Bidirectional on/off switch for controlled targeting of proteins to subcellular compartments.
A regulatable fusion protein was constructed for controlling the localization of plasmid products. A ligand-inducible nuclear localization signal, nuclear export signal (NES) and a truncated form of the ligand binding domain of the progesterone receptor were attached to the desired protein. Enhanced green fluorescent protein (EGFP) was used as a model protein and its trafficking between the nucleus and cytoplasm was studied using fluorescence microscopy in response to the ligand, mifepristone. It was found that the protein trafficking into the nucleus was dose dependent with ligand concentration. Increasing the ligand dose from 1 to 100 nM enhanced import and reduced the rate of export of the fusion protein from the nucleus to the cytoplasm. This study demonstrates the feasibility of using an export signal and a ligand-inducible nuclear import signal as a bi-directional on/off switch with potential use for controlled targeting of therapeutic proteins to subcellular compartments.
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 421 Wakara Way #318, Salt Lake City, UT 84108, USA.
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D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006727:Hormone Antagonists / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015735:Mifepristone / Q000494:pharmacology; D019913:Nuclear Localization Signals / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*
D006727:Hormone Antagonists; D008024:Ligands; D019913:Nuclear Localization Signals; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D015735:Mifepristone