Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

✍ Scribed by James Sinnett-Smith; Ian Zachary; Enrique Rozengurt

• Publisher: John Wiley and Sons
• Year: 1988
• Tongue: English
• Weight: 949 KB
• Volume: 38
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.240380403

✦ Synopsis

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 [ 11.

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,OOO-85 ,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with '251-labelled gastrin-releasing peptide ( '251-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,OOO crosslinked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit '251-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 12%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-0-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.