Identification of the bombesin receptor on murine and human cells by cross-linking experiments.
The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.
D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D019886:Gastrin-Releasing Peptide; D005910:Glioma / Q000032:analysis; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000032:analysis* / Q000378:metabolism; D013388:Succinimides
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D013388:Succinimides; C035891:bombesin, Tyr(4)-; D019886:Gastrin-Releasing Peptide; D006026:Glycoside Hydrolases; C020668:glycanase; D001839:Bombesin; C019358:disuccinimidyl suberate