Evidence of an important and direct role for protein kinase C in agonist-induced phosphorylation leading to desensitization of the angiotensin AT(1A) receptor

1 The role of protein kinase C (PKC) in the mechanism underlying rapid agonist-induced desensitization of angiotensin AT(1) receptors remains unresolved. A major problem has been to isolate these receptors in a sufficiently purified form to allow study of their phosphorylation state. 2 A cleavable (His)(6) affinity tag was introduced into the N-terminus of the recombinant AT(1A) receptor and stably expressed in human embryonic kidney cells. This affinity tag allowed rapid isolation, purification and determination of the phosphorylation state of the AT(1A) receptor. Using these cells, we determined the role of PKC in both agonist-induced receptor phosphorylation and desensitization under identical conditions. 3 Agonist-induced phosphorylation of the AT(1A) receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro-31-8220 (a PKC specific inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC-mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase. 4 In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro-31-8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no effect on the level of desensitization observed. 5 These data directly demonstrate an association between PKC-induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT(1) receptor desensitization.