title: Functional mutations of estrogen receptor protein: assay for detection.
abstract: Antiestrogens block the function of estrogen receptor (ER) by binding and misfolding the AF-2 transcriptional activation region in the ligand-binding domain, inhibiting or altering its association with coactivator proteins. We describe a novel assay uniquely configured to identify aberrations in this function that may lead to antiestrogen resistance. The identification of mutations of ER that affect its function is important to current breast cancer therapies. Standard methods to detect these mutations are cumbersome and the number of described mutations is limited, reflecting this difficulty. Conventional ER analysis in the clinic demonstrates the presence of antigenic determinants of the receptor protein or estrogenic ligand binding without reflection on the critical ability of the liganded receptor to interact with transcription cofactors. Here, we describe the use of estrogenic regulation of a site-specific recombinase activity, measuring deletion of a color marker gene via FLP-ER fusion proteins, to detect functional changes in ER protein folding that affects the site where cofactors interact. The assay provides a method to readily detect single amino acid changes in ER, some with biologically important consequences. Without such a functional assay as described, phenotypic changes are likely to remain undetected and under-evaluated. It is probable that some human tumors have antihormone resistance resulting from ER mutations that either block antihormone binding or transmit antihormone binding as a positive transcriptional signal via cofactor interaction. An assay to evaluate functional ER will lead to better predictive tests of treatment modalities.
authors: Department of Pharmacology, University of Pittsburgh Cancer Institute, University of Pittsburgh, PA 15213, USA. mnichols+@pitt.edu
authors: Mark
authors: M
authors: Nichols
authors: Kenneth S
authors: KS
authors: McCarty
mesh_terms: D001681:Biological Assay; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry; D047628:Estrogen Receptor alpha; D005260:Female; D006801:Humans; D008024:Ligands; D009154:Mutation; D009376:Neoplasms, Hormone-Dependent / Q000188:drug therapy / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012334:RNA, Neoplasm / Q000235:genetics; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D020845:Selective Estrogen Receptor Modulators / Q000627:therapeutic use; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004273:DNA, Neoplasm; D047628:Estrogen Receptor alpha; D008024:Ligands; D012334:RNA, Neoplasm; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D020845:Selective Estrogen Receptor Modulators; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase