G protein-coupled receptor kinase specificity for phosphorylation and desensitization of alpha(2)-adrenergic receptor subtypes

The alpha-adrenergic receptor (alpha(2)AR) subtype alpha(2)C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha(2)C10 in an agonist-dependent manner is the beta AR kinase (beta ARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha(2)C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by beta ARK, However, the substrate specificities of the GRKs for phosphorylating alpha(2)AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate alpha(2)C10 and alpha(2)C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors, COS-7 cells were co-transfected with alpha(2)C10 or alpha(2)C4 without or with the following GRKs: beta ARK, beta ARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with P-32(i), exposing some to agonist, and purifying the alpha(2)AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. beta ARK and beta ARK2 were both found to phosphorylate alpha(2)C10 to equal extents (>2-fold over that of the endogenous kinases), On the other hand, GRK5 and GRK6 did not phosphorylate alpha(2)C10, In contrast to the findings with alpha(2)C10, alpha(2)C4 was not phosphorylated by any of these kinases, Functional studies carried out in transfected HEK293 cells expressing alpha(2)C10 or alpha(2)C4 and selected GRKs were consistent with these phosphorylation results, With the marked expression of these receptors, no agonist-promoted desensitization was observed in the absence of GRK co-expression, However, desensitization was imparted to alpha(2)C10 by co-expression of beta ARK but not GRK6, while alpha(2)C4 failed to desensitize with co-expression of beta ARK. These results indicate that short term agonist-promoted desensitization of alpha(2)ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.