The estrogen receptor in MCF-7 cells: evidence from dense amino acid labeling for rapid turnover and a dimeric model of activated nuclear receptor.
Upon treatment of MCF-7 cells with estradiol at 37 C, practically all of the estrogen receptor is found in the nuclear compartment. This nuclear estrogen receptor form sediments close to 5S on high salt sucrose density gradients, similar to receptor activated in vitro by gentle heating. Upon extraction of the nuclear receptor, this 5S form is not stable at 0-4 C under our buffer conditions and is converted to a 4S form in 10-14 h. Incubation of cells in the presence of dense amino acids for various amounts of time in the absence of estradiol, followed by a 20- to 30-min labeling period with 3 nM [3H]estradiol at 37 C, permits separation of the newly synthesized estrogen receptor on sucrose gradients by means of its different sedimentation properties. With the fresh nuclear extract, we observe a progressive shift to a heavier form on sucrose gradients in proportion to the time of incubation in dense amino acids. The 5S nuclear receptor gradually becomes denser as the time of incubation of cells in dense medium increases and the peak moves from light to heavy without resolving into discrete heavy and light forms at intermediary time points. In contrast, when the nuclear extract, prepared after varying periods of incubation of the cells with dense amino acids and brief treatment with [3H]estradiol, is left standing at 0-4 C for a period of 10-14 h, we observe discrete peaks of heavy and light receptors. The resolution into discrete peaks of heavy and light forms at each time point permits a calculation of the turnover rate for this receptor (t 1/2 = approximately 2.25 h). These findings are consistent with a model in which the 5S nuclear receptor is an oligomer of randomly associated heavy and light monomers, whereas the aged 4S nuclear receptor is a monomer. We conclude from these data that estrogen receptor synthesis is extremely rapid in log growth phase MCF-7 cells; and the activated nuclear receptor appears to be an oligomer of at least two monomers closely related in size and metabolized at a similar rate. The random association between different monomers (heavy and light) results in a broad peak of nuclear receptor and is at least consistent with a dimerization reaction occurring as part of receptor activation.
D000596:Amino Acids / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D008954:Models, Biological; D011960:Receptors, Estrogen / Q000378:metabolism*; D013997:Time Factors
D000596:Amino Acids; D046911:Macromolecular Substances; D011960:Receptors, Estrogen