BOMBESIN RECEPTOR FROM SWISS 3T3-CELLS - AFFINITY-CHROMATOGRAPHY AND RECONSTITUTION INTO PHOSPHOLIPID-VESICLES

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 [lys1]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 [13S]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 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. © 1990.