OLIGOMERIC STRUCTURES OF CYTOSOLUBLE ESTROGEN-RECEPTOR COMPLEXES AS STUDIED BY ANTIESTROGEN RECEPTOR ANTIBODIES AND CHEMICAL CROSS-LINKING OF INTACT-CELLS

The structure of estrogen-receptor complexes recovered in cytosolic ex tracts of MCF-7 cells treated with hormone at 20 degrees was probed by chemical crosslinking of intact cells and sample analysis with four m onoclonal anti-estrogen receptor antibodies. When MCF-7 cells were tre ated 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 hig h 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 H22 6 antibodies could interact with any form we detected, the D75 and D54 7 monoclonals could only recognize those showing sedimentation coeffic ients lower than 7 S. When cytosolic extracts from [S-35]-methionine-l abeled cells were subjected to immunoprecipitation with H222 and D75 a nti-estrogen receptor antibodies, electrophoretic analysis of material extracted from immunoprecipitates revealed the presence of 65 kDa est rogen receptors. If extracts were prepared from crosslinked cells, ins tead, two more components with estimated molecular masses of 220 and 1 00 kDa were specifically immunoprecipitated by the H222 antibody, wher eas only the 100 kDa component and the estrogen receptor were found in immunoprecipitates obtained with the D75 monoclonal. When estrogen-re ceptor complexes were immunopurified from extracts prepared after cell s had been crosslinked with dithiobis(succinimidyl propionate), and th e oligomers were dissociated by treatment with beta-mercaptoethanol, e lectrophoretic analysis of our samples showed that only the 65 kDa est rogen receptor and a 50 kDa protein were selectively immunoprecipitate d by anti-estrogen receptor antibodies. We concluded that the structur es of cytosoluble estrogen-receptor complexes in MCF-7 cells treated w ith hormone at 2 degrees C, include oligomeric forms which contain a 5 0 kDa non-steroid binding protein.