NORMAL GENOMIC

• Alfaras-Melainis K, Gomes I, Rozenfeld R, Zachariou V, Devi L
• Modulation of opioid receptor function by protein-protein interactions.
• Front Biosci. 2009; 14: 3594-607
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• Opioid receptors, MORP, DORP and KORP, belong to the family A of G proteincoupled receptors (GPCR), and have been found to modulate a large numberof physiological functions, including mood, stress, appetite, nociceptionand immune responses. Exogenously applied opioid alkaloids produceanalgesia, hedonia and addiction. Addiction is linked to alterations infunction and responsiveness of all three opioid receptors in the brain.Over the last few years, a large number of studies identifiedprotein-protein interactions that play an essential role in opioidreceptor function and responsiveness. Here, we summarize interactionsshown to affect receptor biogenesis and trafficking, as well as thoseaffecting signal transduction events following receptor activation. Thisarticle also examines protein interactions modulating the rate of receptorendocytosis and degradation, events that play a major role in opiateanalgesia. Like several other GPCRs, opioid receptors may form homo orheterodimers. The last part of this review summarizes recent knowledge onproteins known to affect opioid receptor dimerization.

• Martemyanov KA, Krispel CM, Lishko PV, Burns ME, Arshavsky VY
• Functional comparison of RGS9 splice isoforms in a living cell.
• Proc Natl Acad Sci U S A. 2008; 105: 20988-93
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• Two isoforms of the GTPase-activating protein, regulator of G proteinsignaling 9 (RGS9), control such fundamental functions as vision andbehavior. RGS9-1 regulates phototransduction in rods and cones, and RGS9-2regulates dopamine and opioid signaling in the basal ganglia. To determinetheir functional differences in the same intact cell, we replaced RGS9-1with RGS9-2 in mouse rods. Surprisingly, RGS9-2 not only supported normalphotoresponse recovery under moderate light conditions but alsooutperformed RGS9-1 in bright light. This versatility of RGS9-2 resultsfrom its ability to inactivate the G protein, transducin, regardless ofits effector interactions, whereas RGS9-1 prefers the G protein-effectorcomplex. Such versatility makes RGS9-2 an isoform advantageous for timelysignal inactivation across a wide range of stimulus strengths and mayexplain its predominant representation throughout the nervous system.

• Heximer SP, Blumer KJ
• RGS proteins: Swiss army knives in seven-transmembrane domain receptorsignaling networks.
• Sci STKE. 2007; 2007: 2-2
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• Coordinated regulation of heterotrimeric guanine nucleotide-bindingprotein (G protein) activity is critical for the integration ofinformation from multiple intracellular signaling networks. The humanregulator of G protein signaling (RGS) protein family contains more than35 members that are well suited for this purpose. Although all RGSproteins contain a core ~120-amino acid Galpha-interacting domain (calledthe RGS domain), they differ widely in size and organization of otherfunctional domains. Architecturally complex RGS proteins contain multiplemodular protein-protein interaction domains that mediate their interactionwith diverse signaling effectors. Architecturally simple RGS proteinscontain small amino-terminal domains; however, they show surprisingversatility in the number of intracellular partners with which theyinteract. This Perspective focuses on RGS2, a simple RGS protein with thepotential to integrate multiple signaling networks. In three recentstudies, the amino-terminal domain of RGS2 was shown to interact with andregulate three different effector proteins: adenylyl cyclase, tubulin, andthe cation channel TRPV6. To explain this growing list of RGS2-interactingpartners, we propose two models: (i) The amino-terminal domain of RGS2comprises several short effector protein interaction motifs; (ii) theamino-terminal domain of RGS2 adopts distinct structures to bind varioustargets. Whatever the precise mechanism controlling its targetinteractions, these studies suggest that RGS2 is a key point ofintegration for multiple intracellular signaling pathways, and theyhighlight the role of RGS proteins as dynamic, multifunctional signalingcenters that coordinate a diverse range of cellular functions.

