Heart disease and stroke have remained the major causes of death and disability in the United States and the world despite continuing efforts in cardiovascular research and drug development. Hypertension places individuals at a higher risk for such diseases. Multiple gene candidates and genetic variants have been identified via genome-wide approaches with the hope to better understand the pathophysiology of hypertension and to develop more individualized therapeutic strategies. Often, however, the significance of identified mutant alleles is unknown.Among those are genetic variants identified in regulator of G protein signaling 2 (RGS2). RGS2 has been strongly implicated in cardiovascular regulation. RGS2 selectively attenuates Gq-mediated signaling through its canonical GTPase-activating protein (GAP) activity, thereby suppressing vasoconstrictor action. It has also been shown to inhibit Gs-mediated signaling through direct actions on adenylate cyclase. Homozygous and heterozygous RGS2-deficient mice exhibit hypertension and are prone to heart failure. Some rare mutations in RGS2 identified in hypertensive human subjects result in either a low level of protein expression or functional deficiency. This evidence suggests that genetic changes affecting RGS2 protein expression or function may be novel risk factors contributing to the development of hypertension and exacerbation of heart failure.Genome wide sequencing efforts have now identified over 100 missense mutations in the RGS2 coding sequence. These RGS2 mutations (in ~1.6% of individuals) represent 5 million people in the US. Several mutations are found in a disease context, however, their functional significance and consequences on vascular function are generally unknown. In this thesis work, I experimentally elucidate the functional effects on GPCR-mediated signaling and consequences on vascular reactivity of these RGS2 mutants. Among 16 mutations tested, four mutations lead to loss-of-function proteins that fail to inhibit AT1R-mediated Ca2+ mobilization. These mutant proteins also showed deficits in attenuating angiotensin II-mediated vasoconstriction in mesenteric arteries. Furthermore, these mutant proteins differentially regulate acetylcholine-mediated relaxation in vascular smooth muscle cells through a pathway involving cGMP-PKG activation. I also provide evidence of an association between a loss-of-function mutation in RGS2 with high blood pressure and cardiovascular disease. This study provides a molecular and physiological understanding of different RGS2 alleles in vitro as the first step to identify key candidate alleles for further analysis in in vivo models and in human hypertension and heart failure.
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