Alpha1a-Adrenoceptor Genetic Variant-Triggered Hyperproliferation In Cardiovascular Cells Is Mediated By Novel Interacting Protein Spinophilin

Objectives: Alpha1-Adrenergic Receptors (α1ARs), members of the G protein-coupled receptor (GPCR) superfamily, play a major role in regulating cardiovascular (CV) function. Recently, we discovered that naturally occurring human α1aAR-G247R (247R) genetic variant, identified in the 3rd intracellular loop (3iL) of the receptor in highly hypertensive patient, triggers constitutive hyperproliferation in fibroblasts, cardiomyoblasts and smooth muscle cells (SMC). Specific proteins mediating this signaling remain unknown. Spinophilin (SPL) is a ubiquitously expressed protein controlling GPCR signaling by binding its 3rd intracellular loop (3iL). We hypothesize that SPL mediates α1aAR signaling and examined whether SPL directly interacts with α1aAR-WT (WT) and 247R. Methods: Cells were co-transfected with HA-α1ARs and full length Myc-SPL or its fragments to determine SPL domains responsible for binding to α1ARs. Cell lysates were co-immunoprecipitated with HA-tag antibodies. SPL levels were analyzed by Western blotting. SPL knockdown experiments were performed by transiently transfecting cells with SPL or scrambled siRNA, cell proliferation was determined by cell counting. Results: We demonstrate a distinct interaction of SPL with WT and 247R, WT interaction being the strongest. Different domains of SPL differentially interact with WT or 247R. SPL 1-480aa fragment interacts stronger with WT indicating interaction with 3iL, while SPL 480-817 fragment interacts stronger with 247R. Endogenous SPL levels are increased in 247R cells compared to WT or control cells. Inhibition of SPL expression in SMC with siRNA reduces 247R-triggered hyperproliferation to near normal levels and has no effect in WT cells. Conclusions: We identified SPL as a novel interacting protein involved in mediating intracellular signaling of α1aAR and its genetic variant in CV cells. Different domains of SPL differentially bind to WT or 247R indicating that SPL has a distinct role in regulation of their signaling pathways. Our findings also reveal that SPL is critical for 247R-triggered EGFR transactivation pathways. Thus SPL could be considered as a potentially novel target in α1aAR-mediated cardiovascular disorders.