• Ajit SK, Young KH
• Analysis of chimeric RGS proteins in yeast for the functional evaluationof protein domains and their potential use in drug target validation.
• Cell Signal. 2005; 17: 817-25
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• For the identification of regulators of G-protein signaling (RGS)modulators, previously, we developed a luciferase based yeast pheromoneresponse (YPhR) assay to functionally investigate RGS4 (K.H. Young, Y.Wang, C. Bender, S. Ajit, F. Ramirez, A. Gilbert, B.W. Nieuwenhuijsen, in:D.P. Siderovski (Ed.), Meth. Enzymol. 389 Regulators of G_proteinSignaling, Part A, 2004.). To extend the diversity of this assay,additional RGS proteins were evaluated for functional complementation in aRGS (sst2Delta) knockout yeast strain. For RGS proteins that did notfunction in their native form, a series of chimeric constructs weregenerated with the N terminus of RGS4 fused in frame with the partial orfull-length RGS cDNA of interest. RGS4 N terminus fused to eitherfull-length or the C terminus of RGS7 successfully complemented sst2Delta.On the contrary, the RGS7N/RGS4C chimera (N terminus of RGS7 in frame withRGS domain of RGS4) was not effective, showing that N terminus of RGS4helps in targeting. RGS10 exists as two splice variants, differing only by8 amino acids (aa) in the N terminus, being either 168 aa (RGS10S), or 174aa (RGS10). While RGS10 was functional in yeast, RGS10S required thepresence of the N terminus of RGS4 for its activity. Although the sameRGS4 N terminus domain was present in chimeras generated, the GTPaseaccelerating protein (GAP) function observed was not similar, suggestingdifferences in the RGS domain function. In conclusion, the use of RGS4 Nterminus chimeric constructs enabled us to develop a selectivity assay fordifferent RGS proteins.

• Yildirim N, Hao N, Dohlman HG, Elston TC
• Mathematical modeling of RGS and G-protein regulation in yeast.
• Methods Enzymol. 2004; 389: 383-98
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• G-protein-activated signaling pathways are capable of adapting to apersistent external stimulus. Desensitization is thought to occur at thereceptor level as well as through negative feedback by a family ofproteins called regulators of G-protein signaling (RGS). The pheromoneresponse pathway in yeast is a typical example of such a system, and therelative simplicity of this pathway makes it an attractive system ininvestigating the regulatory role of RGS proteins. Two studies have usedcomputational modeling to gain insight into how this pathway is regulated(Hao et al., 2003; Yi et al., 2003). This article provides an introductionto computational analysis of signaling pathways by developing amathematical model of the pheromone response pathway that synthesizes theresults of these two investigations. Our model qualitatively captures manyfeatures of the pathway and suggests an additional mechanism for pathwayinactivation. It also illustrates that a complete understanding ofsignaling pathways requires an investigation of their time-dependentbehavior.

• Clark MJ, Harrison C, Zhong H, Neubig RR, Traynor JR
• Endogenous RGS protein action modulates mu-opioid signaling throughGalphao. Effects on adenylyl cyclase, extracellular signal-regulatedkinases, and intracellular calcium pathways.
• J Biol Chem. 2003; 278: 9418-25
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• RGS (regulators of G protein signaling) proteins are GTPase-activatingproteins for the Galpha subunits of heterotrimeric G proteins and act toregulate signaling by rapidly cycling G protein. RGS proteins mayintegrate receptors and signaling pathways by physical or kineticscaffolding mechanisms. To determine whether this results in enhancementand/or selectivity of agonist signaling, we have prepared C6 cells stablyexpressing the mu-opioid receptor and either pertussis toxin-insensitiveor RGS- and pertussis toxin-insensitive Galpha(o). We have compared theactivation of G protein, inhibition of adenylyl cyclase, stimulation ofintracellular calcium release, and activation of the ERK1/2 MAPK pathwaybetween cells expressing mutant Galpha(o) that is either RGS-insensitiveor RGS-sensitive. The mu-receptor agonist[d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine weremuch more potent and/or had an increased maximal effect in inhibitingadenylyl cyclase and in activating MAPK in cells expressingRGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases inintracellular calcium were less affected. The results are consistent withthe hypothesis that the GTPase-activating protein activity of RGS proteinsprovides a control that limits agonist action through effector pathwaysand may contribute to selectivity of activation of intracellular signalingpathways.

