title: Characterization of ligand binding and processing by gastrin-releasing peptide receptors in a small-cell lung cancer cell line.
abstract: The ligand-binding properties of the gastrin-releasing peptide (GRP) receptor and the cellular processing of GRP have been studied in the small-cell lung cancer (SCLC) cell line COR-L42. Scatchard analysis of GRP receptor expression indicated a single class of high-affinity receptors (Kd 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a Ki of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with Ki values of 22.7 and 59.1 nM respectively. Binding of 125I-GRP to COR-L42 cells increased rapidly at 37 degrees, achieved a maximum at 10 min and declined rapidly thereafter. At 4 degrees C, maximum binding was achieved at 30 min and the subsequent decline in cell-associated radioactivity was slower than that seen at 37 degrees C. Acid/salt extraction, to separate surface-bound ligand from internalized GRP, indicated that after receptor binding 125I-GRP was rapidly internalized. To determine the pathway of 125I-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10 microM), an inhibitor of the membrane-bound enzyme (EC 3.4.24.11). Both agents markedly inhibited the degradation of GRP, indicating that this process involves a lysosomal pathway and a phosphoramidon-sensitive pathway, possibly involving the EC 3.4.24.11 enzyme. GRP receptor down-regulation was observed following a 10 min exposure to 100 nM-GRP. With longer pretreatment times the number of binding sites recovered to 80% of control values. Treatment with 5 mM-chloroquine plus GRP or cycloheximide (10 micrograms/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of 125I-GRP, suggesting that these peptides do not down-regulate GRP receptors.
authors.affiliation: Clinical Oncology and Radiotherapeutics Unit, Medical Research Council, Cambridge, U.K.
authors.forename: C
authors.initials: C
authors.lastname: Cardona
authors.forename: N M
authors.initials: NM
authors.lastname: Bleehen
authors.forename: J G
authors.initials: JG
authors.lastname: Reeve
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001667:Binding, Competitive; D001839:Bombesin / Q000378:metabolism; D018288:Carcinoma, Small Cell; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D019886:Gastrin-Releasing Peptide; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008175:Lung Neoplasms; D010455:Peptides / Q000187:drug effects / Q000378:metabolism*; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D006020:Glycopeptides; D008024:Ligands; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D019886:Gastrin-Releasing Peptide; D002738:Chloroquine; D001839:Bombesin; C008890:phosphoramidon