Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain.
Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100 nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins.
Kakar
Mudit
M
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84108, USA.
Davis
James R
JR
Kern
Steve E
SE
Lim
Carol S
CS
D021581:Active Transport, Cell Nucleus; D000230:Adenocarcinoma / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003907:Dexamethasone; D018274:Electroporation; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D051379:Mice; D015735:Mifepristone; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D049790:Nuclear Export Signals / Q000502:physiology*; D010957:Plasmids; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D015694:Gene Products, rev; D008024:Ligands; D049790:Nuclear Export Signals; D011506:Proteins; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D015735:Mifepristone; D003907:Dexamethasone; D020929:Mitogen-Activated Protein Kinase Kinases