Differential Regulation of Alpha1a-adrenergic Receptor and its Genetic Variant Signaling by Novel Spinophilin-RGS2 Signalosome in Human Cells

α 1- adrenergic receptors (α 1a , α 1b , α 1d ), members of G protein-coupled receptor (GPCR) family, regulate stress-facilitated changes in the cardiovascular (CV) system and human blood pressure (BP) via smooth muscle cell (SMC) proliferation and vasoconstriction. The α 1a AR is the predominant subtype in human heart and resistance vessels. The α 1a AR G247R (247R) genetic variant was discovered in a patient with severe hypertension and in a cohort of patients enriched for CV disorders. We reported a unique coupling of this genetic variant to βarrestin/MMP/EGFR transactivation pathway in rat fibroblasts, cardiomyoblasts and vascular SMCs leading to hyperproliferation and hypertrophy. Here we present that differential interaction of 247R or α 1a AR (WT) with scaffolding/regulatory proteins mediate unique signaling of 247R. Physiological levels of 247R but not WT upregulate endogenous levels of scaffolding protein spinophilin (SPL) in rat SMCs and cardiomyoblasts. SPL knockdown or overexpression of RGS2 (negative R egulators of G -protein S ignaling) inhibits 247R-mediated hyperproliferation. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that SPL binds RGS2 and prevents 247R-RGS2 interaction. To determine if 247R hyperproliferation is in effect in human cells, we generated human iPSCs from primary fibroblasts and differentiated them to SMCs with characteristic morphology and expression of SMC markers (RT-PCR at various passages). Consistent with our findings in rat cells, expression of 247R but not WT in hiPSC-derived SMCs triggered ~70% increased proliferation. Agonist treatment resulted in increased hypertrophy in 247R cells compared with WT. Co-immunoprecipitation from HEK293 cells expressing HA-α 1 ARs and full length Myc-SPL or Flag-RGS2 revealed that SPL exhibits ~2-fold stronger interaction with WT than with 247R. Consequently, SPL recruits RGS2 and inhibits receptor signaling. In contrast, weaker SPL-247R interaction results in diminished RGS2 inhibitory effect allowing hyperproliferation of 247R cells. Thus, we identify novel SPL/RGS2 signalosome responsible for α 1a AR signaling in human cells and identify SPL as a potential novel target for treatment of α 1 AR-mediated human CV disorders.