Protein kinase C regulates alpha(2A/D)-adrenoceptor constitutive activity

We investigated a possible role for protein kinases in the constitutive activity of alpha(2A/D) adrenoceptors in membranes from transfected PC12 cells, using a [35S]GTPgammaS binding assay. After treatment of intact cells with various protein kinase inhibitors, constitutive activity was assessed by the reduction in basal GTP binding caused by the inverse agonist rauwolscine (RAU). Inhibitors of protein kinase C (PKC) caused the loss of RAU-sensitive GTP binding, while inhibitors of other protein kinases were ineffective. Anti-G(alpha) antibody treatments showed that constitutive alpha(2A/D)-receptor activity is directed toward different G proteins than agonist-stimulated activity. T373A mutant receptors exhibited increased constitutive activity, including a component that was insensitive to PKC inhibition. Since T373 is located within a putative G(i/o) activator sequence, these results suggest that PKC-dependent phosphorylation of T373 increases alpha(2A/D)-adrenergic receptor constitutive activity and causes a switch in G protein preference.