Figure 5.

PNRC interacts with AF1 and LBD domains of human ERα and functions as coactivator for both transactivation domains. (A) A schematic diagram of human ERα domains and the representation of the various ERα deletion constructs prepared and used. (B) Yeast strain Y187 was cotransformed with pACT2 (for the expression of AD_Gal4) or pACT2–PNRC_270–327 (for the expression of AD_Gal4–PNRC_270–327 fusion protein) and an yeast expression plasmid for the fusion proteins of DBD_Gal4 and various deletion mutants of ERα as indicated. The yeast transformants bearing both plasmids were cultured in the absence (DMSO) or presence of 10 nM E₂. β-Galactosidase activities were determined and expressed as the mean (units) ± SD of three independent colonies. (C) HeLa cells were transfected with pGL3 (ERE)₃_SV40_Luciferase reporter (0.25 μg) alone or along with pSG5–ERα wt, pSG5–hERα_1–251, or pSG5–hERα_185–595 (50 ng each) and with (+PNRC) or without pSG5–PNRC (1.0 μg) (−PNRC). Four hours after transfection, cells were cultured in the absence (DMSO, vehicle control) presence of 10 nM E₂ for additional 20 h. Twenty-four hours after transfection, cells were harvested, lysed and the luciferase activities in the cell lysate from triplicate wells were measured as described in the Materials and Methods, and expressed as mean ± SD of three independent assays.