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 /sup 125/I-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 /sup 125/I-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 /sup 125/I-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.
- Research Organization:
- Meloy Laboratories Inc., Rockville, MD
- OSTI ID:
- 6025398
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 262:23
- Country of Publication:
- United States
- Language:
- English
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GLUCOPROTEINS
CROSS-LINKING
PEPTIDES
RECEPTORS
BIOCHEMICAL REACTION KINETICS
CLONE CELLS
ELECTROPHORESIS
FIBROBLASTS
IODINE 125
MAN
MICE
MOLECULAR WEIGHT
TRACER TECHNIQUES
TUMOR CELLS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
CARBOHYDRATES
CELL CULTURES
CHEMICAL REACTIONS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
POLYMERIZATION
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RODENTS
SACCHARIDES
SOMATIC CELLS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques