Identification of a specific domain in the β-adrenergic receptor required for phorbol ester-induced inhibition of catecholamine-stimulated adenylyl cyclase

The molecular basis for the effects of 4β-phorbol 12-myristate 13-acetate (PMA) on adenylyl cyclase activation was examined using site-directed mutants of the hamster β-adrenergic receptor (βAR) expressed in L cells. Phorbol ester activation of protein kinase C (PKC) in L cells transfected with wild-type βAR 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 K(act) for epinephrine stimulation, (ii) a 2-3-fold increase in the maximal level (V(max)) of hormonal stimulation, and (iii) a decrease in the G(i)-mediated inhibition of forskolin stimulation. Deletion from the βAR of amino acid residues 259-262, which removes one of the two consensus sites for phosphorylation by PKC, eliminated (>90%) the PMA-induced increase in the K(act), whereas the PMA-induced increase in the V(max) and loss of G(i)-mediated inhibition were not affected by the deletion. Neither deletion of the other PKC consensus site in the βAR (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 G(i)-mediated inhibition and the V(max) closely mimicked the action of islet-activating protein, consistent with a direct effect of PMA-activated PKC on G(i). In contrast, the effects on the K(act) appear to be receptor specific. These results demonstrate that the consensus site for phosphorylation by PKC, found in the third intracellular loop of the βAR, is required for the PMA-induced increase in the K(act) for epinephrine stimulation. Use of L cells transfected with D(259-262)βAR allowed the characterization of the postreceptor effects of PMA without interference from receptor-level effects.