The coactivator binding site of the estrogen receptor (ER) must be generated by hormone binding to the ER, folding the C-terminal helix 12 over the ligand pocket and forming the interaction surface for the LXXLL motif of coactivator proteins, for estrogen hormone signals to properly activate responsive genes in target tissues. Antihormones block the action of estrogens by binding the ER LBD and misfolding helix 12 into a position that occludes coactivator binding. Similarly, the presence and abundance of specific coregulator gene products are also proposed to explain tissue-specific ligand differences, e.g. tamoxifen antagonism in breast and partial agonism in endometrium. Differential activity of estrogenic ligands, including selective estrogen receptor modulators (SEAMs), probably derive from varied cofactor interaction. Single amino acid changes in ER, some with significant phenotypic changes, currently remain under-evaluated. We have developed an assay in yeast to report normal, hormone-induced folding of the ER AF-2 that is very sensitive to mutationa' disruption. Using this assay, ER LBDs have been recovered from tissues and tumors that indeed show altered protein folding. In preliminary data from tumors so far analyzed, 3 have demonstrated changes in ER affecting the p160 coactivator binding site and tamoxifen folded structure. This proposal hypothesizes that significant alterations in estrogen stimulated transcription result from these naturally occurring changes in the estrogen receptor, its co-regulator proteins, or the interaction between them. This has important implications with respect to estrogen and antiestrogen functions in women, particularly those with cancers in estrogen responsive tissues. The project aims are: 1) to determine mechanistic properties of the recovered estrogen receptors, using transcription assays in yeast and mammalian cells; 2) to test cofactor binding to recovered ER alleles with purified GST fusion proteins and in vitro transcription and translation experiments; 3) to isolate via RT-PCR and assay transcription with ER interacting! p160 coactivator clones SRC1, GRIP1, and NCOR cDNAs recovered from tissue samples, and 4) to isolate and identify further naturally occurring ER mutations in vivo, using assays sensitive to ER protein conformation of the AF-2 coactivator binding domain.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DK059516-02
Application #
6517899
Study Section
Reproductive Endocrinology Study Section (REN)
Program Officer
Margolis, Ronald N
Project Start
2001-06-01
Project End
2004-05-31
Budget Start
2002-06-01
Budget End
2004-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$149,542
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Nichols, Mark (2015) New directions for drug-resistant breast cancer: the CDK4/6 inhibitors. Future Med Chem 7:1473-81
Kim, Sang Woo; Fishilevich, Elane; Arango-Argoty, Gustavo et al. (2015) Genome-wide transcript profiling reveals novel breast cancer-associated intronic sense RNAs. PLoS One 10:e0120296
Nichols, Mark; Cheng, Peng; Liu, Yue et al. (2010) Breast cancer-derived M543V mutation in helix 12 of estrogen receptor alpha inverts response to estrogen and SERMs. Breast Cancer Res Treat 120:761-8
Cheng, Peng; Kanterewicz, Beatriz; Hershberger, Pamela A et al. (2004) Inhibition of estrogen receptor alpha-mediated transcription by antiestrogenic 1,1-dichloro-2,2,3-triarylcyclopropanes. Mol Pharmacol 66:970-7