RGS domain of GRK2 mediates beta3-adrenergic receptor desensitization.

GRANJA GALEANO G; FERNANDEZ NC; CABRERA M; MONCZOR F; ECHEVERRIA E; SHAYO C

Buenos Aires

Congreso; Reunión Conjuntas de Sociedades de Biociencias; 2017

SAIC

Beta3-adrenergic receptor (B3AR) mediates some of the effects of adrenaline and noradrenaline on several tissues,mainly the cardiovascular system.B3AR belongs to the superfamily of G protein-coupled receptors (GPCRs) increasing cAMP intracellular levels upon activation. While previous works showed that pre-treatment with isoproterenol leads to receptor desensitization, the mechanism involved remains unidentified since B3AR lacks the PKA- or GRKs-mediated phosphorylation consensus sites known to participate in the desensitization of other GPCRs.Our aim was to evaluate the mechanism of B3AR desensitization using HEK293T transfected cells.In thesecells, theB3 specific ligand BRL37344 significantly increased cAMP levels, and after a 1h pre-treatment, the response was significantly reduced by 40%. When different GRKs were co-expressed, GRK2 and 3, but not GRK5 or 6 potentiated receptor desensitization. Structurally, GRKs possess an N-terminal RGS-homology domain responsible for G-protein activity regulation, a kinase central domain engaged in receptor phosphorylation, and a less conserved C-terminal region responsible for membrane localization. We co-transfected the cells with different dominant negative mutants of kinase and RGS domains.While the expression of kinase inactive mutant K220R, had no effect, receptor desensitization was impeded in the presence of the mutant of the RGS domain, D110A.Since overexpression systems may not be representative for native tissues, we studied B3AR response in cultured rat cardiomyocytes, endogenously expressing B3AR. In this system,transfection with GRK2 dominant negative mutant D110A increased in almost twice the cAMP response to BRL37344 (483 ± 87 versus 879 ± 156; p