SUBUNIT STRUCTURE OF THE NONACTIVATED HUMAN ESTROGEN-RECEPTOR

The nonactivated estrogen receptor of human MCF-7 mammary carcinoma ce lls was investigated with respect to stoichiometry of protein subunits . The native receptor complex stabilized by molybdate had a molecular mass of approximate to 300 kDa. Chemical cross-linking with several bi functional reagents resulted in complete stabilization of the same rec eptor form of approximate to 300 kDa and was achieved both in cell ext racts and in intact cells. Incubation of the cross-linked receptor wit h a receptor-specific monoclonal IgG1 antibody increased the molecular mass by approximate to 135 kDa-i.e., no more than one immunoglobulin molecule bound to the complex, Partial and progressive cross-linking o f affinity-labeled receptors revealed patterns of labeled bands upon d enaturing gel electrophoresis indicative of a heteromeric structure, T he completely cross-linked receptor was purified to homogeneity and an alyzed for protein components, In addition to the receptor polypeptide of approximate to 65 kDa, we detected the beat shock proteins hsp90 a nd p59; the hsp90 band was roughly twice as intense as the p59 band, T he heat shock protein hsp70 and the 40-kDa cyclophilin were not detect ed as components of the highly purified cross-linked receptor of appro ximate to 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 approximate to 300 kDa. Thus, the nonactivated estrogen receptor has a molecular architecture homol ogous to those of glucocorticoid and progesterone receptors, even thou gh phylogenetically the estrogen receptor. gene forms a distinct subgr oup within the gene family of nuclear hormone receptors,