Estrogen Receptor Accessory Factors in Breast Cancer Cells.
Annual rept. 12 Jul 95-24 Jul 96,
CHICAGO UNIV IL
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The goal of this investigation is to test the hypothesis that estrogen agonists and antagonists promote differential transcriptional activity of the estrogen receptor ER by altering accessory protein interactions. We have shown that one or more ER-associated proteins hsp70, PDI, p48, p45 may be required for maximal interaction of ER with specific DNA sites EREs in responsive genes. In addition, circular permutation and phasing analyses demonstrated that the same proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. We have also used the bacterially expressed ligand binding domain of the human estrogen receptor ER-LBD to capture and characterize proteins from T47D human breast cancer cell extracts that selectively associate with the ER-LBD in the presence or absence of estradiol or two estrogen antagonists, 4-hydroxytamoxifen partial antagonist and ICI 182,780 complete antagonist. Several agonist-specific associated proteins were isolated. At least one of these proteins, which phosphorylated the ER-LBD in an in vitro kinase assay, was identified as a serinethreonine kinase. Additional data indicated that the kinase activity represents a protein or complex of greater than 200 kDa and that it is present in both nuclear and cytosolic extracts. Because the isolated kinase activity is agonist-specific and associated with the AF-2 region of ER, it ma be important for the transcriptional activity of the receptor.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research