The Molecular Mechanism Of Testosterone-mediated Androgen Receptor Trafficking In Mouse Sertoli Cells

Background:Testosterone and AR(Androgen Receptor)are critical for maintaining spermatogenesis and male fertility.It is reported that the AR located on membrane may play a potential roleof the non-classical pathway of androgen,yet there is still a controversy about the presence of AR located on the membrane.Purpose: To explore the molecular mechanism of Testosterone-mediating androgen receptor trafficking in mouse Sertoli cells.Methods: Western blot and immunofluorescence technique were employed to detect the localization of AR in TM4 cells after stimulation with testosterone.Path Scan Antibody Array were used to analyze the phosphorylation of proteins involved in the androgen receptor trafficking towards membrane.Kinases inhibitors were used to study the interaction among these protein kinases and relationships between those protein kinases and the trafficking of AR towards membrane.Results: In this study,we found that physiological testosterone(10 nM)induced fast membrane association of the classical AR.Using phosphorylation antibody arrays,several phosphorylation sites including MEK1/2(Ser217/221),Akt(Ser473)and Erk1/2(Thr202/Tyr204)were fast phosphorylated within 5 minutes.Inhibition of the MEK and Akt signaling pathway prevented AR trafficking.Block of AR by flutamide abolished the stimulation effect of testosterone on kinase phosphorylation.Testosterone induced kinase Srcphosphorylation and inihibition of kinase Src decreased AR translocated to membrane and nucleus.Conclusions: These data suggested that this membrane association of AR was mediated by MEK and Akt pathway,which resulted in activation of kinase Srcand initiated by testosterone binding to the membrane localized AR.