title: Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration.
abstract: The vasoactive intestinal polypeptide type-1 (VPAC(1)) receptor is a class II G protein-coupled receptor, distinct from the adrenergic receptor superfamily. The mechanisms involved in the regulation of the VPAC(1) receptor are largely unknown. We examined agonist-dependent VPAC(1) receptor signaling, phosphorylation, desensitization, and sequestration in human embryonic kidney 293 cells. Agonist stimulation of cells overexpressing this receptor led to a dose-dependent increase in cAMP that peaked within 5-10 min and was completely desensitized after 20 min. Cells cotransfected with the VPAC(1) receptor (VPAC(1)R) and G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6 exhibited enhanced desensitization that was not evident with GRK 4. Immunoprecipitation of the epitope-tagged VPAC(1) receptor revealed dose-dependent phosphorylation that was increased with cotransfection of any GRK. Agonist-stimulated internalization of the VPAC(1)R peaked in 10 min, and neither overexpressed beta-arrestin nor its dominant-negative mutant altered internalization. However, a dynamin-dominant negative mutant did inhibit VPAC(1) receptor internalization. Interestingly, VPAC(1)R specificity in desensitization was not evident by study of the overexpressed receptor; however, we determined that human embryonic kidney 293 cells express an endogenous VPAC(1)R that did demonstrate dose-dependent GRK specificity. Therefore, VPAC(1) receptor regulation involves agonist-stimulated, GRK-mediated phosphorylation, beta-arrestin translocation, and dynamin-dependent receptor internalization. Moreover, study of endogenously expressed receptors may provide information not evident in overexpressed systems.
authors.affiliation: Department of Cell Biology, Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina 27710, USA. shetz001@mc.duke.edu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Shetzline
authors.forename: Julia K L
authors.initials: JK
authors.lastname: Walker
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Valenzano
authors.forename: Richard T
authors.initials: RT
authors.lastname: Premont
mesh_terms: D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D021381:Protein Transport; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D000071557:beta-Arrestins