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G Protein Signaling (g + protein_signaling)
Selected AbstractsRegulators of G Protein SignalingJOURNAL OF NEUROCHEMISTRY, Issue 4 2000A Bestiary of Modular Protein Binding Domains Abstract: Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of G, protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C- and N-terminal modular protein-binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific G,-coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross-talk between different G,-coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C- and N-terminal domains and their roles in the biology of the brain-enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed. [source] Alanine screening of the intracellular loops of the human bradykinin B2 receptor , effects on receptor maintenance, G protein activation and internalizationFEBS JOURNAL, Issue 13 2009Alexander Faussner The bradykinin B2 receptor is coupled to G protein Gq/11 and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY , AAA, retained the ability to specifically bind [3H]bradykinin. The binding affinities at 4 or 37 °C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [3H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in Gq/11 activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability. [source] Novel Mechanisms for Feedback Regulation of Phospholipase C-, ActivityIUBMB LIFE, Issue 5 2002Irene Litosch Abstract The receptor-regulated phospholipase C- ,(PLC- ,) signaling pathway is an important component in a network of signaling cascades that regulate cell function. PLC- ,signaling has been implicated in the regulation of cardiovascular function and neuronal plasticity. The G q family of G proteins mediate receptor stimulation of PLC- ,activity at the plasma membrane. Mitogens stimulate the activity of a nuclear pool of PLC- ,. Stimulation of PLC- ,activity results in the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, with production of inositol-1,4,5-trisphosphate and diacylglycerol, intracellular mediators that increase intracellular Ca 2+ levels and activate protein kinase C activity, respectively. Diacylglycerol kinase converts diacylglycerol to phosphatidic acid, a newly emerging intracellular mediator of hormone action that targets a number of signaling proteins. Activation of the G q linked PLC- ,signaling pathway can also generate additional signaling lipids, including phosphatidylinositol-3-phosphate and phosphatidylinositol-3,4,5-trisphosphate, which regulate the activity and/or localization of a number of proteins. Novel feedback mechanisms, directed at the level of G q and PLC- ,, have been identified. PLC- ,and regulators of G protein signaling (RGS) function as GTPase-activating proteins on G q to control the amplitude and duration of stimulation. Protein kinases phosphorylate and regulate the activation of specific PLC- ,isoforms. Phosphatidic acid regulates PLC- ,1 activity and stimulation of PLC- ,1 activity by G proteins. These feedback mechanisms coordinate receptor signaling and cell activation. Feedback mechanisms constitute possible targets for pharmacological intervention in the treatment of disease. [source] Reversible translocation of p115-RhoGEF by G12/13 -coupled receptorsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Bruno H. Meyer Abstract G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, Gs, Gi, Gq, and G12, engage in their linkage to activation of receptor-specific signal transduction pathways. G12 proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G12/13 -Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active G,12 and G,13 mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G12/13 -coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G12/13 -linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G12/13 -related disease context. J. Cell. Biochem. 104: 1660,1670, 2008. © 2008 Wiley-Liss, Inc. [source] RGS9-2 mediates specific inhibition of agonist-induced internalization of D2 -dopamine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Jeremy Celver J. Neurochem. (2010) 114, 739,749. Abstract Regulator of G protein signaling 9-2 (RGS9-2), a member of the RGS family of GTPase accelerating proteins, is expressed specifically in the striatum, a brain region involved in controlling movement, motivation, mood and addiction. RGS9-2 can be found co-localized with D2 -class dopamine receptors in medium spiny striatal neurons and altered functioning of both RGS9-2 and D2 -like dopamine receptors have been implicated in schizophrenia, movement disorders and reward responses. Previously we showed that RGS9-2 can specifically co-localize with D2 -dopamine receptors (D2R). Here we provide further evidence of the specificity of RGS9-2 for regulating D2R cellular functions: the expression of RGS9-2 inhibits dopamine-mediated cellular internalization of D2R, while