Desensitization of the human vasoactive intestinal peptide receptor (hVIP2/PACAP R): evidence for agonist-induced receptor phosphorylation and internalization.

To investigate the role of phosphorylation and internalization in the desensitization of the hVIP2/PACAP receptor, we expressed a C-terminal epitope-tagged (hemagglutinin; YPYDVPDYASL) receptor in COS7 and HEK293 cell lines. Radiolabeling experiments demonstrated that exposure to agonist induced receptor phosphorylation significantly above basal levels. This receptor phosphorylation was greater than that induced by receptor-independent activation of PKA with forskolin and that induced by co-application of forskolin and agonist. This suggests that receptor occupancy promotes phosphorylation and also that receptor phosphorylation may involve a specific G protein-coupled receptor kinase in addition to PKA. Immunocytochemical analysis showed that the receptor was internalized in response to agonist to a single site of accumulation within the cell and this was dependent on temperature, agonist concentration, and time. Further studies will focus on identifying phosphorylation sites and endocytic signals within the hVIP2/PACAP R.