FIG. 1.

Affinity selection of ERα binding motifs by using phage display technology. Baculovirus-expressed full-length ERα was treated with 10⁻⁶ M 17β-estradiol and immobilized on 96-well Immulon 4 plates as a screening target. The LXXLL motif-containing phage peptide library was constructed as described in Materials and Methods. Phage that interacted specifically with estradiol-activated ER were selected, and the peptide sequences were deduced by DNA sequencing. These peptides were classified into three different classes based on sequences flanking the conserved LXXLL motif. Peptide #293 was obtained in a similar manner from random peptide libraries; it bound specifically to estradiol-activated ERβ when analyzed in vitro. Sequences from the center three copies of LXXLL motifs in the SRC-1 and GRIP-1 coactivators are also included for comparison. For reference, we have defined the first conserved leucine as position 1.