INVOLVEMENT OF RGS DOMAIN OF GRK2 IN THE DESENSITIZATION OF BETA3-ADRENERGIC RECEPTOR

ECHEVERRIA E; DIAZ NEBREDA, A; CABRERA M; DAVIO C; SHAYO C; FERNANDEZ N

Bariloche

Simposio; The Third South American Symposium in Signal Transduction and Molecular Medicine; 2015

3-adrenergic receptor (3AR) belongs to the superfamily of G protein-coupled receptors and when activated mediates intracellular cAMP increase. It is mainly expressed in adipose tissues and bladder and has been postulated as target for the treatment of overactive bladder syndrome (OBS). Previous work in endogenously expressing 3AR SK-N-MC cells, showed that pre-treatment with isoproterenol led to a significant desensitization of the receptor. However, 3AR lacks potential sites for PKA or GRKs mediated phosphorylation, which have been demonstrated to be involved in 1AR and 2AR desensitization.The aim of the present work was to evaluate the mechanism of 3AR desensitization using HEK293T transfected cells. In this system, concentration-response assays to the specific 3AR ligand BRL37344, significantly increased cAMP levels. After pre-treatment with BRL37344, 3AR response was significantly reduced by 40%. When different GRKs were co-expressed, GRK2 and 3, but not GRK5 or 6 increased receptor desensitization. GRKs proteins possess several structural and functional domains, an N-terminal RGS-homology domain, a kinase central domain, 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, that could be involved in receptor desensitization. When desensitization experiments were performed in the presence of GRK2 dominant negative mutants, receptor desensitization was impeded only in the presence of the mutant D110A of the RGS domain, while expression of kinase inactive mutant, K220R, had no effect. Present results indicate that 3AR response can be regulated by GRK2, where the RGS domain plays a crucial role.