title: Role of receptor and protein kinase C activation in the internalization of the gastrin-releasing peptide receptor.
abstract: The mechanisms regulating receptor internalization are not well understood and vary among different G protein-coupled receptors. The bombesin (Bn)/gastrin-releasing peptide receptor GRP-R, which is coupled to phospholipase C via the Gq family of transducing proteins, is internalized rapidly after Bn binding. Agonist stimulation leads to rapid receptor phosphorylation, as does activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA). However, agonist- and PMA-induced phosphorylation occur at different receptor sites. Here, we examined the role of PKC in GRP-R internalization after agonist and antagonist binding. We synthesized [D-Tyr6]Bn(6-13)propylamide ([D-Tyr6]Bn(6-13)PA) and found that it potently inhibited Bn-stimulated insulin release and [125I-Tyr4]Bn binding (Ki = 4.72 nM) in the HIT-T15 pancreatic cell line. The radiolabeled antagonist peptide, [125I-D-Tyr6]Bn(6-13)PA, bound with high affinity (KD = 0.29 nM at 4 degrees) to a single class of receptor sites, and competition binding studies exhibited the analog specificity expected for the GRP-R subtype. Although the agonist [125I-Tyr4]Bn was internalized rapidly at 37 degrees and subsequently degraded, [125I-D-Tyr6]Bn(6-13)PA was not internalized and was released into the medium mainly as intact peptide. The lysosomal inhibitor chloroquine (200 microM) increased the intracellular accumulation of [125I-Tyr4]Bn but had no effect on the subcellular distribution of [125I-D-Tyr6]Bn(6-13)PA. Consistent with these observations, the treatment of cells with 100 nM Bn at 37 degrees reduced cell surface receptors within minutes, whereas [D-Tyr6]Bn(6-13)PA had no effect. The addition of PMA did not induce the internalization of antagonist-occupied receptors, but pharmacological inhibition of PKC decreased the rate of agonist-induced receptor internalization. These results therefore demonstrate that although PKC contributes to agonist-induced internalization of the GRP-R, it does not elicit receptor internalization of the antagonist-occupied receptor.
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77225, USA.
authors.forename: B Y
authors.initials: BY
authors.lastname: Williams
authors.forename: S B
authors.initials: SB
authors.lastname: Dion
authors.forename: A
authors.initials: A
authors.lastname: Schonbrunn
mesh_terms: D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007700:Kinetics; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D000075370:Receptors for Activated C Kinase; D018004:Receptors, Bombesin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002273:Carcinogens; D007328:Insulin; D000075370:Receptors for Activated C Kinase; D018004:Receptors, Bombesin; D011956:Receptors, Cell Surface; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D001839:Bombesin