Nongenomic Activity of Ligands in the Association of Androgen Receptor with Src

Abstract

Androgen receptor (AR) induces cell proliferation by increasing the kinase activity of Src. We describe an approach for discriminating agonist and antagonist in a nongenomic steroid-signaling pathway using an association of AR with Src. We constructed a pair of genetically encoded indicators, where N- and C-terminal fragments of split firefly luciferase (FLuc) were fused to AR and Src, respectively. The fusion proteins with AR and Src are localized in the cytoplasm and on the plasma membrane, respectively. Upon being activated with androgen, AR undergoes an intramolecular conformational change and binds with Src. The association causes the complementation of the split FLuc and recovery of FLuc activity. The resulting luminescence intensities were taken as a measure of the rapid hormonal activity of steroids in the nongenomic AR signaling. Ten minutes are required for the AR-Src association by 5alpha-dihydroxytestosterone (DHT), which was completely inhibited by an antagonist, cyproterone acetate. The activities of ligands in the nongenomic pathway of AR were compared with those in the genomic pathway obtained on the basis of the nuclear trafficking of AR in mammalian cells. The comparison revealed that DHT and testosterone activate both genomic and nongenomic pathways of AR. 17beta-Estradiol and progesterone were found to be specific activators only for the genomic signaling pathway of AR. On the other hand, procymidone exhibited a specific activity only for the nongenomic signaling pathway of AR. The present approach is the first example addressing the agonistic and antagonistic activities of ligands in a nongenomic pathway of AR.