Signal sequences for targeting of gene therapy products to subcellular compartments: the role of CRM1 in nucleocytoplasmic shuttling of the protein switch.
OBJECTIVE
The purpose of this study was to understand the mechanism of nuclear export of the protein switch, used for controlled intracellular delivery of gene products, by studying the involvement of classical export receptor CRM1.


METHODS
Transient transfections of protein switch constructs, isolated nuclear export and import signals were carried out. Effect of leptomycin B (inhibitor of export receptor) and geldanamycin (inhibitor of Hsp90) on localization of these constructs was studied using fluorescence microscopy. Putative nuclear export signals in the glucocorticoid and progesterone receptor ligand binding domains were identified and studied.


RESULTS
It was observed that treatment with leptomycin B caused nuclear accumulation of the protein switch constructs. However, geldanamycin did not have any pronounced effect on the localization. The isolated nuclear export signal from glucocorticoid receptor localized mostly in the cytoplasm, while its mutated version was present everywhere.


CONCLUSIONS
The localization controlled protein switch constructs are exported out of the nucleus by the classical CRM1 receptors. The ligand binding domain of these protein switch constructs plays an important role in maintaining these constructs in the cytoplasm in the absence of ligand, as well the re-export back to the cytoplasm from the nucleus after ligand washout.
Kakar
Mudit
M
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 421 Wakara Way #318, Salt Lake City, UT 84108, USA.
Cadwallader
Amy B
AB
Davis
James R
JR
Lim
Carol S
CS
D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D015316:Genetic Therapy; D028884:Karyopherins / Q000502:physiology*; D051379:Mice; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D021382:Protein Sorting Signals; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D000097606:Exportin 1 Protein
D028884:Karyopherins; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein