Description
The Colony Stimulating Factor 1 receptor (CSF-1 receptor) is a protein tyrosine kinase that controls the survival, proliferation, and differentiation of mononuclear phagocytes and regulates cells of the female reproductive tract. Receptor deficient mice show defects in bone formation; development of the mammary gland and lack of macrophages. CSF-1 receptor overexpression has been linked to cancers of the breast, ovaries, and endometrium. We have shown previously that the CSF-1 receptor is subject to regulated intramembrane proteolysis or RIP. RIP involves cleavage at two separate sites in the receptor and results in ectodomain shedding, followed by the release of the intracellular domain (ICD) into the cytosol. We propose that the ICD travels to the nucleus where it is likely to be involved in regulation of proinflammatory gene transcription. To investigate this we have to increase the stability of the ICD by adding an amino-terminal V5-epitope tag. In addition, we have mutated a stretch of leucine and/or conserved lysine residues in the context of the epitopetagged ICD, to investigate their role in localization of the ICD. Normal and mutant proteins were expressed in 293 cells and analyzed for subcellular localization by subcellular fractionation followed by immunoblotting. The normal and mutant protein expressing cell lines were later analyzed by immunofluorescence. The V5-tagged ICD protein has been found in the cytoplasmic fraction and to a lesser extent in nucleus. Mutation of the leucine and conserved lysine residues resulted in an increase in the level of nuclear ICD whereas mutation of the conserved lysine residues resulted in a decrease in the levels of nuclear ICD and also the stability of the overall protein. We used confocal immunofluorescence to confirm the results obtained using subcellular fractionation. While most of the CICD is present in the cytoplasm, the ICD can also be detected in the nucleus. Mutation of the conserved lysines results in a decrease in the levels of the ICD seen in the nucleus and hence may be a part of the export signal. Mutation of a stretch of leucines and conserved lysines results in an increase in the levels of the ICD seen in the nucleus and hence may be a part of the import signal.
