PHOSPHORYLATION AND FUNCTIONAL DESENSITIZATION OF THE ALPHA(2A)-ADRENERGIC RECEPTOR BY PROTEIN-KINASE-C

We have investigated the potential for protein kinase C (PKC) to phosp horylate and desensitize the alpha(2A)-adrenergic receptor (alpha(2A)A R). In whole-cell phosphorylation studies, recombinantly expressed hum an alpha(2A)AR displayed an increase in phosphorylation after short-te rm exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was b locked by preincubation with a PKC inhibitor. This increase in recepto r phosphorylation over basal amounted to 172 +/- 40% in COS-7 cells an d 201 +/- 40% in Chinese hamster ovary cells. In permanently transfect ed Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of alpha(2A)A R function, amounting to a 68 +/- 4% decrease in the maximal agonist ( UK14304)-stimulated intracellular calcium release. Such desensitizatio n was blocked by the PKC inhibitor bisindolylmaleimide I and was not e voked by an inactive phorbol ester. The desensitization of this agonis t response was not caused by PKC-mediated augmentation of G protein-co upled receptor kinase activity, because PMA-promoted desensitization o f a mutated alpha(2A)AR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type alpha(2A)AR. To test whether PKC phosphorylation is a mechanism by which alpha(2A)A R can be regulated by other receptors, the alpha(1b)AR was cc-expresse d with the alpha(2A)AR in Chinese hamster ovary cells. Upon selective activation of alpha(1b)AR, the function of alpha(2A)AR underwent a 53 +/- 5% desensitization. Thus, cellular events that result in PKC activ ation promote phosphorylation of the alpha(2A)AR and lead to substanti al desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the alpha (2A)AR and other receptors can occur.