title: Bombesin receptor from Swiss 3T3 cells. Affinity chromatography and reconstitution into phospholipid vesicles.
abstract: Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys3]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [35S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and the eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed [125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity.
authors.affiliation: Imperial Cancer Research Fund, London, UK.
authors.forename: A
authors.initials: A
authors.lastname: Coffer
authors.forename: J
authors.initials: J
authors.lastname: Sinnett-Smith
authors.forename: E
authors.initials: E
authors.lastname: Rozengurt
mesh_terms: D000818:Animals; D001710:Biotin; D001839:Bombesin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D019886:Gastrin-Releasing Peptide; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D010743:Phospholipids; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D010743:Phospholipids; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D001710:Biotin; D019886:Gastrin-Releasing Peptide; D001839:Bombesin; D002118:Calcium