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

Abstract

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...

N-Terminal Residues Control Proteasomal Degradation of RGS2, RGS4, and RGS5 in Human Embryonic Kidney 293 Cells

“…Our negative results for M 1 /M 3 mAChR selectivity of RGS2 reported here do not invalidate literature on receptor/RGS specificity (Zeng et al, 1998;Xu et al, 1999;Saitoh et al, 2002;Wang et al, 2002); rather, they suggest that mechanisms may be more complex than just receptor/RGS binding. It is clear that cell-type specific processes, such as scaffold molecules [i.e., G␣-interacting protein C terminus (GIPC)] may play a role.…”

“…RGS2, -3, -4, -5, and -8 have been identified to inhibit G␣ q , yet relatively little is known about determinants of their function. There is substantial published evidence for receptor-dependent specificity of RGS proteins (Zeng et al, 1998;Xu et al, 1999;Saitoh et al, 2002;Wang et al, 2002), and direct binding of RGS2 and -4 to the i3 loop of G␣ q/11 -coupled mAChRs was recently demonstrated (Bernstein et al, 2004) with greater binding to the M 1 and M 5 i3 loops than for the M 3 i3 loop. However, association with full-length receptors and the functional significance of this interaction in cells was not investigated.…”

“…There is emerging evidence that RGS proteins can preferentially inhibit signaling through different GPCRs signaling through the same G␣ subunit (Zeng et al, 1998;Xu et al, 1999;Saitoh et al, 2002;Wang et al, 2002). We recently demonstrated that endogenous RGS3 and RGS5 in vascular smooth muscle cells exhibit specificity for the M 3 muscarinic acetylcholine receptor (mAChR) and angiotensin AT 1A receptor, respectively (Wang et al, 2002).…”

The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins

“…This has been reported for different RGS proteins in regulating Gq proteins activated by distinct receptors (Xu et al, 1999); RGS3 and muscarinic m3 receptor; RGS5 and angiotensin AT1a receptor (Wang et al, 2002); RGS8 towards muscarinic m1 and substance P receptors (Saitoh et al, 2002); RGS-R4 subfamily and subtypes of muscarinic acetylcholine receptors (Roy et al, 2003;Bernstein et al, 2004). In the present study, we show that both RGSZ1 and RGSZ2 co-precipitate with m-opioid receptors, and that the association between RGSZ and Ga subunits is altered by a morphine challenge.…”

Modulation of μ-Opioid Receptor Signaling by RGS19 in SH-SY5Y Cells

“…Thus, the apparent selectivity of RGS19 as a GAP for MOR signaling and RGS4 as a GAP for DOR signaling is unexpected. However, there is accumulating evidence for GPCRs contributing to the specificity of RGS protein action (Xu et al, 1999;Saitoh et al, 2002;Wang et al, 2002Wang et al, , 2009Bernstein et al, 2004), and we (Wang et al, 2009) and others (Georgoussi et al, 2006;Xie et al, 2007) have suggested a role for the C-terminus of opioid receptors in providing specificity of RGS action at DOR versus MOR. Moroever, outside the RH domain, RGS19 has a more complex structure than RGS4 with an N-terminal cysteine string that binds to the N-terminal leucine-rich region of GAIP-interacting protein N-terminus (Fischer et al, Fig.…”