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Title: 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, treatmentmore » 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.« less
Authors:
; ;
Publication Date:
OSTI Identifier:
6101675
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Cell. Biochem.; (United States); Journal Volume: 38:4
Research Org:
Imperial Cancer Research Fund, London (England)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PEPTIDES; RECEPTORS; BIOCHEMICAL REACTION KINETICS; AFFINITY; CELL CULTURES; CELL MEMBRANES; CROSS-LINKING; GLYCOPROTEINS; GLYCOSYL HYDROLASES; IODINE 125; MICE; MOLECULAR WEIGHT; TRACER TECHNIQUES; ANIMALS; BETA DECAY RADIOISOTOPES; CELL CONSTITUENTS; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; MAMMALS; MEMBRANE PROTEINS; MEMBRANES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; POLYMERIZATION; PROTEINS; RADIOISOTOPES; REACTION KINETICS; RODENTS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques