G Protein-coupled Receptor Kinase Specificity for Phosphorylation and Desensitization of α2-Adrenergic Receptor Subtypes*

The α2-adrenergic receptor (α2AR) subtype α2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate α2C10 in an agonist-dependent manner is the βAR kinase (βARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the α2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by βARK. However, the substrate specificities of the GRKs for phosphorylating α2AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate α2C10 and α2C4 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 α2C10 or α2C4 without or with the following GRKs: βARK, βARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the α2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. βARK and βARK2 were both found to phosphorylate α2C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate α2C10. In contrast to the findings with α2C10, α2C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing α2C10 or α2C4 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 α2C10 by co-expression of βARK but not GRK6, while α2C4 failed to desensitize with co-expression of βARK. These results indicate that short term agonist-promoted desensitization of α2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.