Evidence for the existence of a third progesterone receptor protein in human breast cancer cell line T47D.
We have used a new monoclonal antibody, designated C-262, directed against the last 14 amino acids of the carboxy-terminus of human progesterone receptors (N.L. Weigel et al., Mol. Endocrinol., 6: 1585-1597, 1992) to analyze progesterone receptor structure. This new antibody recognizes the previously described B-receptors (M(r) 120,000) and the naturally occurring N-terminal truncated A-receptor (M(r) 94,000). In addition to B- and A-receptors, C-262 detects a third progestin-binding protein with a molecular weight of approximately 60,000 in the progestin-responsive human breast cancer cell line, T47D. The 60,000 dalton protein is predominantly found in the cytosolic fraction of untreated T47D cells and binds tightly to the nucleus following progesterone or R5020 treatment of T47D cells. These dynamics are similar to the previously described progesterone receptor isoforms. The 60,000 dalton protein binds the synthetic progestin, [3H]R5020, which competes with cold R5020 as determined with the technique of in situ photoaffinity labeling. Prolonged incubation of nuclear extracts at elevated temperatures does not result in accumulation of the 60,000 dalton protein, yet the level of photoaffinity-labeled B- and A-receptors declines. These data support our hypothesis that the 60,000 dalton protein is not a degradation product of the two larger progesterone receptor isoforms but a distinct progestin-binding protein. This is further supported by our previous study identifying at least two progesterone receptor mRNAs that do not code B- or A-receptors. These two transcripts are not unique to T47D cells and also are present in human breast cancer cells, MCF-7, and normal human endometrium. Taken together, these data provide evidence for the existence of a third progesterone receptor isoform in progestin-responsive tissues.
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Department of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033.
Miner
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D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D006801:Humans; D008970:Molecular Weight; D011372:Progestins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D011980:Receptors, Progesterone / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011372:Progestins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011980:Receptors, Progesterone