The actions of progesterone, a hormone of central importance in controlling the function and growth of female reproductive tissues, such as the breast and uterus, appear to be mediated via interaction with an intracellular protein, the progesterone receptor (PR). Although much is known about the nature of this protein, little is known about the synthesis and degradation of PR and factors that regulate these rates. Our initial studies have provided evidence for a biosynthetic, non hormone-binding precursor of PR and for differences in the form of PR when it is associated with the antiprogestin RU486 vs progestin.
Our aims are to study hormonal factors involved in regulation of PR levels and turnover, to investigate biosynthetic precursors of PR, and to examine the nature of nuclear PR complexes. We will use the density shift technique to examine how turnover kinetics are affected by the nature of the ligand (progestin vs antiprogestin) and the level of receptor occupancy. We will compare systems in which PR is under estrogen control (MCF-7 human breast cancer and rat uterine cells) and independent of estrogen (T47D human brease cancer cells). We will utilize a kinetic model, from which the PR precursor pool size and biosynthetic, activation and degradation rate constants can be derived, and we will attempt to characterize the precursor using monoclonal antibodies to PR. These approaches will also enable us to address the important issue of whether estradiol increases PR levels by changes in receptor synthesis, precursor activation, or stabilization of PR. Since we have found that nuclear PR complexes with antiprogestin sediment as 6 S species under conditions where the complexes with progestin are exclusively 4 S, we will examine factors that affect the 6 S/4 S ratio, determine the subunit composition of the 6 S species by crosslinking and photoaffinity labelling, and evaluate whether the 6 S species represents an """"""""unactivated"""""""" non-DNA binding form of PR. These studies should provide new information on the biosynthesis and degradation of this important regulatory protein, and on the receptor interactions that may underlie progestin antagonist action.
