Abstract
We have used the technique of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids to study the synthesis and degradation rates of the progesterone receptor in MCF-7 human breast cancer cells. In cells grown in the absence of progestin, sucrose gradient shift analyses reveal that it takes 17 h for the normal density progesterone receptor levels to be reduced to half the initial value, whereas in the presence of 10 nM of the synthetic progestin [3H]R5020, the receptor turns over more rapidly, such that the normal density R5020-occupied progesterone receptor complexes are reduced to half in 12 h. The accelerated progesterone receptor turnover in the presence of [3H]R5020 reflects increased turnover rates of both the A (Mr-85,000) and B (Mr-115,000) subunits, as determined by sodium dodecyl sulfate gel analyses of dense and light receptors photoaffinity labeled with [3H]R5020. In both control and progestin-exposed cells, the time course of progesterone receptor turnover shows a lag of approximately 6 h after dense (15N, 13C, 2H) amino acid exposure, before dense hormone binding receptor species are seen and before normal density progestin binding activity starts decreasing. Since our evaluations of progesterone receptor depend upon its binding of radiolabeled ligand ([3H]R5020), this lag in the density shift kinetics would be consistent with the presence of a non-hormone binding biosynthetic precursor, from which the hormone-binding form of progesterone receptor is derived. A kinetic model is used to analyze the lag-decay profiles and to determine the rate constants for progesterone receptor synthesis, activation to the hormone-binding form, and degradation.
Highlights
From the Department of Physiology and Biophysics, University of Illinois, and University of Illinois College of Medicine, Urbana, Illinais 61801
We have used a well characterized human breast cancer sence of progestin, sucrose gradient shift analysesreveal that it takes17 h for the normal density progesterone receptor levels tobe reduced to half the initial value, whereas in the preseonfc1e0nM of the synthetic progestin[3H]R5020, thereceptorturnsovermore rapidly, such that the normal densiRty5OZO-occupied progesterone receptor complexes are reduced to half in 12 h
Whereas cells grown without estradiol have only low, basal P R levels (9), cells treated with estradiol havemarkedly increased P R levels (8), and with continued estradiol treatmentP, R remains elevated at a constant, steady-statelevel
Summary
The lag-decay profiles and to determine the ratecon- Chemicals-Radioactive R5020 Nor-pregna-4,g-diene 3,20-dione-[17a-methyL38H7],Ci/mmol) and radioinert R5020 (New England Nuclear Corp.); l7fl-estradiol, progesterone, cortisol, leupeptin, phenylmethylsulfonyl flouride, soybean trypsin inhibitor, TrisE, DTA, thioglycerol (Sigma).All chemicals for the SDS-polyacrylamide gel electrophoresis analysis were obtained from Bio-Rad. The Triton/xylene scintillation fluid contained 3 g/. For amino acids; R5020, 17,21-dimethyl-19 nor-pregna-4,9-diene 3,20- experiments, cells were grown in normal amino acid medium suppledione; estradiol, 1,3,5(10)-estratriene-3,17fl-diolC;DCS, charcoal- mented with 15% dialyzed charcoal-dextran-treated serum, 0.5%. Synthesis and Degradation harvesting the cells, the normal medium was replaced with medium described (17) using seven 8-W black light bulbs with a 350-nm containing dense amino acids. The protein was extracted from the gel by incubating indicated times in NAA medium + estradiol or in DAA medium + the gel slices with 5 ml of 7.5% protosol in toluene-based scintillation estradiol. Buffer) and homogenized in 300 pl of TETG buffer containing protease inhibitors(soybean trypsin inhibitor, 5mg/ml; leupeptin, 1mg/
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