Subunit composition of the estrogen receptor: Involvement of the hormone-binding domain in the dimeric state

Abstract

The purified estrogen receptor (ER) whether in 9 or 5 S molecular form, binds more than one molecule of the monoclonal antibody JS 34/32 (Redeuilh, G., Moncharmont, B., Secco, C., and Baulieu, E.-E. (1987) J. Biol. Chem. 262, 6969-6975). We now have investigated the effects of controlled trypsin proteolysis and of a dissociating chaotropic salt (NaSCN) on the structure of the estrogen receptor covalently labeled with radioactive tamoxifen aziridine. When the DNA-binding transformed 5S ER was dialyzed against a buffer containing 0.5 M NaSCN, it was converted into a form sedimenting at 3.7 S +/- 0.1 (n = 3). It reverted to the 5 S molecular form when NaSCN was dialyzed away. Fluorographic analysis of both the 5 and 3.7 S ER following SDS-gel electrophoresis revealed one main band corresponding to Mr congruent to 66,000. After limited trypsin treatment of the 5 S ER, tamoxifen aziridine-binding protein sedimented at 4.3 S +/- 0.1 (n = 5), had a Stokes radius of 3.6 nm (calculated Mr = 65,000), and did not bind DNA. The same form was obtained after limited trypsin digestion of the ER bound to DNA-cellulose or to hsp 90 (the nontransformed 8-9 S ER molecular form). This 4.3 S trypsinized ER was reversibly dissociated by NaSCN into a congruent to 3 S +/- 0.1 (n = 3) molecular form. Fluorographic analysis of both the 4.3 and 3 S ER after SDS-gel electrophoresis showed one main radioactive band of Mr congruent to 30,000. Taken together our results suggest that 1) the 5 S ER is a homodimer of two Mr congruent to 66,000 hormone binding subunits which may be released as such from the nontransformed 8-9 S ER, 2) the trypsin digestion products yield two carboxyl-terminal fragments of Mr congruent to 30,000 that remain in the form of a dimer having lost their DNA-binding region, and 3) the trypsin cleavage would occur within the region located between the hormone-binding domain and the DNA-binding domain. These data indicate that the dimerization of the receptor occurs through hydrophobic interaction of its hormone-binding domain but cannot exclude that other part(s) of the receptor may also contribute to the dimer formation. The dimerization may be critically involved in the mechanism by which estradiol-receptor complexes promote change of gene transcription.