, a site distant from the Gα interface. Additionally, one hit, an isothiazaolone, interacts with RGS17 in a covalent manner, showing that RGS17, like other RGS family members RGS4 and RGS8, is subject to inhibition via cysteine‐modification. Subsequent experimental and computational efforts will focus on structure‐activity relationship optimization of these hits to increase their affinities for RGS17 to further explore the druggabiliy of this allosteric site, as well as to facilitate the determination of the structure of the RGS17:fragment complex with atomic resolution.Support or Funding InformationThis work was supported by NIH 5R01CA160470 (DLR) and an American Foundation for Pharmaceutical Education Predoctoral Fellowship (MPH).

GRK3 suppresses L-DOPA-induced dyskinesia in the rat model of Parkinson’s disease via its RGS homology domain

Degeneration of dopaminergic neurons causes Parkinson’s disease. Dopamine replacement therapy with L-DOPA is the best available treatment. However, patients develop L-DOPA-induced dyskinesia (LID). In the hemiparkinsonian rat, chronic L-DOPA increases rotations and abnormal involuntary movements modeling LID, via supersensitive dopamine receptors. Dopamine receptors are controlled by G protein-coupled receptor kinases (GRKs). Here we demonstrate that LID is attenuated by overexpression of GRK3 in the striatum, whereas knockdown of GRK3 by microRNA exacerbated it. Kinase-dead GRK3 and its separated RGS homology domain (RH) suppressed sensitization to L-DOPA, whereas GRK3 with disabled RH did not. RH alleviated LID without compromising anti-akinetic effect of L-DOPA. RH binds striatal Gq. GRK3, kinase-dead GRK3, and RH inhibited accumulation of ∆FosB, a marker of LID. RH-dead mutant was ineffective, whereas GRK3 knockdown exacerbated ∆FosB accumulation. Our findings reveal a novel mechanism of GRK3 control of the dopamine receptor signaling and the role of Gq in LID.

Identification of Gα12‐specific binding determinants in AKAP‐Lbc (802.8)

The Rho family of small GTP‐binding proteins are key regulators of cytoskeletal dynamics and cell growth. Rho activation is triggered by guanine nucleotide exchange factors (RhoGEFs), and a specific subset of these proteins, including leukemia‐associated RhoGEF (LARG), are stimulated by trimeric G protein α subunits of the G12/13 class. This interaction is mediated by a regulator of G protein signaling (RGS) homology domain common to these RhoGEFs; however, a RhoGEF lacking an RGS homology domain, termed AKAP‐Lbc, is also activated by the G12/13 class in a signaling pathway required in cardiac development. To identify AKAP‐Lbc regions involved in Gα12 binding, we compared AKAP‐Lbc to other Gα12 targets and found a C‐terminal region with weak homology to axin. A GST‐fusion of this region co‐precipitated Gα12 in constitutively active and inactive forms, in contrast to the activation‐dependent interaction of Gα12 with LARG. In addition, binding experiments utilizing Gα12 and Gα13 harboring a common epitope tag revealed this AKAP‐Lbc domain as preferential for Gα12 interaction, whereas the RGS homology domain of LARG bound Gα13 with greater affinity than Gα12. These findings reveal a novel, Gα12‐specific mechanism for engagement of a non‐RGS RhoGEF by the G12/13 class of trimeric G proteins.Grant Funding Source: Supported by the GlaxoSmithKline Foundation and Lineberger Comprehensive Cancer Center

Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins

The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs.

Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

Mitochondrial localization unveils a novel role for GRK2 in organelle biogenesis

Metabolic stimuli such as insulin and insulin like growth factor cause cellular accumulation of G protein coupled receptor kinase 2 (GRK2), which in turn is able to induce insulin resistance. Here we show that in fibroblasts, GRK2 is able to increase ATP cellular content by enhancing mitochondrial biogenesis; also, it antagonizes ATP loss after hypoxia/reperfusion. Interestingly, GRK2 is able to localize in the mitochondrial outer membrane, possibly through one region within the RGS homology domain and one region within the catalytic domain. In vivo, GRK2 removal from the skeletal muscle results in reduced ATP production and impaired tolerance to ischemia. Our data show a novel sub-cellular localization of GRK2 in the mitochondria and an unexpected role in regulating mitochondrial biogenesis and ATP generation.

Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

Activation of G protein-coupled receptors at the cell surface leads to the activation or inhibition of intracellular effector enzymes, which include various Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs activate small molecular weight GTPases at the plasma membrane (PM). Many of the known G protein-coupled receptor-regulated RhoGEFs are found in the cytoplasm of unstimulated cells, and PM recruitment is a critical aspect of their regulation. In contrast, p63RhoGEF, a Gα(q)-regulated RhoGEF, appears to be constitutively localized to the PM. The objective of this study was to determine the molecular basis for the localization of p63RhoGEF and the impact of its subcellular localization on its regulation by Gα(q). Herein, we show that the pleckstrin homology domain of p63RhoGEF is not involved in its PM targeting. Instead, a conserved string of cysteines (Cys-23/25/26) at the N terminus of the enzyme is palmitoylated and required for membrane localization and full basal activity in cells. Conversion of these residues to serine relocates p63RhoGEF from the PM to the cytoplasm, diminishes its basal activity, and eliminates palmitoylation. The activity of palmitoylation-deficient p63RhoGEF can be rescued by targeting to the PM by fusion with tandem phospholipase C-δ1 pleckstrin homology domains or by co-expression with wild-type Gα(q) but not with palmitoylation-deficient Gα(q). Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by Gα(q), as opposed to other known Gα-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their high basal activity.

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.

Intracardiac Injection of AdGRK5-NT Reduces Left Ventricular Hypertrophy by Inhibiting NF-κB–Dependent Hypertrophic Gene Expression

Several studies underline the role of the transcription factor NF-κB in the development of left cardiac hypertrophy (LVH). We have demonstrated recently that the RGS homology domain within the amino terminus of GRK5 (GRK5-NT) is able to inhibit NF-κB transcription activity and its associated phenotypes. The aim of this study was to evaluate the ability of GRK5-NT to regulate LVH through the inhibition of NF-κB both in vitro and in vivo. In cardiomyoblasts, GRK5-NT inhibits phenylephrine-induced transcription of both NF-κB and atrial natriuretic factor promoters, assessed by luciferase assay, thus confirming a role for this protein in the regulation of cardiomyocyte hypertrophy. In vivo, we explored 2 rat models of LVH, the spontaneously hypertensive rat and the normotensive Wistar Kyoto rat exposed to chronic administration of phenylephrine. Intracardiac injection of an adenovirus encoding for GRK5-NT reduces cardiac mass in spontaneously hypertensive rats and prevents the development of phenylephrine-induced LVH in Wistar Kyoto rats. This associates with inhibition of NF-κB signaling (assessed by NF-κB levels), transcriptional activity and phenotypes (fibrosis and apoptosis). Such phenomenon is independent from hemodynamic changes, because adenovirus encoding for GRK5-NT did not reduce blood pressure levels in spontaneously hypertensive rats or in Wistar Kyoto rats. In conclusion, our study supports the regulation of LVH based on the GRK5-NT inhibition of the NF-κB transduction signaling.

Activation of Leukemia-associated RhoGEF by Gα13 with Significant Conformational Rearrangements in the Interface

The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).

Distinct regions of Gα13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs

Gα12 and Gα13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Gα12 and Gα13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Gα13 than in Gα12. At the same time, Gα13, but not Gα12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Gα13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Gα13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Gα12 and Gα13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Gα12 (residues 267–379) with that of Gα13 (residues 264–377) conferred gain-of-function to the resulting chimeric subunit, Gα12C13. The inverse chimera, Gα13C12, exhibited basal RhoA activation which was similar to Gα12. In contrast to GEF assays, GAP assays showed that Gα12C13 or Gα13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Gα12 or Gα13, respectively. We conclude from these results that the carboxyl-terminal region of Gα13 (residues 264–377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal α helical and switch regions of Gα12 and Gα13 are responsible for their differential GAP responses to the RH domain.

G Protein-coupled Receptor (GPCR) Kinase 2 Regulates Agonist-independent Gq/11 Signaling from the Mouse Cytomegalovirus GPCR M33

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.