Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown , Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adr energic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta -arrestin, and dynamin, However, other class I GPCRs, for example, the angi otensin II type 1A receptor (AT(1A)R), exhibit different internalization pr operties than the beta(2)-AR, The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process, In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) t runcated secretin receptor using flow cytometry and fluorescence imaging, a nd compare the properties of secretin receptor internalization to that of t he beta(2)-AR, In HEK 293 cells, sequestration of both the wild-type and CO OH-terminal truncated secretin receptors was unaffected by GRK phosphorylat ion, whereas inhibition of cAMP-dependent protein kinase mediated phosphory lation markedly decreased sequestration, Addition of secretin to cells resu lted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by e xpression of dominant negative beta-arrestin, Thus, like the AT(1A)R, secre tin receptor internalization is not inhibited by reagents that interfere wi th clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in end ocytic vesicles. This study demonstrates that the ability of secretin recep tor to undergo GRK phosphorylation and beta-arrestin binding is not suffici ent to facilitate or mediate its internalization. These results suggest tha t other receptors may undergo endocytosis by mechanisms used by the secreti n and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.