title: Vasoactive intestinal peptide receptor/adenylate cyclase system: differences between agonist- and protein kinase C-mediated desensitization and further evidence for receptor internalization.
abstract: In this study we have characterized and compared the regulation of the HT29 cell vasoactive intestinal peptide receptor/adenylate cyclase system (VIP-R/AC) by the VIP-R agonist peptide histidineisoleucineamide (PHI) and by activators of protein kinase C (PKC) including phorbol 12-myristate, 13-acetate (PMA) and mezerein. Preincubation with either PHI or PKC activator decreased maximum VIP-stimulated AC activity and decreased the number of cell surface VIP-R. A [125I]VIP binding assay using solubilized VIP-R of the plasma membrane and light vesicle fractions from sucrose density step gradients was developed as a more direct measure of VIP-R internalization. Preincubation with PHI or PMA decreased plasma membrane fraction [125I]VIP binding and increased binding in the light vesicle fraction, thus providing the most direct evidence to date for translocation of VIP-R per se from the plasma membrane to another, presumably intracellular, compartment. Two experimental approaches differentiated between agonist and PKC activator regulation of VIP-R/AC. The protein kinase inhibitors H-7 and staurosporine blocked mezerein-, but not PHI-, induced losses of cell surface VIP-R. Also, down-regulation of PKC did not block PHI-induced loss of cell surface VIP-R. Thus, although both agonist and PKC activators can lead to desensitization and internalization of VIP-R, PKC is apparently not involved in the mechanisms of agonist-induced desensitization.
authors.affiliation: Department of Pharmacology, School of Medicine, University of Missouri-Columbia.
authors.forename: J T
authors.initials: JT
authors.lastname: Turner
authors.forename: D W
authors.initials: DW
authors.lastname: Bollinger
authors.forename: M L
authors.initials: ML
authors.lastname: Toews
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D007546:Isoquinolines / Q000494:pharmacology; D010451:Peptide PHI / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
chemical_list: D000470:Alkaloids; D007546:Isoquinolines; D010451:Peptide PHI; D010879:Piperazines; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D005576:Colforsin; D014660:Vasoactive Intestinal Peptide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000242:Cyclic AMP; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate