Abstract
Regulator of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling. The N termini of some RGS4-family proteins provide receptor specificity and also contain an N-end rule determinant that results in ubiquitylation and decreased protein expression. The relevance of these mechanisms to other RGS proteins is not fully understood. Thus we examined function, receptor specificity, and expression of R4 subfamily RGS proteins (RGS2, -3, -4, -5, and -8). Although the N terminus plays a key role in protein stability in human embryonic kidney (HEK) 293 cells, we were unable to demonstrate specificity of RGS2, -3, -4, -5, or -8 for muscarinic receptors (M1, M3, and M5). However, cellular RGS activity (8 = 3 > 2) was strongly correlated with expression; RGS4 and -5 had minimal expression and activity. Stabilizing mutations of RGS4 and -5 (C2S) enhanced expression and function with a greater influence on RGS4 than on RGS5. We were surprised to find that a predicted destabilizing mutation in RGS8 (A2C) did not markedly affect expression and had no effect on function. In contrast, a destabilizing mutation in RGS2 (RGS2-Q2L) recently identified as a rare N-terminal genetic variant in a Japanese hypertensive cohort (J Hypertens23:1497–1505, 2005) showed significantly reduced expression and inhibition of angiotensin II (AT1) receptor-stimulated accumulation of inositol phosphates. We were surprised to find that RGS2-Q2R, also predicted to be destabilizing, showed nearly normal expression and function. Thus, proteasomal regulation of RGS expression in HEK293 cells strongly controls RGS function and a novel RGS2 mutation with decreased protein expression could be relevant to the pathophysiology of hypertension in humans.
Footnotes
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This work was supported by National Institutes of Health Research grant R01-GM39561 (to R.R.N.).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.029397.
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ABBREVIATIONS: RGS, regulator of G protein signaling; mAChR, muscarinic acetylcholine receptor; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; PBS, phosphate-buffered saline; [3H]NMS, N-methyl-[3H]scopolamine; MG-132, N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal; WT, wild type; AT1, angiotensin II type 1; HA, hemagglutinin; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; InsPx, inositol mono-, bis-, or trisphosphate; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; ANOVA, analysis of variance; fluo-4 AM, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-difluoro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxoethyl], (acetyloxy)methyl ester.
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↵1 Current affiliation: Department of Pharmacology, School of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban, South Africa.
- Received July 27, 2006.
- Accepted January 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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