CHARACTERIZATION OF LIGAND-BINDING AND PROCESSING BY GASTRIN-RELEASING PEPTIDE RECEPTORS IN A SMALL-CELL LUNG-CANCER CELL-LINE

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 (K(d) 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a K(i) of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with K(i) values of 22.7 and 59.1 nM respectively. Binding of I-125-GRP to COR-L42 cells increased rapidly at 37-degrees-C, 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 I-125-GRP was rapidly internalized. To determine the pathway of I-125-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10-mu-M), 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-mu-g/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of I-125-GRP, suggesting that these peptides do not down-regulate GRP receptors.