EVIDENCE FOR THE EXISTENCE OF A 3RD PROGESTERONE-RECEPTOR PROTEIN IN HUMAN BREAST-CANCER CELL LINE-T47D

We have used a new monoclonal antibody, designated C-262, directed aga inst the last 14 amino acids of the carboxy-terminus of human progeste rone receptors (N. L. Weigel et al., Mol. Endocrinol., 6. 1585-1597, 1 992) to analyze progesterone receptor structure. This new antibody rec ognizes the previously described B-receptors (M(r) 120,000) and the na turally occurring N-terminal truncated A-receptor (M(r) 94,000). In ad dition to Band A-receptors, C-262 detects a third progestin-binding pr otein with a molecular weight of approximately 60,000 in the progestin -responsive human breast cancer cell line, T47D. The 60,000 dalton pro tein is predominantly found in the cytosolic fraction of untreated T47 D cells and binds tightly to the nucleus following progesterone or R50 20 treatment of T47D cells. These dynamics are similar to the previous ly described progesterone receptor isoforms. The 60,000 dalton protein binds the synthetic progestin, [H-3]R5020, which competes with cold R 5020 as determined with the technique of in situ photoaffinity labelin g. Prolonged incubation of nuclear extracts at elevated temperatures d oes not result in accumulation of the 60,000 dalton protein, vet the l evel of photoaffinity-labeled B- and A-receptors declines. These data support our hypothesis that the 60,000 dalton protein is not a degrada tion product of the two larger progesterone receptor isoforms but a di stinct progestin-binding protein. This is further supported by our pre vious 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 evide nce for the existence of a third progesterone receptor isoform in prog estin-responsive tissues.