the expression of another RGS protein, RGS4, had no effect. In addition, the agonist-mediated internalization of the G protein coupled delta opioid receptor was unaffected by RGS9-2 expression. We utilized mutant constructs of RGS9-2 to show that the RGS9-2 DEP (for Disheveled, EGL-10, Pleckstrin homology) domain and the GTPase accelerating activity of RGS9-2 were necessary for mediating specific inhibition of D2R internalization. [source] RGS7 Is Palmitoylated and Exists as Biochemically Distinct FormsJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Jeremy J. Rose Abstract:Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners G, and G,5 are enriched in brain, but biochemical mechanisms governing RGS7/G,/G,5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and G,5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. G,5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with G,5, are equally effective stimulators of G,o GTPase activity, suggesting that palmitoylation does not prevent RGS7/G,o interactions. The isolated core RGS domain of RGS7 selectively binds activated G,i/o in brain extracts and is an effective stimulator of both G,o and G,i1 GTPase activities in vitro. In contrast, the RGS7/G,5 complex selectively interacts with G,o only, suggesting that features outside the RGS domain and/or G,5 association dictate RGS7-G, interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated. [source] Regulators of G Protein SignalingJOURNAL OF NEUROCHEMISTRY, Issue 4 2000A Bestiary of Modular Protein Binding Domains Abstract: Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of G, protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C- and N-terminal modular protein-binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific G,-coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross-talk between different G,-coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C- and N-terminal domains and their roles in the biology of the brain-enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed. [source] Identification of RGS1 as a candidate biomarker for undifferentiated spondylarthritis by genome-wide expression profiling and real-time polymerase chain reactionARTHRITIS & RHEUMATISM, Issue 11 2009Jieruo Gu Objective To compare gene expression profiles between ankylosing spondylitis (AS) and undifferentiated spondylarthritis (uSpA) patients with inflammatory low back pain. Methods Peripheral blood mononuclear cells (PBMCs) from patients with AS, patients with uSpA, and healthy subjects were screened using genome-wide microarrays, followed by validation by real-time polymerase chain reaction (PCR). Results Microarray profiling and real-time PCR assays showed only minor differences between AS patients and healthy subjects. In contrast, 20 genes were strikingly more highly expressed in uSpA patients. Regulator of G protein signaling 1 (RGS1) was identified as the most useful biomarker for distinguishing uSpA patients, and to a lesser extent AS patients, from control subjects (P = 2.3 × 10,7 and 6.7 × 10,3, respectively). These findings were verified in an independent cohort that also included patients with rheumatoid arthritis and patients with mechanical low back pain. The receiver operating characteristic area under the curve values in the first and second cohorts of uSpA patients were 0.99 and 0.93, respectively (P = 1 × 10,4). To evaluate the possible derivation of RGS1, we cultured a monocyte-derived cell line with a panel of cytokines and chemokines. RGS1 was significantly induced either by tumor necrosis factor , (TNF,) or by interleukin-17 (IL-17). Conclusion Our findings indicate that uSpA PBMCs carry strikingly more highly expressed genes compared with PBMCs from AS patients or healthy subjects, and that TNF,- and IL-17,inducible RGS1 is a potential biomarker for uSpA, and to a lesser extent for AS, with inflammatory low back pain. [source] Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound LibraryCHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2008Rebecca A. Roof Regulators of G protein signaling accelerate GTP hydrolysis by G, subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of regulators of G protein signaling proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation, and structure,activity relationships of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac -Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu- NH2, S-S) (Roof et al., Chem Biol Drug Des, 67, 2006, 266). Using a focused one-bead, one-compound peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2 (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp- NH2, S-S with a free or acetylated N -terminus) inhibited RGS4-stimulated G,o GTPase activity at 25,50 ,m. They selectively inhibit RGS4 but not RGS7, RGS16, and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation. 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