NMR Structure of Free RGS4 Reveals an Induced Conformational Change upon Binding Gα

Moy, Fred; Chanda, Pranab K.; Cockett, Mark I.; Edris, Wade; Jones, Philip; Mason, Kim; Semus, Simon F.; Powers, Robert

doi:10.1021/bi992760w

Cited by 39 publications

(33 citation statements)

References 57 publications

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“…Canonical RGS domains consist of a nine-helix bundle comprising two lobes formed by the ␣I, ␣II, ␣III, ␣VIII, and ␣IX helices and the ␣IV, ␣V, ␣VI, and ␣VII helices, respectively. The majority of apo-RGS domain structures we obtained conform to the structural archetype established by RGS4 (6,28). Crystal structures of RGS6 and RGS7 (PDB IDs 2ES0 and 2A72) are atypical, domain-swapped dimers.…”

Section: Resultsmentioning

confidence: 63%

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

Soundararajan

Willard

Kimple

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

187

239

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Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with G␣ when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of G␣, RGS domain binding consequently accelerates G␣-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domaincontaining proteins with varied G␣ substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential G␣ selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/G␣ complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the G␣ substrate, suggests that unique structural determinants specific to particular RGS protein/G␣ pairings exist and could be used to achieve selective inhibition by small molecules.GTPase-accelerating proteins ͉ NMR structure ͉ RGS proteins ͉ x-ray crystallography G protein-coupled receptors (GPCRs) are critical for many physiological processes including vision, olfaction, neurotransmission, and the actions of many hormones (1). As such, GPCRs are the largest fraction of the ''druggable proteome,'' and their ligand-binding and signaling properties remain of considerable interest to academia and industry (2). GPCRs catalyze activation of heterotrimeric G proteins comprising a guanine nucleotide-binding G␣ subunit and an obligate G␤␥ dimer (3). Receptor-promoted activation of G␣␤␥ causes exchange of GDP for GTP by G␣ and resultant dissociation of G␤␥. GTP-bound G␣ and freed G␤␥ then regulate intracellular effectors such as adenylyl cyclase, phospholipase C, ion channels, RhoGEFs, and phosphodiesterases (1, 4). This ''G protein cycle'' is reset by the intrinsic GTP hydrolysis activity of G␣, producing G␣⅐GDP that favors heterotrimer reformation and, consequently, signal termination. Thus, a major determinant of the duration and magnitude of GPCR signaling is the lifetime of G␣ in the GTP-bound state.Regulators of G protein signaling are GTPase-accelerating proteins (GAPs) for G␣ subunits and thus facilitate GPCR signal termination (5). GAP activity is conferred by an RGS domain present in one or more copies within members of this protein superfamily (5). The archetypal RGS domain is composed of nine ␣-helices (6) and binds most avidly to G␣ in the transi...

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Section: Resultsmentioning

confidence: 63%

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

Soundararajan

Willard

Kimple

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

187

239

View full text Add to dashboard Cite

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“…The three-dimensional structures of several RH domains have been determined by x-ray crystallography (RGS4, RGS9, axin, PDZRhoGEF, and p115RhoGEF) (23,24,(52)(53)(54), and solution NMR (GAIP and RGS4) (55,56). Together these studies show that these RH domains share a very similar fold (two four-helix bundles, see Fig.…”

Section: Identification Of Grk2 Rgs Domain Mutants That Are Defective Inmentioning

confidence: 95%

G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction

Sterne‐Marr

Tesmer

Day

et al. 2003

Journal of Biological Chemistry

114

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G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G␣ q/ll and inhibiting G␣ q stimulation of the effector phospholipase C␤. The binding site for G␣ q/ll resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the G␣ q/ll binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G␣ q/ll . These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G␣ q/ll binding defects impair recruitment to the plasma membrane by activated G␣ q and regulation of G␣ q -stimulated phospholipase C␤ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G␣ binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices ␣3 and ␣4, ␣5 and ␣6, and ␣7 and ␣8. The adenomatous polyposis coli (APC) binding site of axin involves residues on ␣ helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G␣ q/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its ␣5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.

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“…The RGSZ1-His 6 protein was purified from E. coli via a nickel-nitrilotriacetic resin-based chromatography. The RGS4 core domain peptide (166 amino acids) and G␣ i1 -His 6 protein were purified as described previously (36). A RGS7 core domain (residues 330 -448) was a gift from Dr. Philip Jones (Wyeth, Princeton, NJ).…”

Section: Methodsmentioning

confidence: 99%

Regulator of G Protein Signaling Z1 (RGSZ1) Interacts with Gαi Subunits and Regulates Gαi-mediated Cell Signaling

Wang

Zhang

et al. 2002

Journal of Biological Chemistry

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Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein ␣ subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for G␣ z subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with G␣ z, but also with G␣ i , as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pulldown assays, glutathione S-transferase-RGSZ1 protein was shown to bind 35 S-labeled G␣ i1 protein in an AlF 4 ؊ dependent manner. The interaction between RGSZ1 and G␣ i was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of G␣ i1 in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast G␣ i -like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an ␣ 2 -adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for G␣ i and regulates G␣ i -mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.Trimeric guanine nucleotide-binding proteins (G proteins) 1 are key components in transducing extracellular signals into intracellular responses (1-5). The functional activity of G protein ␣ subunit is critically governed by the rates of GDP/GTP exchange stimulated by the receptor activation, and the intrinsic GTPase activity that hydrolyzes the GTP to GDP. However, the in vitro measured intrinsic GTPase activity of G protein ␣ subunits is too slow to account for the rapid signal termination that occurs in vivo. A new family of proteins, regulators of G protein signaling (RGS), have been identified to accelerate the intrinsic GTPase activity of G␣ subunits and mechanistically impact the kinetics of G protein physiological function (6 -12). To date, over 20 mammalian proteins are known to contain the RGS domain, a homologous core domain of 120 amino acids. The RGS domain-containing proteins dramatically enhance the endogenous GTPase activity of G␣ subunits by binding to and stabilizing the flexible switch regions of G␣ to resemble that of the transition state, thereby lowering the activation energy barrier of the hydrolysis reaction (13-16). RGS proteins have been shown to play an essential role in desensitizing and negatively regulating G protein-mediated responses or accelerating the k...

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NMR Structure of Free RGS4 Reveals an Induced Conformational Change upon Binding Gα

Cited by 39 publications

References 57 publications

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein α-subunits

G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction

Regulator of G Protein Signaling Z1 (RGSZ1) Interacts with Gαi Subunits and Regulates Gαi-mediated Cell Signaling

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