Subunit structure of the nonactivated human estrogen receptor.
The nonactivated estrogen receptor of human MCF-7 mammary carcinoma cells was investigated with respect to stoichiometry of protein subunits. The native receptor complex stabilized by molybdate had a molecular mass of approximately 300 kDa. Chemical cross-linking with several bifunctional reagents resulted in complete stabilization of the same receptor form of approximately 300 kDa and was achieved both in cell extracts and in intact cells. Incubation of the cross-linked receptor with a receptor-specific monoclonal IgG1 antibody increased the molecular mass by approximately 135 kDa--i.e., no more than one immunoglobulin molecule bound to the complex. Partial and progressive cross-linking of affinity-labeled receptors revealed patterns of labeled bands upon denaturing gel electrophoresis indicative of a heteromeric structure. The completely cross-linked receptor was purified to homogeneity and analyzed for protein components. In addition to the receptor polypeptide of approximately 65 kDa, we detected the heat shock proteins hsp90 and p59; the hsp90 band was roughly twice as intense as the p59 band. The heat shock protein hsp70 and the 40-kDa cyclophilin were not detected as components of the highly purified cross-linked receptor of approximately 300 kDa. We suggest a heterotetrameric structure consisting of one receptor polypeptide, two hsp90 molecules, and one p59 subunit, for which the molecular mass adds up to approximately 300 kDa. Thus, the nonactivated estrogen receptor has a molecular architecture homologous to those of glucocorticoid and progesterone receptors, even though phylogenetically the estrogen receptor gene forms a distinct subgroup within the gene family of nuclear hormone receptors.
Segnitz
B
B
Institut für Biologische Chemie, Universität Heidelberg, Germany.
Gehring
U
U
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms; D002460:Cell Line; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G; D007700:Kinetics; D046911:Macromolecular Substances; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013388:Succinimides; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D011960:Receptors, Estrogen; D013388:Succinimides; D013629:Tamoxifen; C028722:ethylene glycolylbis(succinimidyl succinate); C031859:tamoxifen aziridine