Gβ5·RGS7 Inhibits Gαq-mediated Signaling via a Direct Protein-Protein Interaction

Witherow, D. Scott; Tovey, Steven C.; Wang, Qiang; Willars, Gary B.; Slepak, Vladlen Z.

doi:10.1074/jbc.m212884200

Cited by 40 publications

(44 citation statements)

References 36 publications

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“…1B, (i) and (ii)). We did not find it necessary to coexpress G␤5 with RGS7 to observe pronounced functional effects and presumably RGS7 complexes with endogenously expressed G␤5 (33,34). It is also known that G␤5 containing G␤␥ complexes can inhibit GIRK channels (35), and overexpression of this protein would potentially complicate our electrophysiological analysis.…”

Section: Resultsmentioning

confidence: 94%

Regulators of G-protein Signaling Form a Quaternary Complex with the Agonist, Receptor, and G-protein

Benians¹,

Nobles²,

Hosny

et al. 2005

Journal of Biological Chemistry

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Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the G␣ subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K ؉ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein ␣ subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein ␣ subunit.The G-protein cycle is initiated by binding of an agonist to its target seven-helical G-protein-coupled receptor (GPCR), 1 which associates with a heterotrimeric G-protein on the cytoplasmic side of the cell membrane. Once assembled, this "ternary complex" promotes GDP release and stimulates GTP binding on the G-protein ␣ subunit and dissociation of the G-protein subunits G␣-GTP and G␤␥. Both subunits can activate downstream signaling molecules, including enzymes and ion channels (1, 2). Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. Cloning studies have identified a large RGS gene family, each endowed with a conserved RGS domain of 120 -130 amino acids that is flanked by N and C termini of varying lengths (3-7). By itself, the RGS domain is capable of interacting with G-protein ␣ subunits to accelerate the GTP hydrolysis rate of the G␣ subunit, thereby promoting termination of the G-protein signal (3-7). Based on primary sequence similarities, mammalian RGS proteins have been grouped into five subfamilies (7). In this study we focus largely on RGS8, belonging to the R4 subfamily of RGS proteins that are generally considered prototypical in that they appear to have little function other than to act as GTPase-activating proteins (GAPs) on G i/o and G q/11 G-protein ␣ subunits. However, we also examine RGS7, which belongs to the R7 family, and GAIP, which belongs to the RZ family. RGS7 is particularly interesting because it contains a number of protein-protein interaction domains in the N terminus, but it is of particular relevance for our study because it has a substantially attenuated GAP activity compared with other RG...

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Section: Resultsmentioning

confidence: 94%

Regulators of G-protein Signaling Form a Quaternary Complex with the Agonist, Receptor, and G-protein

Benians¹,

Nobles²,

Hosny

et al. 2005

Journal of Biological Chemistry

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“…Others have used wide-field fluorescence microscopy to measure FRET signals by YFP/CFP ratio change. [25][26][27][45][46][47] However, this approach suffers from complications caused by bleed-through of CFP fluorescence into the YFP channel. The correction for signal cross-talk must be carried out first, and therefore FRET cannot be analyzed easily.…”

Section: Discussionmentioning

confidence: 99%

Monitoring Caspase Activity in Living Cells Using Fluorescent Proteins and Flow Cytometry

Meylan

et al. 2004

The American Journal of Pathology

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“…5A) (59). We exploited this phenomenon to test whether the G␤ 5 subunit is essential for RGS7 localization and/or oligomerization.…”

Section: Role Of G␤ 5 and Domains Of Rgs7 In Its Localization Andmentioning

confidence: 99%

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein

Tayou

Wang

Jang

et al. 2016

Journal of Biological Chemistry

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RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical G␤ subunit, G␤ 5 . They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ϳ150 kDa on SDS-PAGE and did not contain G␤ 5 . Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the G␤ 5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homooligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active G␣ o prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners. G protein-coupled receptors (GPCRs)2 regulate many functions in eukaryotic cells by responding to chemically diverse molecules such as biogenic amines, lipids, and peptides. A large fraction of therapeutic drugs act through GPCRs. Although ligand-bound GPCRs directly interact with several proteins, the canonical signaling mechanism involves heterotrimeric (G␣␤␥) G proteins. The receptor promotes the exchange of GDP for GTP on the G␣ subunit and the subsequent dissociation of G␣-GTP from G␤␥. Both G␣-GTP and G␤␥ modulate the activity of effector enzymes or ion channels, thereby causing alterations in second messenger concentrations, which in turn generate cellular responses (1-3).GPCR signaling can be terminated by several mechanisms, one of which involves hydrolysis of the G␣-bound GTP and reassembly of the inactive G␣␤␥ trimer (1). The intrinsic GTPase activity of the G␣ subunit is very slow, and for most G proteins it is accelerated by regulator of G protein signaling (RGS) proteins. The GTPase activating (GAP) function of RGS proteins is mediated by the characteristic ϳ120-amino acid domain ("RGS box") present in all members of the RGS family (4 -8). The role of RGS proteins in the inactivation of GPCR signaling was demonstrated in numerous studies using mice lacking RGS proteins or harboring RG...

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Gβ5·RGS7 Inhibits Gαq-mediated Signaling via a Direct Protein-Protein Interaction

Cited by 40 publications

References 36 publications

Regulators of G-protein Signaling Form a Quaternary Complex with the Agonist, Receptor, and G-protein

Regulators of G-protein Signaling Form a Quaternary Complex with the Agonist, Receptor, and G-protein

Monitoring Caspase Activity in Living Cells Using Fluorescent Proteins and Flow Cytometry

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein

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