Abstract

Effects of the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu) on the binding behavior of the alpha-1 adrenergic receptor were determined from radioligand binding assays at 25 and 2 degrees C. Membranes prepared from PDBu-treated bovine aorta exhibited a 16% reduction in [3H]prazosin binding capacity, whereas [3H]prazosin affinity was unchanged. This may reflect a role for protein kinase C-mediated receptor phosphorylation in determining receptor turnover and surface density. After PDBu treatment, the affinity of epinephrine for [3H]prazosin sites was altered in two respects. Control membranes exhibited both high and low affinity epinephrine binding (KDH, 20 nM; KDL, 1086 nM) whereas, PDBu-treated membranes exhibited only a single class of low affinity sites (KDL, 655 nM). The inclusion of 5'-guanylylimidodiphosphate caused the loss of high affinity sites in control membranes but had no effect on PDBu-treated membranes (KDL, 681 nM). Thus, protein kinase C blocks the ability of the agonist-receptor complex to couple to a GTP binding regulatory protein. In binding studies conducted at 2 degrees C epinephrine also bound to high (KDH, 34 nM) and low affinity (KDL, 1920 nM) sites although the percentage of high affinity sites was higher (percentage of RH, 80) than at 25 degrees C (percentage of RH, 19). PDBu-treated membranes also exhibited two agonist affinity states in 2 degrees C studies although affinity was slightly reduced (KDH, 74 nM; KDL, 2405 nM). 5'-Guanylylimidodiphosphate was without effect at 2 degrees C. These results indicate that a high affinity agonist binding state can still be achieved after PDBu treatment.(ABSTRACT TRUNCATED AT 250 WORDS)