The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR.
In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.
Takahashi
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Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Breitman
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TR
D001386:Azides / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism*; D011959:Receptors, Estradiol / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001386:Azides; D009363:Neoplasm Proteins; D011959:Receptors, Estradiol; C017042:8-azidoadenosine-3',5'-monophosphate; D011374:Progesterone; D004958:Estradiol; D014212:Tretinoin; D000242:Cyclic AMP