Estrogen receptor-associated proteins and their influence on receptor function

• Main Author: Landel, Carolyn Church
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1995
• Subjects: Biochemistry; Molecular biology
• ISBN: 9798209450689

The estrogen receptor (ER) is a member of the superfamily of nuclear receptors that are activated by ligand and subsequently bind specifically to DNA in the region of responsive genes, resulting in transcriptional regulation. Little information is available concerning the role of accessory proteins required to mediate receptor function. We have used immuno-, steroid-, and site-specific DNA-affinity chromatography to identify proteins associated with ER in extracts from MCF-7 and CHO-ER cells. A similar pattern of eluted proteins was observed in SDS-PAGE analysis from each approach, with the exception of two proteins, p45 and p48, preferentially retained by DNA-affinity chromatography. In addition to ER, a 70-kDa band, identified by Western blot as an hsp70 family member, was observed. A 55-kDa protein, detected by all three approaches, was identified by microsequencing as protein disulfide isomerase. p45 and p48, preferentially retained by DNA-affinity chromatography, await identification. $\sp{35}$S-methionine labeling of cells in presence of agonists or antagonists was used to examine the effect of these agents on ER associated proteins following immuno- or DNA-affinity chromatography. Although neither agonists nor antagonists had an effect on the ER-associated proteins isolated by DNA affinity-chromatography, immunoadsorption revealed a reduction in the level of ER-associated hsp70 following treatment with estradiol or hydroxy-tamoxifen suggesting that ER-ERE interaction requires the presence of hsp70. Mobility shift analysis was used to examine the interaction of ER complexes with DNA in the presence of accessory proteins, revealing that maximal DNA binding of ER to the ERE occurred in the presence of all four associated proteins isolated by DNA-affinity chromatography. Removal of p45/p48 and/or hsp70 led to a substantial reduction in ER-ERE binding. Addition of hsp70 or p45/p48 could restore full ER-ERE binding activity. The nitrocellulose filter binding assay demonstrated that the rate of ER-ERE association and dissociation of the ER complexes were not significantly different. Scatchard analysis clearly demonstrated that the major difference between the ER complexes was the absolute binding capacities. Finally, circular permutation analysis indicated that some of these proteins also contribute to the bend angle induced by ER-ERE interaction.