Identification of the bombesin receptor on murine and human cells by cross-linking experiments

R. M. Kris, R. Hazan, J. Villines, T. W. Moody, J. Schlessinger

Research output: Contribution to journalArticle

65 Scopus citations

Abstract

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 ~100,000 and ~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.

Original languageEnglish (US)
Pages (from-to)11215-11220
Number of pages6
JournalJournal of Biological Chemistry
Volume262
Issue number23
StatePublished - Oct 19 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Kris, R. M., Hazan, R., Villines, J., Moody, T. W., & Schlessinger, J. (1987). Identification of the bombesin receptor on murine and human cells by cross-linking experiments. Journal of Biological Chemistry, 262(23), 11215-11220.