• Wang Y et al.
• Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha isubunits and regulates Galpha i-mediated cell signaling.
• J Biol Chem. 2002; 277: 48325-32
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• Regulator of G protein signaling (RGS) proteins constitute a family ofover 20 proteins that negatively regulate heterotrimeric G protein-coupledreceptor signaling pathways by enhancing endogenous GTPase activities of Gprotein alpha subunits. RGSZ1, one of the RGS proteins specificallylocalized to the brain, has been cloned previously and described as aselective GTPase accelerating protein for Galpha(z) subunit. Here, weemployed several methods to provide new evidence that RGSZ1 interacts notonly with Galpha(z,) but also with Galpha(i), as supported by in vitrobinding assays and functional studies. Using glutathione S-transferasefusion protein pull-down assays, glutathione S-transferase-RGSZ1 proteinwas shown to bind (35)S-labeled Galpha(i1) protein in anAlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) wasconfirmed further by co-immunoprecipitation studies and yeast two-hybridexperiments using a quantitative luciferase reporter gene. Extending theseobservations to functional studies, RGSZ1 accelerated endogenous GTPaseactivity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediatedyeast pheromone response when expressed in a SST2 (yeast RGS protein)knockout strain. In PC12 cells, transfected RGSZ1 blockedmitogen-activated protein kinase activity induced by UK14304, analpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2dopamine receptor agonist-induced serum response element reporter geneactivity in Chinese hamster ovary cells. In summary, these data suggestthat RGSZ1 serves as a GTPase accelerating protein for Galpha(i) andregulates Galpha(i)-mediated signaling, thus expanding the potential roleof RGSZ1 in G protein-mediated cellular activities.

• Hollinger S, Taylor JB, Goldman EH, Hepler JR
• RGS14 is a bifunctional regulator of Galphai/o activity that exists inmultiple populations in brain.
• J Neurochem. 2001; 79: 941-9
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• Members of the regulators of G protein signaling (RGS) family modulateGalpha-directed signals as a result of the GTPase-activating protein (GAP)activity of their conserved RGS domain. In addition to its RGS domain,RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To definethe cellular and biochemical properties of RGS14 we utilized two differentaffinity purified antisera that specifically recognize recombinant andnative RGS14. In brain, we observed two RGS14-like immunoreactive bands ofdistinct size (60 kDa and 55 kDa). Both forms are present in brain cytosoland in two, biochemically distinct, membrane subpopulations: onedetergent-extractable and the other detergent-insensitive. RecombinantRGS14 binds specifically to activated Galphai/o, but not Galphaq/11,Galpha12/13, or Galphas in brain membranes. In reconstitution studies, wefound that RGS14 is a non-selective GAP for Galphai1 and Galphao and thatfull-length RGS14 is an approximately 10-fold more potent stimulator ofGalpha GTPase activity than the RGS domain alone. In contrast, neitherfull-length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 isalso a highly selective guanine nucleotide dissociation inhibitor (GDI)for Galphai but not Galphao, and this activity is restricted to theC-terminus containing the GoLoco domain. These findings highlightpreviously unknown biochemical properties of RGS14 in brain, and provideone of the first examples of an RGS protein that is a bifunctionalregulator of Galpha actions.

• Hepler JR
• Emerging roles for RGS proteins in cell signalling.
• Trends Pharmacol Sci. 1999; 20: 376-82
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• Regulators of G-protein signalling (RGS proteins) are a family of highly diverse, multifunctional signalling proteins that share a conserved 120 amino acid domain (RGS domain). RGS domains bind directly to activated Galpha subunits and act as GTPase-activating proteins (GAPs) to attenuate and/or modulate hormone and neurotransmitter receptor-initiated signalling by both Galpha-GTP and Gbetagamma. Apart from this structural domain, which is shared by all known RGS proteins, these proteins differ widely in their overall size and amino acid identity and possess a remarkable variety of structural domains and motifs. These biochemical features impart signalling functions and/or enable RGS proteins to interact with a growing list of unexpected protein-binding partners with diverse cellular roles. New appreciation for the broader cellular functions of RGS proteins challenges established models of G-protein signalling and serves to identify these proteins as central participants in receptor signalling and cell physiology.

• Kehrl JH
• Heterotrimeric G protein signaling: roles in immune function andfine-tuning by RGS proteins.
• Immunity. 1998; 8: 1-10