Phosphorylation of Ser360 in the third intracellular loop of the alpha2A-adrenoceptor during protein kinase C-mediated desensitization.

The alpha2A-adrenoceptor undergoes desensitization in response to protein kinase C activation. Using mutagenesis and recombinant expression we sought to determine the specific sites within the receptor which are phosphorylated by protein kinase C and are responsible for this desensitization. Ser/Thr in the third intracellular loop were substituted with Ala to create mutant receptors T272A, S258A, S324A and S360A. These mutations had no effect on ligand binding or functional coupling to inositol phosphate accumulation and intracellular Ca2+ release. Three of the four mutant receptors displayed wild-type desensitization (approximately 60% loss of function) in response to 0.1 microM phorbol-12-myristate-13-acetate (PMA) exposure for 2 min. However, the S360A mutant had only approximately 24% desensitization. In whole cell phosphorylation studies, S360A failed to undergo detectable PMA promoted phosphorylation. We conclude that protein kinase C-mediated desensitization of alpha2A-adrenoceptor function is primarily due to phosphorylation of Ser at amino acid 360. This thus represents one mechanism by which these receptors undergo regulation by heterologous means, such as pathologic processes which activate protein kinase C or crosstalk with other receptors.