title: Utilizing the estrogen receptor ligand-binding domain for controlled protein translocation to the insoluble fraction.
abstract: OBJECTIVE
  The estrogen receptor forms insoluble aggregates in the insoluble cytoskeletal subcellular fraction when bound to the antagonist fulvestrant. The ligand-binding domain was isolated and fused to signal sequences to target subcellular compartments. Sequestering a pro-apoptotic peptide tested the utility of a protein targeted to the insoluble fraction.
  
  
  METHODS
  The ligand-binding domain of the estrogen receptor was isolated and fused with signal sequences, either a nuclear localization signal or nuclear export signal. The subcellular localization when bound to drug fulvestrant was examined, specifically its interaction with cytokeratins 8 and 18. The ability to target a therapeutic peptide to the insoluble fraction was tested by fusing a therapeutic coiled-coil from Bcr-Abl in K562 cells.
  
  
  RESULTS
  The estrogen receptor ligand-binding domain responds to fulvestrant by translocating to the insoluble fraction. Adding a signal sequence significantly limited the translocation to either the nucleus or cytoplasm. The cytokeratin 8/18 status of the cell did not alter this response. The therapeutic coiled-coil fused to ERLBD was inactivated upon ligand induction.
  
  
  CONCLUSIONS
  Isolating the ligand-binding domain of the estrogen receptor creates a ligand-controllable protein capable of translocation to the insoluble fraction. This can be used to sequester an active peptide to alter its function.
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry College of Pharmacy, University of Utah, Salt Lake City, Utah, USA. james.r.davis@utah.edu
authors.forename: James R
authors.initials: JR
authors.lastname: Davis
authors.forename: Mohanad
authors.initials: M
authors.lastname: Mossalam
authors.forename: Carol S
authors.initials: CS
authors.lastname: Lim
mesh_terms: D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology*; D000077267:Fulvestrant; D006801:Humans; D007633:Keratins / Q000378:metabolism; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility
chemical_list: D020847:Estrogen Receptor Modulators; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D011960:Receptors, Estrogen; D000077267:Fulvestrant; D004958:Estradiol; D007633:Keratins