Structural analyses of progesterone receptors

Progesterone receptors of T47Dco human breast cancer cells consist of two equimolar hormone-binding proteins of mol. wt approximately 85,000 (A protein) and 115,000 (B protein). Both proteins can be demonstrated in intact cells by in situ photoaffinity labeling; that is, in cells treated with the synthetic progestin 3H]R5020, irradiated 2 min with 300 nm u.v., solubilized directly in SDS and subjected to electrophoresis under denaturing conditions. These proteins are 6000-10,000 dalton heavier than the corresponding proteins of chick oviducts. This difference has been measured by direct comparison of photolabeled chick and human receptors on SDS-PAGE and by immunoblotting with the 9G10 antibody prepared against chick protein B. The antibody binds to a protein of mol. wt approximately 106,000 in human cells that is smaller than the hormone-bound B protein and larger than the hormone-bound A. In T47Dco cells, in situ photolabeled, untransformed receptors, as well as transformed nuclear-bound receptors, have equimolar amounts of A and B proteins. This ratio remains stable during a 1 h 37 degrees C in vitro incubation. Analysis of the in situ labeled receptors on gradient gels shows that the untransformed B protein exists as a doublet of mol. wt approximately 115,000 and 119,000 while the A protein is a singlet. After 3H]R5020 treatment, nuclear receptors change further: during the first 30 min in the nucleus the B protein shifts entirely to the heavier, mol. wt = 119,000 form. Between 30 and 60 min after nuclear binding, the A protein first becomes a doublet of 85,000 and 89,000 dalton then shifts entirely to the 4000 dalton heavier form. Later, nuclear processing leads to the simultaneous disappearance of both proteins without generation of smaller molecular weight fragments. Cleveland mapping studies show that the A and B proteins are closely related; despite the initial difference in the molecular weight of A and B, digestion with S. aureaus V8 protease yields identical fragmentation patterns for each, with sequential peptides of mol. wt approximately 49,000, 39,000, 26,000 and 14,000.(ABSTRACT TRUNCATED AT 400 WORDS)