Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Abstract

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.

Selection of Radiolabeled Gastrin Analogs for Peptide Receptor-Targeted Radionuclide Therapy

“…After labeling these peptides with 111 In, we compared their biodistribution with that of 3 comparators—described by Reubi et al (10), Behe et al (9), and Bernard et al (15). We studied the biodistribution in 3 nude mouse models using tumors expressing differing levels of gastrin/CCK-2 receptors: AR42J (16), CA20948 (17), and HT29-CCK2R, a human colorectal cell line transfected with relatively low levels of gastrin receptors (Academic Unit of Cancer Studies). We chose to study the biodistribution of these radioligands 4 h after intravenous administration.…”

Circular Dichroism Study on Fully Bioactive CCK-Peptides of Increasing Chain Length

“…The tracer used for the competitive binding assays on CCK-receptor containing systems was 125I-BH-(Thr,Nle)-CCK-9; this was prepared fol lowing the procedure previously described [28]. The CCK-peptides were analyzed for their binding affinities to intact A R 4 -2 J cells [29] as well as to membrane preparations of these cells and to mem branes from normal pancreas as described pre viously [30]. Binding to intact rat pancreatic acini was determined following known protocols [31].…”

Visualization of G Protein-coupled Receptor Trafficking with the Aid of the Green Fluorescent Protein

“…The CCKAR was well characterized with respect to the mechanisms of signal transduction (10) and phosphorylation upon ligand binding (11). It was shown to undergo sequestration upon binding of agonists (12)(13)(14)(15)(16)(17) like many other GPCR. It was suggested that internalization of the CCKAR and many other GPCRs is ligand-dependent, but direct proof has been missing.…”