Oligomeric structures of cytosoluble estrogen-receptor complexes as studied by anti-estrogen receptor antibodies and chemical crosslinking of intact cells

The structure of estrogen-receptor complexes recovered in cytosolic extracts of MCF-7 cells treated with hormone at 20 degrees was probed by chemical crosslinking of intact cells and sample analysis with four monoclonal anti-estrogen receptor antibodies. When MCF-7 cells were treated with either glutaraldehyde or dithiobis(succinimidyl propionate), cytosoluble estrogen-receptor complexes consisted of two major forms sedimenting as 4 S monomers and 8-9 S salt-resistant oligomers. By high salt sucrose density gradient centrifugation, we could observe that the four monoclonal anti-estrogen receptor antibodies bound different forms of receptor complexes from crosslinked cells. While H222 and H226 antibodies could interact with any form we detected, the D75 and D547 monoclonals could only recognize those showing sedimentation coefficients lower than 7 S. When cytosolic extracts from [S-35]-methionine-labeled cells were subjected to immunoprecipitation with H222 and D75 anti-estrogen receptor antibodies, electrophoretic analysis of material extracted from immunoprecipitates revealed the presence of 65 kDa estrogen receptors. If extracts were prepared from crosslinked cells, instead, two more components with estimated molecular masses of 220 and 100 kDa were specifically immunoprecipitated by the H222 antibody, whereas only the 100 kDa component and the estrogen receptor were found in immunoprecipitates obtained with the D75 monoclonal. When estrogen-receptor complexes were immunopurified from extracts prepared after cells had been crosslinked with dithiobis(succinimidyl propionate), and the oligomers were dissociated by treatment with beta-mercaptoethanol, electrophoretic analysis of our samples showed that only the 65 kDa estrogen receptor and a 50 kDa protein were selectively immunoprecipitated by anti-estrogen receptor antibodies. We concluded that the structures of cytosoluble estrogen-receptor complexes in MCF-7 cells treated with hormone at 2 degrees C, include oligomeric forms which contain a 50 kDa non-steroid binding protein.