Saitoh O, etal., Nature 1997 Dec 4;390(6659):525-9.
Transmembrane signal transduction via heterotrimeric G proteins is reported to be inhibited by RGS (regulators of G-protein signalling) proteins. These RGS proteins work by increasing the GTPase activity of G protein alpha-subunits (G alpha), thereby driving G proteins into their inactive GDP-bound
form. However, it is not known how RGS proteins regulate the kinetics of physiological responses that depend on G proteins. Here we report the isolation of a full-length complementary DNA encoding a neural-tissue-specific RGS protein, RGS8, and the determination of its function. We show that RGS8 binds preferentially to the alpha-subunits G(alpha)o and G(alpha)i3 and that it functions as a GTPase-activating protein (GAP). When co-expressed in Xenopus oocytes with a G-protein-coupled receptor and a G-protein-coupled inwardly rectifying K+ channel (GIRK1/2), RGS8 accelerated not only the turning off but also the turning on of the GIRK1/2 current upon receptor stimulation, without affecting the dose-response relationship. We conclude that RGS8 accelerates the modulation of G-protein-coupled channels and is not just a simple negative regulator. This property of RGS8 may be crucial for the rapid regulation of neuronal excitability upon stimulation of G-protein-coupled receptors.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was
transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling.
Itoh M, etal., Biochem Biophys Res Commun 2001 Sep 14;287(1):223-8.
RGS8 was originally identified as an RGS protein specifically expressed in neuronally differentiated P19 cells. We generated a polyclonal antibody specific to rat RGS8 using a synthetic peptide. When nonneural cells (DDT1MF2
, CHO, and NIH3T3) transfected with rat RGS8 cDNA were immuno-stained with this antibody, the RGS8 protein was mainly detected in the nuclei. Since RGS8 mRNA was exclusively expressed in Purkinje cells of the cerebellum in the rat brain, we further examined the cellular distribution of the RGS8 protein in Purkinje cells using cultured cerebellar cells and tissue sections of the cerebellum. The RGS8 protein was excluded from the nuclei and distributed in the cell body and dendrites, but not in the axons of Purkinje cells. These results demonstrate the presence of a mechanism controlling the distribution of RGS8 protein in cerebellar Purkinje cells.
The regulator of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 was identified as a brain-specific RGS protein of 180 amino acids. Biochemical studies indicated that RGS8 binds to Galpha
o and Galphai3, and that it functions as a GTPase-activating protein (GAP) for Galpha subunits. Physiological investigations demonstrated that RGS8 is not a simple negative regulator, but accelerates the G-protein-coupled responses. In situ hybridization analysis showed a highly dense expression of RGS8 mRNA in Purkinje cells of the cerebellum in rat brain. When the cellular distribution of RGS8 was examined in non-neural cells transfected with RGS8 cDNA, the protein was found to be concentrated in nuclei. Further, co-expression of constitutively active Galphao resulted in the translocation of RGS8 protein to the plasma membrane. The cellular distribution of the RGS8 protein in cerebellar Pukinje cells was also studied in detail. It was shown that the protein is excluded from the nuclei and distributed in the cell body and dendrites except the axons of Purkinje cells. Thus, it is evident that there is a novel mechanism controlling the distribution of RGS8 protein in cerebellar Purkinje cells.
Saitoh O, etal., J Biol Chem 1999 Apr 2;274(14):9899-904.
The recently discovered family of RGS (regulators of G protein signaling) proteins acts as GTPase activating proteins which bind to alpha subunits of heterotrimeric G proteins. We previously showed that a brain-specific RGS, RGS8 speeds up the activation and dea
ctivation kinetics of the G protein-coupled inward rectifier K+ channel (GIRK) upon receptor stimulation (Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529). Here we report the isolation of a full-length rat cDNA of another brain-specific RGS, RGS7. In situ hybridization study revealed that RGS7 mRNA is predominantly expressed in Golgi cells within granule cell layer of cerebellar cortex. We observed that RGS7 recombinant protein binds preferentially to Galphao, Galphai3, and Galphaz. When co-expressed with GIRK1/2 in Xenopus oocytes, RGS7 and RGS8 differentially accelerate G protein-mediated modulation of GIRK. RGS7 clearly accelerated activation of GIRK current similarly with RGS8 but the acceleration effect of deactivation was significantly weaker than that of RGS8. These acceleration properties of RGS proteins may play important roles in the rapid regulation of neuronal excitability and the cellular responses to short-lived stimulations.
Saitoh O, etal., Proc Natl Acad Sci U S A 2002 Jul 23;99(15):10138-43.
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, RGS8
8S, 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 Gi/o-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.
Taylor VG, etal., J Biol Chem. 2016 Mar 4;291(10):5138-45. doi: 10.1074/jbc.M115.712075. Epub 2016 Jan 11.
Regulator of G protein signaling (RGS) proteins interact with activated Galpha subunits via their RGS domains and accelerate the hydrolysis of GTP. Although the R4 subfamily of RGS proteins generally accepts both Galphai/o and Galphaq/11 subunits as substrates, the R7 and R12 subfamilies select agai
nst Galphaq/11. In contrast, only one RGS protein, RGS2, is known to be selective for Galphaq/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of RGS2 in complex with Galphaq revealed a non-canonical interaction that could be due to interfacial differences imposed by RGS2, the Galpha subunit, or both. To resolve this ambiguity, the 2.6 A crystal structure of RGS8, an R4 subfamily member, was determined in complex with Galphaq. RGS8 adopts the same pose on Galphaq as it does when bound to Galphai3, indicating that the non-canonical interaction of RGS2 with Galphaq is due to unique features of RGS2. Based on the RGS8-Galphaq structure, residues in RGS8 that contact a unique alpha-helical domain loop of Galphaq were converted to those typically found in R12 subfamily members, and the reverse substitutions were introduced into RGS10, an R12 subfamily member. Although these substitutions perturbed their ability to stimulate GTP hydrolysis, they did not reverse selectivity. Instead, selectivity for Galphaq seems more likely determined by whether strong contacts can be maintained between alpha6 of the RGS domain and Switch III of Galphaq, regions of high sequence and conformational diversity in both protein families.
