Identification of protein kinase C phosphorylation sites in the angiotensin II (AT(1A)) receptor

Protein kinase C (PKC) phosphorylates the C-terminus of the type 1 angioten sin II receptor (AT(1)), although the exact site(s) of phosphorylation are unidentified. In the present study, we examined the phosphorylation of epit ope-tagged wild-type AT(1A) receptors, transiently expressed in Chinese ham ster ovary K1 cells, in response to angiotensin II (AngII) and following se lective activation and inhibition of PKC. This phosphorylation was compared with mutant receptors where C-terminal serine residues (Ser(331), Ser(338) and Ser(348)) within three putative PKC consensus sites were replaced with alanine, either individually or in combination. Stimulation by AngII or th e phorbol ester PMA to activate PKC induced an increase in phosphorylation of the wild-type AT(1A) receptor, which was prevented by truncation of the receptor C-terminus to remove the last 34 amino acids, including Ser(331), Ser(338) and Ser(348). Whereas single alanine mutation (Ser(331)Ala, Ser(33 8)Ala and Ser(348)Ala) resulted in decreased receptor phosphorylation, no s ingle mutant completely inhibited either AngII- or PMA-induced phosphorylat ion. Combined mutation of the three PKC consensus sites caused an approxima te to 70 % reduction in PMA-mediated phosphorylation. The approximate to 60 % reduction in AngII (1 mu M)-induced phosphorylation of this triple mutan t and the partial inhibition of wild-type receptor phosphorylation by bisin dolylmaleimide, a specific PKC inhibitor, suggest a significant contributio n of PKC to agonist-stimulated regulation. The ratio of PKC to total recept or phosphorylation was greatest at low doses of AngII (1 nM), consistent wi th the idea that PKC phosphorylates and regulates receptor function at low levels of stimulation, whereas phosphorylation by other kinases is more pre valent at high levels of agonist stimulation. To determine if a single PKC site is favoured when the contribution of PKC varies, the phosphorylation o f wild-type and mutant receptors was examined over a range of AngII concent rations (0, 1, 10 and 100 nM). At all AngII concentrations, single mutation of Ser(331), Ser(338) or Ser348 was incapable of completely preventing rec eptor phosphorylation, suggesting no clear preference for PKC consensus-sit e utilization. Together, these results indicate a redundancy in PKC phospho rylation of the AT(1A) receptor, whereby all three consensus sites are util ized to some degree following homologous (AngII) and heterologous (PMA) sti mulation. The contribution of PKC phosphorylation to receptor regulation is unclear, but multiple PKC phosphorylation of the AT(1A) receptor may allow independent and/or complementary events to occur at the three separate sit es of the C-terminus.