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 [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.
Coffer
A
A
Imperial Cancer Research Fund, London, UK.
Sinnett-Smith
J
J
Rozengurt
E
E
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*
D008565:Membrane Proteins; D010455:Peptides; D010743:Phospholipids; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D001710:Biotin; D019886:Gastrin-Releasing Peptide; D001839:Bombesin; D002118:Calcium