gastrins and oxophenylarsine

Endocytosis of the gastrin releasing peptide receptor (GRP-R) may regulate cellular responses to GRP. We observed endocytosis in transfected epithelial cells by confocal microscopy using cyanine 3-GRP (cyanine 3.18-labeled gastrin releasing peptide) and GRP-R antibodies. At 4 degrees C, cy3-GRP and GRP-R were confined to the plasma membrane. After 5 min at 37 degrees C, ligand and receptor were internalized into early endosomes with fluorescein isothiocyanate-transferrin. After 10 min, cy3-GRP and GRP-R were in perinuclear vesicles, and at 60 min cy3-GRP was in large, central vesicles, while GRP-R was at the cell surface. We quantified surface GRP-R using an antibody to an extracellular epitope and an 125I-labeled secondary antibody. After exposure to GRP, there was a loss and subsequent recovery of surface GRP-R. Recovery was unaffected by cycloheximide, and thus independent of new protein synthesis, but was attenuated by acidotropic agents, and therefore required endosomal acidification. Internalization of 125I-GRP, assessed using an acid wash, was maximal after 10-20 min, and was clathrin-mediated since it was inhibited by hyperosmolar sucrose and phenylarsine oxide. Thus, GRP and its receptor are rapidly internalized into early endosomes and then dissociate in an acidified compartment. GRP is probably degraded whereas the GRP-R recycles.

Topics: Amino Acid Sequence; Animals; Arsenicals; Endocytosis; Gastrin-Releasing Peptide; Gastrins; Humans; Mice; Molecular Sequence Data; Osmolar Concentration; Peptides; Protein Binding; Receptors, Bombesin; Sucrose; Transferrin

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.

Topics: Animals; Arsenicals; Binding, Competitive; Cell Membrane; Chloroquine; Cholecystokinin; Cytochalasin D; Dinitrophenols; Endocytosis; Gastrins; Hydrogen-Ion Concentration; Iodine Radioisotopes; Kinetics; Pancreas; Pancreatic Neoplasms; Rats; Sincalide; Succinimides; Temperature; Tumor Cells, Cultured