title: Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.
abstract: 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)-adrenergic 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 angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties 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) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.
authors.affiliation: Howard Hughes Medical Institute, Department of Cell Biology, Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Walker
authors.forename: R T
authors.initials: RT
authors.lastname: Premont
authors.forename: L S
authors.initials: LS
authors.lastname: Barak
authors.forename: M G
authors.initials: MG
authors.lastname: Caron
authors.forename: M A
authors.initials: MA
authors.lastname: Shetzline
mesh_terms: D019390:Arrestins / Q000378:metabolism; D001692:Biological Transport; D002451:Cell Compartmentation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D009842:Oligopeptides; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010455:Peptides; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012633:Secretin / Q000494:pharmacology*; D017384:Sequence Deletion; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D007546:Isoquinolines; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D044140:Receptor, Angiotensin, Type 1; D018343:Receptors, Adrenergic, beta-2; D011945:Receptors, Angiotensin; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D000071557:beta-Arrestins; C029937:secretin receptor; D012633:Secretin; C085706:FLAG peptide; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D034281:Dynamins; D019311:Staurosporine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide