Abstract

We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the α_2A-adrenergic receptor (α_2AAR). In whole-cell phosphorylation studies, recombinantly expressed human α_2AAR displayed an increase in phosphorylation after short-term exposure to 100 nmphorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 ± 40% in COS-7 cells and 201 ± 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of α_2AAR function, amounting to a 68 ± 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated α_2AAR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type α_2AAR. To test whether PKC phosphorylation is a mechanism by which α_2AAR can be regulated by other receptors, the α_1bAR was co-expressed with the α_2AAR in Chinese hamster ovary cells. Upon selective activation of α_1bAR, the function of α_2AAR underwent a 53 ± 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the α_2AAR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the α_2AAR and other receptors can occur.