Abstract

The molecular basis for the effects of 4 beta-phorbol 12-myristate 13-acetate (PMA) on adenylyl cyclase activation was examined using site-directed mutants of the hamster beta-adrenergic receptor (beta AR) expressed in L cells. Phorbol ester activation of protein kinase C (PKC) in L cells transfected with wild-type beta AR caused at least three discernible effects on adenylyl cyclase activity, each with an EC50 of 20 to 50 nM, (i) a 2-3-fold increase in the Kact for epinephrine stimulation, (ii) a 2-3-fold increase in the maximal level (Vmax) of hormonal stimulation, and (iii) a decrease in the Gi-mediated inhibition of forskolin stimulation. Deletion from the beta AR of amino acid residues 259-262, which removes one of the two consensus sites for phosphorylation by PKC, eliminated (greater than 90%) the PMA-induced increase in the Kact, whereas the PMA-induced increase in the Vmax and loss of Gi-mediated inhibition were not affected by the deletion. Neither deletion of the other PKC consensus site in the beta AR (residues 343-348) nor truncation of the Ser/Thr-rich C-terminal domain (residues 354-418) affected the PMA-induced changes in adenylyl cyclase. The effects of PMA on Gi-mediated inhibition and the Vmax closely mimicked the action of islet-activating protein, consistent with a direct effect of PMA-activated PKC on Gi. In contrast, the effects on the Kact appear to be receptor specific. These results demonstrate that the consensus site for phosphorylation by PKC, found in the third intracellular loop of the beta AR, is required for the PMA-induced increase in the Kact for epinephrine stimulation. Use of L cells transfected with D(259-262)beta AR allowed the characterization of the postreceptor effects of PMA without interference from receptor-level effects.