{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/86771"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/86771","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Reengineering Estrogen Receptor Hormone Specificity Through Coordinated Changes at Key Contact Sites Between Estrogen Receptor and Estradiol","abstract":"The specificity of hormone action arises principally from the specific interaction that a hormonal ligand has with its cognate receptor. This interaction is defined by steric and electronic complementarity at certain sites at which portions of the ligand contact specific residues in its receptor. In the nuclear hormone receptors, mutational mapping studies, and more recently, X-ray structures, provide information on these key, specificity-defining contact sites. Based on this information, we have made a series of coordinated changes in the A-ring of estrogens (E) and in the A-ring binding sub-pocket of the estrogen receptor alpha (ER). This is the site at which the 3-phenolic hydroxyl of estrogens is hydrogen bonded to the side chains of Glu353 and Arg394. These changes are designed to create a favorable interaction when both E and ER are changed, but to disfavor interaction when only E is changed or only ER is changed. We have evaluated several altered ligand (E') and receptor (ER') pairs in quantitative ligand binding and reporter gene assays. The most successful coordinated changes have involved the change of Glu353 to a serine or alanine residue coupled with a change of the 3-OH of estradiol to a carboxymethyl or an ethoxy group, respectively. In these cases, the rematched interaction E'-ER' is more favorable than the mismatched ones. These new ligand receptor pairs represent the creation of new hormone specificities. To examine the utility of these ligand receptor rematches for the regulation of transgenes, we have made chimeric receptors that combine the DNA binding domain of Gal4 with the ligand binding domain of wild type, E353S or E353A mutant ER and the activation domain of VP16. These chimeric proteins were tested in mammalian cell transfection assays, and although these chimeric receptors required higher levels of ligand than the full length receptor, the specificity shifts mirrored those we had seen previously. These chimeric receptor-ligand pairs do show promise for use in artificial regulation of gene activity, but some additional work would be required to optimize the chimeric receptor-altered ligand pairs for this function.","abstract_html":"The specificity of hormone action arises principally from the specific interaction that a hormonal ligand has with its cognate receptor. This interaction is defined by steric and electronic complementarity at certain sites at which portions of the ligand contact specific residues in its receptor. In the nuclear hormone receptors, mutational mapping studies, and more recently, X-ray structures, provide information on these key, specificity-defining contact sites. Based on this information, we have made a series of coordinated changes in the A-ring of estrogens (E) and in the A-ring binding sub-pocket of the estrogen receptor alpha (ER). This is the site at which the 3-phenolic hydroxyl of estrogens is hydrogen bonded to the side chains of Glu353 and Arg394. These changes are designed to create a favorable interaction when both E and ER are changed, but to disfavor interaction when only E is changed or only ER is changed. We have evaluated several altered ligand (E') and receptor (ER') pairs in quantitative ligand binding and reporter gene assays. The most successful coordinated changes have involved the change of Glu353 to a serine or alanine residue coupled with a change of the 3-OH of estradiol to a carboxymethyl or an ethoxy group, respectively. In these cases, the rematched interaction E'-ER' is more favorable than the mismatched ones. These new ligand receptor pairs represent the creation of new hormone specificities. To examine the utility of these ligand receptor rematches for the regulation of transgenes, we have made chimeric receptors that combine the DNA binding domain of Gal4 with the ligand binding domain of wild type, E353S or E353A mutant ER and the activation domain of VP16. These chimeric proteins were tested in mammalian cell transfection assays, and although these chimeric receptors required higher levels of ligand than the full length receptor, the specificity shifts mirrored those we had seen previously. These chimeric receptor-ligand pairs do show promise for use in artificial regulation of gene activity, but some additional work would be required to optimize the chimeric receptor-altered ligand pairs for this function.","abstract_has_math":false,"creators":["Thomas, James Adrian"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Microbiology","degree_department":null,"school":null,"contributors":["Katzenellenbogen, Benita S."