FIG. 4.

Fli-I is a secondary coactivator for NRs. (A) Dependence of Fli-I coactivator activity on GRIP1 and ER. CV-1 cells were transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and with expression vectors encoding ER (100 ng), GRIP1 (250 ng), CARM1 (500 ng), and Fli-I (500 ng) as indicated elsewhere. Transfected cells were grown with E2, and luciferase activities of the transfected-cell extracts were determined. (B) Increasing coactivator activity with increasing Fli-I. Transfections were performed as done for panel A except that 200 ng of CARM1 vector and 200 to 600 ng of Fli-I vector were used. (C) Partial coactivator function by the Fli-I LRR domain. Transfections were performed with MMTV(TRE)-LUC reporter plasmid (250 ng) and expression vectors encoding TR (100 ng), GRIP1 (250 ng), CARM1 (500 ng), and full-length Fli-I or the LRR or gelsolin-like fragment (500 ng). Transfected cells were grown with T3 before harvesting for luciferase assays. Similar results were obtained with ER and the MMTV(ERE)-LUC reporter plasmid (data not shown). (D) Autonomous activation function in the Fli-I LRR domain. CV-1 cells were transfected with GK1 reporter plasmid (250 ng) encoding luciferase controlled by Gal4 response elements and expression vectors encoding Gal4 DBD or Gal4 DBD fused to the LRR domain (250 ng) and ER (500 ng). Transfected cells were grown with E2 as indicated. Results portrayed in panels A to D are representative of three or more independent experiments.