Endocytosis of gastrin in cancer cells expressing gastrin/CCK-B receptor

Abstract

Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4-7 min) and internalization of fluorescent ligand upon binding at room temperature and 37 degrees C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37 degrees C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.

Regulation of Membrane Cholecystokinin-2 Receptor by Agonists Enables Classification of Partial Agonists as Biased Agonists

“…Biased ligands identified in the current study should be able to block ␤-arrestin-dependent signaling and effects downstream of the CCK2R. This is of high importance because the CCK2R is expressed in a large variety of cancers and, according to a previous study (19), undergoes internalization in gastric, pancreatic, and colonic cell lines that endogenously express this receptor.…”

“…Moreover, we delineated the role and mechanism of action of RGS-2 (regulator of G-protein signaling-2) in CCK2R-induced inositol phosphate production (17). However, the mechanism and consequences of CCK2R regulation at the cell surface by its natural ligands or by synthetic ligands are not yet precisely known (18,19).…”

Cholecystokinin and Gastrin Receptors

“…Both CCK1R and CCK2R have been shown to undergo internalization in pancreatic acini and transfected NIH 3T3 or CHO cells (177,418,522). Interestingly, in the case of CCK1R, agonist and antagonistinduced desensitization of CCK1R were described (417).…”

Cytotoxic agents directed to peptide hormone receptors: Defining the requirements for a successful drug

“…The receptors and ligands are sorted in endosomes (11). Both CCK-A and CCK-B receptors are recycled to the cell surface with very high efficiency, whereas the peptide ligands end up in lysosomes, where presumably they are degraded (11,29). This paper reports on the synthesis, processing, and selective toxicity of drugs directed to CCKBR-positive cells.…”

“…Subsequent studies in our laboratory, which used fluorescent-labeled heptagastrin and CCK-8 (11,28,29), showed that both CCKBR and CCK-A receptor agonists undergo internalization by endocytosis upon binding to the receptors. The receptors and ligands are sorted in endosomes (11).…”