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-28T15:17:58Z","date_published":"2015-09-28T15:17:58Z","updated_at":"2026-07-22T22:26:27Z","subjects":["Biology, Molecular"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI9996690"],"render_values":[{"text":"(MiAaPQ)AAI9996690","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/86771","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Katzenellenbogen, Benita S."]},{"key":"dc:creator","label":"Author","values":["Thomas, James Adrian"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-28T15:17:58Z","10000-01-01","2001"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology, Molecular"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/86771","(MiAaPQ)AAI9996690"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The specificity of hormone action arises principally from the specific interaction that a hormonal ligand has with its cognate receptor. This interaction is defined by steric and electronic complementarity at certain sites at which portions of the ligand contact specific residues in its receptor. In the nuclear hormone receptors, mutational mapping studies, and more recently, X-ray structures, provide information on these key, specificity-defining contact sites. Based on this information, we have made a series of coordinated changes in the A-ring of estrogens (E) and in the A-ring binding sub-pocket of the estrogen receptor alpha (ER). This is the site at which the 3-phenolic hydroxyl of estrogens is hydrogen bonded to the side chains of Glu353 and Arg394. These changes are designed to create a favorable interaction when both E and ER are changed, but to disfavor interaction when only E is changed or only ER is changed. We have evaluated several altered ligand (E') and receptor (ER') pairs in quantitative ligand binding and reporter gene assays. The most successful coordinated changes have involved the change of Glu353 to a serine or alanine residue coupled with a change of the 3-OH of estradiol to a carboxymethyl or an ethoxy group, respectively. In these cases, the rematched interaction E'-ER' is more favorable than the mismatched ones. These new ligand receptor pairs represent the creation of new hormone specificities. To examine the utility of these ligand receptor rematches for the regulation of transgenes, we have made chimeric receptors that combine the DNA binding domain of Gal4 with the ligand binding domain of wild type, E353S or E353A mutant ER and the activation domain of VP16. These chimeric proteins were tested in mammalian cell transfection assays, and although these chimeric receptors required higher levels of ligand than the full length receptor, the specificity shifts mirrored those we had seen previously. These chimeric receptor-ligand pairs do show promise for use in artificial regulation of gene activity, but some additional work would be required to optimize the chimeric receptor-altered ligand pairs for this function.","Made available in DSpace on 2015-09-28T15:17:58Z (GMT). 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This interaction is defined by steric and electronic complementarity at certain sites at which portions of the ligand contact specific residues in its receptor. In the nuclear hormone receptors, mutational mapping studies, and more recently, X-ray structures, provide information on these key, specificity-defining contact sites. Based on this information, we have made a series of coordinated changes in the A-ring of estrogens (E) and in the A-ring binding sub-pocket of the estrogen receptor alpha (ER). This is the site at which the 3-phenolic hydroxyl of estrogens is hydrogen bonded to the side chains of Glu353 and Arg394. These changes are designed to create a favorable interaction when both E and ER are changed, but to disfavor interaction when only E is changed or only ER is changed. We have evaluated several altered ligand (E') and receptor (ER') pairs in quantitative ligand binding and reporter gene assays. The most successful coordinated changes have involved the change of Glu353 to a serine or alanine residue coupled with a change of the 3-OH of estradiol to a carboxymethyl or an ethoxy group, respectively. In these cases, the rematched interaction E'-ER' is more favorable than the mismatched ones. These new ligand receptor pairs represent the creation of new hormone specificities. To examine the utility of these ligand receptor rematches for the regulation of transgenes, we have made chimeric receptors that combine the DNA binding domain of Gal4 with the ligand binding domain of wild type, E353S or E353A mutant ER and the activation domain of VP16. These chimeric proteins were tested in mammalian cell transfection assays, and although these chimeric receptors required higher levels of ligand than the full length receptor, the specificity shifts mirrored those we had seen previously. These chimeric receptor-ligand pairs do show promise for use in artificial regulation of gene activity, but some additional work would be required to optimize the chimeric receptor-altered ligand pairs for this function.","Made available in DSpace on 2015-09-28T15:17:58Z (GMT). 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