Human a 1A ‐adrenergic receptor phosphorylation, internalization and desensitization.

The a 1A ‐adrenergic receptor (a 1A ‐AR) is a G protein coupled‐receptor (GPCR) that signals through the Gq/ phospholipase C/ calcium release pathway. This adrenergic receptor subtype modulates important cellular responses and it is also involved in the pathogenesis of diseases. Phosphorylation of intracellular loops and the carboxyl terminal tail of a 1A ‐AR is part of their fine regulation, generally associated with desensitization/internalization processes, in which different protein kinases participate, Including second messenger‐activated protein kinases, such as protein kinase C isoforms (PKCs) and G protein‐coupled receptor kinases (GRKs). In silico analysis of the human a 1A ‐ AR amino acid sequenced, suggested a series of possible phosphorylation sites targeted by PKCs, GRKs and also for the glycogen synthase protein kinase‐3 (GSK3). GSK3 is an important element into signal transduction of diverse receptor systems, and the phosphorylation on specific substrates can modulates enzyme activity, subcellular localization, stability of the target protein and protein‐protein interactions. We observed using a 1A ‐AR immunopurification and mass spectrometry analysis that in cells treated with noradrenaline, oxymetazoline or phorbol esters four serines that are target of GSK3: S229, S258, S352 and S381, are phosphorylated. However, the specific GSK3 inhibitor, SB216763, did not block the a 1A ‐AR desensitization induced by noradrenaline or oxymetazoline. This suggested that other protein kinases play major roles in such agonist‐induced desensitization. Interestingly, phorbol ester‐induced a 1A ‐AR desensitization was increased by the GSK3 inhibitor. In cellulo colocalization assays of a 1A ‐AR ‐GSK3, showed increased interaction of these proteins in the presence of agonists and phorbol esters. Interestingly, expression of a GSK3 dominant‐negative mutant interferes with a 1A ‐AR internalization in response to NA and OXY, but this effect does not occur when cells are pretreated with PMA. All these data suggest the possibility that GSK3 might play a role in the regulation of a 1A ‐AR function and localization. Partially supported by Grants from Dirección General de Asuntos del Personal Académico (IN200718) and Consejo Nacional de Ciencia y Tecnología (Fronteras 882 and 253156). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .