The Subunit Structure of Human Breast Cancer Progesterone Receptors: Characterization by Chromatography and Photoaffinity Labeling*

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Abstract
We have partially purified the progesterone receptors (PR) from T47D human breast cancer cells and show that they consist of at least two hormone-binding proteins of dissimilar size and unequal DNA-binding forms with electrophoretic properties analogous to the A and B subunits of chick oviduct PR. Cytosols were labeled with the progestin [3H]R5020 in the presence or absence of unlabeled R5020, and samples of the parallel incubations were chromatographed on DEAE-cellulose. A large heterogeneous peak of specific R5020 binding eluted between 0.1 and 0.2 M KCl. Pooled fractions of this peak were resolved on phosphocellulose into two peaks: an acidic moiety eluting at 0.15 M KCl, and a more basic one eluting at 0.2 M KCl. Both sediment at approximately 4.1S on sucrose density gradients. To test DNA binding capacity, two studies were performed. First, ammonium sulfate-precipitated receptors were applied to DNA-cellulose; approximately half of the specific radioactivity was retained. Second, separate DNA-cellulose chromatography of the individual phosphocellulose peaks showed that the basic peak had greater affinity for DNA. Molecular weights were determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis after in vitro covalent photoaffinity linking of the receptors to [3H]R5020, a photoreactive ligand. Radioactive R5020-receptor complexes were precipitated with ammonium sulfate, and the redissolved samples were immediately photolinked and electrophoresed. Two specifically photolinked peaks with molecular weights of 115,000 (B subunit) and 81,000 (A subunit), were invariably present in equimolar ratios (B:A = 1.1; n = 9). Similar values were obtained for receptors irradiated in cells in situ. A third specifically photolabeled peak (mol wt, 206,000) was seen in approximately 20% of gels after in vitro irradiation. It may be an adventitiously crosslinked A-B dimer. We conclude that human breast cancer PR, like chick oviduct PR but unlike other steroi receptors, are oligomers composed of at least two dissimilar subunits.