Abstract P194: A Novel Signalosome in the Alpha1a-Adrenergic Receptor and its Genetic Variant Signaling Pathway

Abstract

Objectives : Human α 1 adrenergic receptors (AR), members of G protein-coupled receptor superfamily (GPCR) regulate blood pressure via smooth muscle cell (SMC) proliferation and vasoconstriction. We showed that α 1a AR-G247R (247R) genetic variant, identified in a hypertensive patient, constitutively couples to β-arrestin1/MMP/EGFR transactivation pathway leading to hyperproliferation in fibroblasts, cardiomyoblasts and SMCs. A scaffolding protein spinophilin (SPL) serves as a docking site for many regulatory proteins including RGS2 (negative R egulators of G -protein S ignaling). SPL also binds 3 rd intracellular loop of some GPCRs. Hypothesis: differential interaction of 247R or α 1a AR-WT (WT) with SPL/RGS2/β-arrestin signalosome mediates unique signaling of 247R and hyperproliferation in cardiovascular cells. Methods: Receptor-protein interactions were determined by co-immunoprecipitation from HEK293 cells expressing HA-α 1 ARs and full length Myc-SPL, its fragments or Flag-RGS2. Protein levels were analyzed by Western. SPL knockdown or RGS2 overexpression was performed using SPL-specific or scrambled siRNA or Flag-RGS2. Results: Our results reveal that in SMCs or cardiomyoblasts 247R but not WT upregulates endogenous SPL. SPL exhibits stronger (~2-fold) interaction with WT compared to 247R or α 1b , recruits RGS2 and inhibits receptor signaling. In contrast, weak SPL-247R interaction diminishes RGS2 inhibitory effect permitting hyperproliferation of 247R cells. SPL knockdown inhibits 247R-induced proliferation by allowing RGS2 directly bind to 247R as observed with RGS2 overexpression. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that in 247R cells SPL binds RGS2 preventing RGS2-247R interaction. Conclusions: We present SPL/RGS2/β-arrestin as a novel signalosome responsible for α 1a AR-247R genetic variant triggered hyperproliferation in different cardiovascular cells. We reveal that SPL regulates α 1a AR signaling by differentially binding WT or 247R receptors and recruiting RGS2 protein to the receptor. These novel findings unravel critical roles of SPL and RGS2 in α 1 AR signaling, as well as identify SPL as a potential novel target for treatment of α 1 AR-mediated cardiovascular disorders.