Bombesin receptor from Swiss 3T3 cells Affinity chromatography and reconstitution into phospholipid vesicles

FEBS Letters

Coffer, Arnold¹  Sinnett-Smith, James¹  Rozengurt, Enrique¹ 

[1] Imperial Cancer Research Fund. PO Box Lincoln's Inn Fields, London WC2A 3PX, UK

关键词: Signal transduction;    Growth control;    Biotinylated bombesin;   

DOI  :  10.1016/0014-5793(90)81462-W

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown