Hormone-dependent covalent modification and processing of human progesterone receptors in the nucleus.

In situ photoaffinity labeling, which minimizes in vitro incubations and proteolytic artifacts, was used to study the structure of progesterone receptors (PR) in intact T47D human breast cancer cells. These cells, rich in PR, were incubated with the photoreactive progestin [3H]R5020 at 0 degrees C for 3 hr to keep PR in their untransformed state, or at 37 degrees C for 5 min to transform PR and convert them to tight chromatin-binding proteins. The cells, still intact, were then irradiated with 300-nm UV light to link the hormone covalently to receptors at any intracellular location. In T47D cells, untransformed PR, as well as transformed nuclear-bound PR, have equimolar amounts of proteins A (Mr approximately 94,000) and B (Mr approximately 120,000). The quantitative relationship between these is stable--no degradation of B to A is seen even if in situ photolabeled receptors are incubated in vitro at 37 degrees C for as long as 1 hr. Analysis of the in situ labeled receptors on gradient NaDodSO4-polyacrylamide gels shows that the untransformed B protein is a doublet of Mr approximately 117,000 and 120,000, while the A protein is a singlet. After R5020 treatment, transformed hormone-receptor complexes rapidly (5 min) translocate to nuclei. During the next 30 min the B protein becomes modified and shifts entirely to the heavier, Mr approximately 120,000 form. Between 30 and 60 min after nuclear binding, the A protein first splits, and then also becomes approximately 3000 daltons heavier. These changes are consistent with asynchronous modification--occurring first in protein B and then in protein A. Four to 8 hr after nuclear residence, receptor "processing" leads to the simultaneous disappearance of both proteins without generation of smaller molecular weight fragments. Peptide mapping shows that proteins A and B are closely related: despite the initial difference in molecular weight of A and B, digestion with Staphylococcus aureus V8 protease yields identical fragmentation patterns for each, with sequential peptides of Mr approximately 49,000, 39,000, 26,000, and 14,000. These data are consistent with the hypothesis that B and A are closely related integral intracellular proteins; that in their untransformed state only B is phosphorylated; that hormone treatment leads to their rapid (5 min) transformation to nuclear and DNA binding states; and that a nuclear phosphoprotein kinase(s) then modifies both proteins further to influence their gene regulatory activities.