Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking.
We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.
Sinnett-Smith
J
J
Imperial Cancer Research Fund, London, England.
Zachary
I
I
Rozengurt
E
E
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001839:Bombesin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000378:metabolism*; D019886:Gastrin-Releasing Peptide; D007457:Iodine Radioisotopes; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D013388:Succinimides; C028722:ethylene glycolylbis(succinimidyl succinate); D019886:Gastrin-Releasing Peptide; C011240:dithiobis(succinimidylpropionate); D001839:Bombesin