Regulator of G Protein Signaling 8 (RGS8) Requires Its NH2 Terminus for Subcellular Localization and Acute Desensitization of G Protein-gated K+ Channels

Saitoh, Osamu; Masuho, Ikuo; Terakawa, Ion; Nomoto, Satoshi; Asano, Taku; Kubo, Yoshihiro

doi:10.1074/jbc.m006917200

Cited by 63 publications

(58 citation statements)

References 27 publications

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“…A bioassay was used to measure the sensitivity of the pheromone response in yeast that expresses RGS proteins as described (6,12). As the expression level of RGS8 protein was not high enough in yeast, a sequence based on the Kozak consensus sequence and a N-terminus myc tag (MEQKLISEEDLSRGS) were fused to RGS8 or RGS8S cDNA to increase the expression level.…”

Section: Materials and Methods Reverse Transcriptase-pcr (Rt-pcr)mentioning

confidence: 99%

“…Acute desensitization of GIRK current observed in the presence of RGS8, however, was not induced. Thus, we clarified that RGS8 requires its N terminus for subcellular localization and full regulatory function (6). On the other hand, Zeng et al (7) reported that the N-terminal domain (1-33 aa) of RGS4 confers receptor-selective inhibition of Gq signaling.…”

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confidence: 93%

“…On the other hand, Zeng et al (7) reported that the N-terminal domain (1-33 aa) of RGS4 confers receptor-selective inhibition of Gq signaling. There is significant sequence conservation in the N-terminal region of RGS4 and RGS8 (6).…”

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confidence: 99%

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Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling

Saitoh

Murata

Odagiri

et al. 2002

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

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The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 is a brain-specific RGS protein of 180 aa. Here we identified a short isoform of RGS8, RGS8S, that arises by alternative splicing. RGS8S cDNA encodes a N terminus of 7 aa instead of amino acids 1-9 of RGS8 and 10 -180 of RGS8. The subcellular distribution of RGS8 and RGS8S did not differ significantly in transfected cells. RGS8S accelerated, not as efficiently as RGS8, the turning on and off of Gi͞o-mediated modulation of G protein-gated inwardly rectifying K ؉ channels in Xenopus oocytes. We next examined the effects of RGS8 and RGS8S on Gq-mediated signaling. RGS8 decreased the amplitude of the response upon activation of m1 muscarinic or substance P receptors, but did not remarkably inhibit signaling from m3 muscarinic receptors. In contrast, RGS8S showed much less inhibition of the response of either of these Gq-coupled receptors. By quantitative analysis of the inhibitory effect and the protein expression level, we confirmed that the difference of inhibitory effect is caused by both the qualitative difference between RGS8 and RGS8S and the quantitative difference of the protein expression level. We also confirmed that the receptor-type specificity of inhibition is not caused by the difference of the expression level of the receptors. In summary, we showed that 9 aa in the N terminus of RGS8 contribute to the function to inhibit Gq-coupled signaling in a receptor type-specific manner and that the regulatory function of RGS8S is especially diminished on Gq-coupled responses. R egulators of G protein signaling (RGS) proteins comprise a large family of more than 20 members, which modulate heterotrimeric G protein signaling. They share a homologous domain, the RGS domain, which is flanked by diverse N and C termini (1-3). The RGS domain alone is sufficient for activating the GTPase of G␣, whereas the flanking domains confer various regulatory properties (3). RGS8 was identified in rat brain and is a small RGS protein along with RGS4, RGS5, and RGS16 (4, 5). We recently showed that RGS8 protein was concentrated in nuclei of cells transfected with cDNA for RGS8 expression and that coexpression of a constitutively active G␣o resulted in the translocation of RGS8 protein to the plasma membrane. The deletion of the N-terminal region (35 aa) of RGS8 abolished its nuclear localization and active G␣o-induced redistribution. This truncated mutant of RGS8, however, is still functional in inhibiting pheromone signaling in yeast to some extent. When coexpressed with G protein-gated inwardly rectifying K ϩ (GIRK) channels, the truncated RGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of GIRK current observed in the presence of RGS8, however, was not induced. Thus, we clarified that RGS8 requires its N terminus for subcellular localization and full regulatory function (6). On the other hand, Zeng et al. (7) reported that the N-terminal domain (1-33 aa) of RGS4 confers receptor-selective inhibi...

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Section: Materials and Methods Reverse Transcriptase-pcr (Rt-pcr)mentioning

confidence: 99%

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Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling

Saitoh

Murata

Odagiri

et al. 2002

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

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“…Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16)(17)(18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).…”

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confidence: 99%

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

Wensel²

2002

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

157

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The regulator of G protein signaling (RGS)-9-1⅐G␤5 complex forms the GTPase accelerating protein for G␣t in vertebrate photoreceptors. Although the complex is soluble when expressed in vitro, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, G␤5, G␣t, and a 25-kDa phosphoprotein, R9AP (RGS9-1-Anchor Protein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and Xenopus laevis. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.T o ensure high efficiency of phototransduction, the essential components of the phototransduction cascade are packed at a high density on photoreceptor outer segment disk membranes (1), where phototransduction occurs. One challenge in photoreceptor biology has been to determine the mechanisms by which these molecules and their modulators are localized to the disk membranes. This question has been particularly puzzling for regulator of G protein signaling (RGS)-9-1, which is the GTPase accelerating protein (GAP) for the visual G protein G ␣t (2-4).RGS9-1 plays an essential role in setting the timing of the recovery phase of light responses in vertebrate photoreceptors (5, 6). It is a tightly bound peripheral membrane protein (7), but has no obvious features that target it to membranes and is largely soluble when expressed in vitro (8, 9). RGS9-1 forms a constitutive complex with its obligate subunit, G ␤5 (5, 8, 10), and it also interacts with the inhibitory subunit of the effector for G ␣t , cGMP phosphodiesterase-␥ (PDE␥) (11)(12)(13)(14). Neither G ␤5 nor PDE␥ can serve as its membrane anchor, because selective removal of either of them does not affect RGS9-1 membrane binding (2, 9, 15). Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16-18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).We describe here the identification by coimmunoprecipitation of a heterotetrameric complex containing RGS9-1, G ␤5L , G ␣t , and a previously unknown photoreceptor-specific protein, R9AP. R9AP interacts with the N-terminal domain of RGS9-1 and has a transmembrane helix at its C terminus, allowing it to serve as the RGS9-1-anchor protein. Materials and MethodsBuffers. The following standard buffers were used: low-salt buffer (5 mM Tris⅐HCl͞0.5 mM MgCl 2 ), GAPN buffer (10 mM Hepes͞ 100 mM NaCl͞2 mM MgCl 2 ), high-salt buffer (10 mM Hepes͞1 M NH 4 Cl͞2 mM MgCl 2 ), urea buffer (4 M urea͞5 mM Tris⅐HCl). The pH of all buffers was adjusted to 7.4-7.5, and 1 mM DTT and Ϸ20 mg/liter solid PMSF were added before use.P...

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“…Because receptor-G protein complexes are membrane bound, cellular mechanisms must direct RGS, usually confined away from signaling components (Hollinger and Hepler, 2002), to target G␣ subunits. Several RGS translocate to the plasma membrane (PM) when exposed to GTPase-deficient G␣ subunits or through mechanisms initiated by G protein activation (Druey et al, 1998;Saitoh et al, 2001). How RGS assemble with the signaling machinery in living cells is a highly debated issue (Hepler, 2003;Roy et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D₂Receptor Signaling

Jeanneteau

Guillin

Díaz³

et al. 2004

MBoC

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Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its G␣ subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D 2 receptor (D 2 R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells. Dynamic translocation of GAIP to the plasma membrane and coassembly in a protein complex in which GIPC was a required component was dictated by D 2 R activation and physical interactions. In addition, two different D 2 R-mediated responses were regulated by the GTPase activity of GAIP at the level of the G protein coupling in a GIPC-dependent manner. Since GIPC exclusively interacted with GAIP and selectively with subsets of GPCR, this mechanism may serve to sort GPCR signaling in cells that usually express a large repertoire of GPCRs, G proteins, and RGS. INTRODUCTIONA general concept of signal transduction establishes that distinct signaling pathways form through the combination of components from a common repertoire of enzymes to evoke distinct physiological responses. For instance, neurotransmitters can induce a wide range of direct effects on target cells through the activation of G protein-coupled receptors (GPCR), which in turn stimulate particular intracellular signaling components. Selective interactions between these components may serve to sort signaling pathways in cells that usually express a wide range of GPCRs, G proteins, and effectors. Regulator of G protein signaling (RGS) proteins exert their GTPase function through direct interactions on activated (GTP-bound) form of G proteins to limit their lifetime and terminate signaling (Berman and Gilman, 1998;Ross and Wilkie, 2000;Hollinger and Hepler, 2002). Although most RGS are promiscuous in their G␣ subunit binding (De Vries et al., 2000), recruitment of a particular RGS in G-mediated signaling cascades may not be dictated by the G␣ subunit itself, but by the receptor that initiates G protein activation. Previous studies support this concept, showing that distinct GPCRs, although coupled to the same G protein, select different RGS to regulate their signaling (Wang et al., 2002;Xu et al., 1999). Because receptor-G protein complexes are membrane bound, cellular mechanisms must direct RGS, usually confined away from signaling components (Hollinger and Hepler, 2002), to target G␣ subunits. Several RGS translocate to the plasma membrane (PM) when exposed to GTPase-deficient G␣ subunits or through mechanisms initiated by G protein activation (Druey et al., 1998;Saitoh et al., 2001). How RGS assemble with the signaling machinery in li...

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Regulator of G Protein Signaling 8 (RGS8) Requires Its NH2 Terminus for Subcellular Localization and Acute Desensitization of G Protein-gated K+ Channels

Cited by 63 publications

References 27 publications

Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling

Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D₂Receptor Signaling

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Regulator of G Protein Signaling 8 (RGS8) Requires Its NH2 Terminus for Subcellular Localization and Acute Desensitization of G Protein-gated K+ Channels

Cited by 63 publications

References 27 publications

Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling

Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D2Receptor Signaling

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GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D₂Receptor Signaling