Purification and Characterization of a Novel Intracellular 17ß-Estradiol Binding Protein in Estrogen-Resistant New World Primate Cells

Hong Chen, Bing Hu, Gang-Hua Huang, Amanda G. Trainor, David H. Abbott and John S. Adams

Division of Endocrinology, Diabetes and Metabolism (H.C., G.-H.H., J.S.A.), and Department of Pathology (B.H.), Cedars-Sinai Burns and Allen Research Institute, UCLA School of Medicine, Los Angeles, California; and Pathology Unit, Department of Obstetrics and Gynecology (A.G.T., D.H.A.), Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin

Address correspondence to: Hong Chen, M.D., Division of Endocrinology, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room B-131, Los Angeles, California 90048. E-mail: chenh{at}cshs.org

Abstract

Compared to Old World primates, including man, New World primates display target-tissue resistance to gonadal steroid hormones. In female New World primates this resistant phenotype is characterized by elevated concentrations of plasma estradiol and progesterone. Here we describe the discovery of an intracellular estrogen binding protein (IEBP) that acts to concentrate 17ß-estradiol (E2) in the cytoplasm of New World primate target cells. IEBP was purified by E2-affinity chromatography from postnuclear extracts of the B95-8 cells established from an E2-resistant New World primate. Compared with unpurified extract, affinity-purified IEBP demonstrated a 300-fold enrichment in specific E2 binding activity; half-maximal displacement of [3H]E2 from affinity-purified IEBP was observed with 0.1 nM E2. Affinity-purified extracts were subjected to SDS-PAGE with isolation of a dominant 27-28 kDa protein. N-terminal sequencing of tryptic peptides of the protein showed sequence homology with human heat shock protein-27 (hsp27). By immunoblot and E2 binding capacity, IEBP was 1] 2–3-fold greater in New World than in Old World primate tissues and cell lines, 2] heat-inducible and 3] up-regulated in vivo in the presence of the functioning female gonad. In conclusion, IEBP is a specific E2-interacting heat shock protein in the hsp-27 family that is relatively overexpressed in estrogen-resistant cells.