The estrogen receptor (ER) is a ligand dependent transcriptional activator that belongs to the steroid/nuclear receptor superfamily. To probe the structure and function of the ER ligand binding domain (LBD), we developed a genetic screen in yeast Saccharomyces cerevisiae using a library of reverse ERs screened with a low affinity estrogen agonist, 2-methoxyestrone. Mutants isolated from this screen demonstrated altered ligand binding and/or transactivation properties. One of these mutants, L536P, showed high levels of constitutive activity in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. This suggests that substitution of a proline at position 536 in the wild type ER (HEGO) induces a reversible conformational change in the region of AF-2 that partially mimics activation of the receptor by hormone binding. / Activation of transcription by the ER requires the recruitment of different classes of coactivators. L536P interacted with coactivators in the absence of hormone and this constitutive interaction can be abolished by antiestrogens. We conclude that constitutive activity of L536P-HEGO is manifested to in part from constitutive coactivator binding. We also demonstrated that different classes of coactivators do not recognize the ER LBD in the same manner and can compete for binding to the ER LBD suggesting that different classes of coactivators recognize distinct but overlapping binding sites. Interestingly, coexpression of RIP140 blocked enhanced transactivation by HEGO observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. / Using a yeast two-hybrid system with the ER LBD as bait, we isolated a novel estrogen receptor cofactor (ERC) that interacts with the ER LBD in a hormone dependent manner. The primary structure of ERC consists of 4 ankyrin repeat domains, 2 LXXLL motifs and an ATP/GTP binding domain. ERC is highly expressed in ovary, testes, and spleen, with moderate levels in heart, brain, and placenta. ERC repressed ER transactivation in several cell lines in the presence of estradiol suggesting that ERC may function as a novel estrogen dependent repressor of the ER. Immunohistochemistry and confocal microscopy localized ERC to the cytoplasm with partial nuclear staining. Recently, synphilin-1, a novel protein that has been implicated in the pathogenesis of Parkinson's disease was isolated and is identical to ERC. A role for ERC in intracellular signaling through a membrane bound ER in the brain is currently being investigated.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.38483 |
| Date | January 2001 |
| Creators | Eng, Frank Chung Sing, 1972- |
| Contributors | White, John (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001944871, proquestno: NQ85705, Theses scanned by UMI/ProQuest. |
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