A 12(S)-hydroxyeicosatetraenoic acid receptor interacts with steroid receptor coactivator-1

Lewis lung carcinoma cells contain specific high-affinity binding sites for the eicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE], These binding sites have a cytosolic/nuclear localization and contain the heat shock proteins hsp70 and hsp90 as components of a high molecular weight cytosolic binding complex. The ligand binding subunit of this complex is a protein with an apparent molecular mass of approximate to 50 kDa as judged by gel permeation chromatography, In this report, we present data showing that the 50-kDa 12(S)-HETE binding protein interacts as a homodimer with steroid receptor coactivator-1 (SRC-1) in the presence of 12(S)-HETE. Two putative interaction domains were mapped. One of these (amino acids 701-781) was within the nuclear receptor interaction domain in SRC-1 required for binding of various steroid and thyroid hormone receptors, It contains the most C-terminal of the three copies of LXXLL motif present in the nuclear receptor interaction domain, The second interaction domain was present in the N-terminal part of SRC-1 (amino acids 1-221), This region has two LXXLL motifs, one does not bind and the other binds only weakly to steroid and thyroid hormone receptors, Glutathione S-transferase (GST) pulldown experiments and far Western analyses demonstrated that the N-terminal region of SRC-1 (amino acids 1-212) alone does not bind the 50-kDa 12(S)-HETE binding protein, whereas GST/Delta SRC-1(1-1138) ligand-dependently pulled down a protein of approximate to 50 kDa in size. Our results suggest that the 50-kDa 12(S)-HETE binding protein is a receptor that may signal through interaction with a nuclear receptor coactivator protein.