In situ photolinked nuclear progesterone receptors of human breast cancer cells: subunit molecular weights after transformation and translocation.
The subunit structure of mammalian cytoplasmic progesterone receptors (PR) has been difficult to study because these proteins are subject to in vitro proteolysis; the structure of nuclear PR is unknown. We have now developed an in situ photoaffinity labeling method for PR that permits study of their subunits with minimal in vitro incubations. The strategy is to use [3H]R5020, a synthetic photoactive progestin, and suitable incubation temperatures to place receptors into their precise intracellular sites in intact cells. The cells, still intact, are then irradiated with UV at 300 nm for 2 min. This irradiation efficiently (approximately 15%) yields covalently linked hormone-receptor complexes at any intracellular location. Cells are than rapidly ruptured, nuclei are separated, and receptors are extracted with salt and/or directly solubilized with detergents before the subunits are displayed on denaturing polyacrylamide gels. With this as well as with modified in vitro methods, we show here that untransformed human breast cancer PR have two dissimilar subunits (mol wt, 115,000 and 81,000) present in equimolar amounts. The same subunits, with apparently unmodified mass, can be demonstrated in nuclei after they have been translocated by progestin treatment. Therefore, PR transformation and acquisition of nuclear binding capacity does not require prior proteolytic processing of subunits or other major structural modifications that are detectable on single dimension gels.
D000345:Affinity Labels / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D009649:Norpregnadienes / Q000378:metabolism*; D011397:Promegestone / Q000378:metabolism* / Q000528:radiation effects; D011980:Receptors, Progesterone / Q000378:metabolism*; D014466:Ultraviolet Rays
D000345:Affinity Labels; D046911:Macromolecular Substances; D009649:Norpregnadienes; D011980:Receptors, Progesterone; D011397:Promegestone