Abstract
Hypertension is a leading risk factor for the development of cardiovascular disease. Data from human and animal studies suggest that RGS2, a potent inhibitor of Gq signaling, is important for blood pressure regulation. Several RGS2 mutations in the Japanese population have been found to be associated with hypertension. The product of one of these alleles, R44H, is mutated within the amino terminal amphipathic α-helix domain, the region responsible for plasma membrane-targeting. The functional consequence of this mutation and its potential link to the development of hypertension, however, are not known. In this study, we showed that R44H was a weaker inhibitor of receptor-mediated Gq signaling than wild-type RGS2. Confocal microscopy revealed that YFP-tagged R44H bound to the plasma membrane less efficiently than wild-type RGS2. R44 is one of the basic residues positioned to stabilize lipid bilayer interaction of the RGS2 amphipathic helix domain. Tryptophan fluorescence and circular dichroism studies of this domain showed that the R44H mutation prevented proper entrenchment of hydrophobic residues into the lipid bilayer without disrupting helix-forming capacity. Together, these data suggest that decreasing the side-chain length and flexibility at R44 prevented proper lipid bilayer association and function of RGS2. Finally, the R44H protein did not behave as a dominant-negative interfering mutant. Thus, our data are consistent with the notion that a R44H missense mutation in human RGS2 produces a hypomorphic allele that may lead to altered receptor-mediated Gq inhibition and contribute to the development of hypertension in affected subjects.
Footnotes
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This work was supported by grants from the Ontario Heart and Stroke Foundation (NA 5291), Ontario Graduate Scholarships in Science and Technology (S.G.), and the Canada Research Chairs Program of the Canadian Institute for Health Research (S.P.H.).
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; Ang II, angiotensin II; ET-1, endothelin-1; RGS, Regulators of G-protein signaling; NTD, amino-terminal domain; YFP; yellow fluorescent protein; HEK, human embryonic kidney; PBS, phosphate-buffered saline; TFE, trifluoroethanol; PM, plasma membrane; FR, fluorescent ratio.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received December 11, 2007.
- Accepted January 23, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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