A new family of regulators of G-protein-coupled receptors?

Siderovski, David P.; Hessel, Andrew; Chung, Shinjae; Mak, Tak W.; Tyers, Michael

doi:10.1016/s0960-9822(02)00454-2

Cited by 204 publications

(140 citation statements)

References 8 publications

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“…Interestingly, Siderovski et al (17) identified sequence homology between RGS domains and an ϳ120 residue region in the N terminus of GRKs through a BLAST search of the NCBI protein data base (17). Indeed, both GRK2 and GRK3 (residues 51-173) are ϳ20% identical and ϳ30% similar to various RGS domains (Fig.…”

Section: Homology Between the N Terminus Of Grks And Rgs Domains-whermentioning

confidence: 99%

“…In general, these RGS proteins interact with the ␣ subunits of the G i and G q families (10,11,16). In addition, a small collection of proteins including GRKs (17), axin (18), D-AKAP (19), and p115 Rho-GEF (20,21) have been identified as having somewhat less conserved RGS domains. Recently, one of these atypical RGS proteins, p115 Rho-GEF, was shown to function as a selective GAP for G␣ 12/13 suggesting that sequence differences in these RGS proteins may correlate with different preferences for G protein-binding partners (20,21).…”

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confidence: 99%

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Selective Regulation of Gαq/11 by an RGS Domain in the G Protein-coupled Receptor Kinase, GRK2

Carman¹,

Parent²,

Day³

et al. 1999

Journal of Biological Chemistry

301

300

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؊ . This revealed the specific ability of bovine brain G␣ q/11 to bind to both GRK2 and GRK3 in an AlF 4 ؊ -dependent manner. In contrast, G␣ s , G␣ i , and G␣ 12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain G␣ q/11 could also bind to an N-terminal construct of GRK2, while no binding of G␣ q/11 , G␣ s , G␣ i , or G␣ 12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified G␣ q revealed significant binding of both G␣ q GDP/AlF 4 ؊ and G␣ q (GTP␥S), but not G␣ q (GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active G␣ q (R183C) but not wild type G␣ q . In vitro analysis revealed that GRK2 possesses weak GAP activity toward G␣ q that is dependent on the presence of a G proteincoupled receptor. However, GRK2 effectively inhibited G␣ q -mediated activation of phospholipase C-␤ both in vitro and in cells, possibly through sequestration of activated G␣ q . These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

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Section: Homology Between the N Terminus Of Grks And Rgs Domains-whermentioning

confidence: 99%

mentioning

confidence: 99%

Selective Regulation of Gαq/11 by an RGS Domain in the G Protein-coupled Receptor Kinase, GRK2

Carman¹,

Parent²,

Day³

et al. 1999

Journal of Biological Chemistry

301

300

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“…Although the mutated residues were spread over the entire NH 2 -terminal region, there are particularly high concentrations of mutations localized to the first 10 residues and to the central portion of the NH 2 -terminal domain (residues 72 to 87) suggesting the functional importance of these regions. Although GRK5 interaction with G␣ subunits has not been reported, this central region falls within the regulator of G protein signaling (RGS) homology domain in GRKs (encompassing residues ϳ40 -180 in GRK5) that mediates GRK2 interaction with G␣ q (31)(32)(33)(34). Glu-72, Pro-75, and Val-87 may be particularly important because multiple clones were identified with these residues mutated.…”

Section: Figmentioning

confidence: 99%

Development of a Yeast Bioassay to Characterize G Protein-coupled Receptor Kinases

Noble

Kallal

Pausch

et al. 2003

Journal of Biological Chemistry

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G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH 2 -terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH 2 -terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH 2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.A diverse array of extracellular stimuli transduce their signals through interaction with G protein-coupled receptors (GPCRs). 1 A critical process that occurs in most cells is the regulation of hormonal responsiveness, a phenomenon often termed desensitization. Whereas multiple mechanisms contribute to the regulation of GPCR function, G protein-coupled receptor kinases (GRKs) and arrestins play an important role in many cells (1-3). GRKs comprise a family of serine/threonine kinases that are uniquely able to associate with the agonistoccupied form of receptors (3-5). Signaling is terminated upon receptor phosphorylation and the subsequent binding of arrestins, effectively uncoupling receptor from G protein. Mammalian GRKs are classified into three subgroups according to their sequence homology: GRK1 and -7; GRK2 and -3; and GRK4, -5, and -6 (4 -6). GRKs share a common structural organization that includes a moderately conserved (ϳ20% identity) NH 2 -terminal domain of ϳ185 residues, a conserved (ϳ50% identity) central catalytic domain of ϳ330 residues, and a poorly conserved COOH-terminal domain of ϳ80 -180 residues (4, 5).An important feature of GRKs is their ability to specifically interact with activated receptor...

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“…All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9)(10)(11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14).…”

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confidence: 99%

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Srinivasa

Bernstein

Blumer

et al. 1998

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

139

154

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RGS4, a mammalian GTPase activating protein for G protein ␣ subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (⌬1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (⌬1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green f luorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.Heterotrimeric G proteins couple receptors for hormones, neurotransmitters, and sensory signals to intracellular effector molecules, thereby eliciting cellular responses (1, 2). Guanine nucleotide exchange and hydrolysis on the G protein ␣ subunit drives the cycle of activation and deactivation of these signaling pathways. The duration of G protein-mediated responses is subject to the intrinsic GTPase rate of the G protein ␣ subunit, but is also modulated by extrinsic factors. A recently appreciated form of regulation has come from the discovery that members of a protein family called regulators of G protein signaling, or RGS proteins, stimulate the rate of GTP hydrolysis by G protein ␣ subunits (3-5). RGS proteins are found in species ranging from yeast to mammals and constitute a family of at least 20 mammalian proteins (6-8).All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9-11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein). In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14, 15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.Although rapid progress has been made in dissecting the biochemical mechanism by which RGS proteins regulate G protein a...

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A new family of regulators of G-protein-coupled receptors?

Cited by 204 publications

References 8 publications

Selective Regulation of Gαq/11 by an RGS Domain in the G Protein-coupled Receptor Kinase, GRK2

Selective Regulation of Gαq/11 by an RGS Domain in the G Protein-coupled Receptor Kinase, GRK2

Development of a Yeast Bioassay to Characterize G Protein-coupled Receptor Kinases

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

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