Molecular cloning,stable expression and cellular localization of human alpha1-adrenergic receptor subtypes:effect of charcoal/dextran treated serum on expression and localization of alpha_(1D)-adrenergic receptor

摘要

The cDNAs encoding for three subtypes of adrenergic receptors,alpha_(1A)-,alpha_(1B)-and alpha_(1D)-ARs,were cloned and expressed in HEK 293 cells.Expression of alpha_(1A)-and alpha_(1B)-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha_(1D)-AR was not.Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha_(1A)-and alpha_(1B)-ARs was primarily localized on the cell membrane whereas expression of alpha_(1D)-AR was predominantly intracellular.Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha_(1D)-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha_(1D)-AR to the cell membrane and thus,significantly improving its stability and availability for ligand binding studies.