Figure 4.

Mutations in the ERβ485 AF-2 surface impair repression of TNF-RE TKLuc. (A) U937 cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of wild-type ERβ485 or human ERβ485 AF-2 mutants (helix 3, K269A, and helix 12, E448K, based on ERβ485, which corresponds to K314A and E493K based on hERβ530, respectively). Cells were treated for 24 h with TNF-α (5 ng/ml) in the absence or presence of 10 nM E₂ and then assayed for luciferase activity. (B) Overexpressing GRIP1 enhances repression activity of ERβ485. Cells were cotransfected with 3 μg of TNF-RE TKLuc and 50 ng of ERβ485 and in the absence or presence of 5 μg of pSG5-GRIP1. All cells were treated for 24 h with TNF-α (5 ng/ml) in the absence or presence of 10 nM E₂. (C) Overexpressing GRIP1 restores repression activity of the helix 3 ERβ485 AF-2 mutant (K269A). Cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of ERβK269A in the presence of increasing amounts of pSG5–wild-type (WT) GRIP1 or pSG5-GRIP1 NR box II and III mutant (mut). All cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of 10 nM E₂. The data are expressed as per cent repression of the TNF-α activation of the TNF-RE. (D) RIP140 blocks E₂ repression of the TNF-RE in the presence of wild-type ERβ485. Cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of ERβ485 in the presence of increasing amounts of an expression vector for RIP140 and 5 μg of pSG5-wild-type (WT) GRIP1 or pSG5-GRIP1 NR box II and III mutant. All cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of 10 nM E₂ and then assayed for luciferase activity.