FIG. 5.
Activity of two signal input and two signal output domains in p160 coactivator fragments. (A) Enhancement of AR AF-1 function by C-terminal p160 coactivator fragments containing NIDAF-1 and AD2. CV-1 cells were transiently transfected with 0.25 μg of pM vector encoding Gal4DBD-ARAF1, 0.5 μg of GK1 reporter gene, and 1.25 μg of pSG5.HA vector (no CoA) or the same vector encoding the indicated coactivator or coactivator fragment; luciferase activity was measured. Functional domains in diagrams of coactivator fragments are represented as in Fig. 1A. The activity of the Gal4 DBD alone was 0.1 × 105 RLU (data not shown). (B) Expression levels of p160 coactivators and their fragments in transfected cells. COS-7 cells were transfected with pSG5.HA vectors encoding the indicated coactivator or fragment. Cell extracts were subjected to immunoblot analysis with antibodies against the HA epitope. (C) AD1-dependent or AD2-dependent enhancement of AR AF-2 function by fragments of p160 coactivators. CV-1 cells were transfected with 0.5 μg of pM vector encoding Gal4DBD-ARAF2, 0.5 μg of GK1 reporter gene, and 1.0 μg of pSG5 vector encoding no coactivator (no CoA) or the indicated coactivator fragment or mutant, with or without DHT treatment as indicated. Functional domains in diagrams of coactivator fragments are represented as in Fig. 1A. GRIP1.NRBIIm+IIIm is full-length GRIP1 with LL-to-AA mutations in NR boxes II and III.