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.
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.
Purpose
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.
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.