A signal sequence trap based on a constitutively active cytokine receptor.
Targeting of secreted and cell-surface proteins to the cell membrane is mediated by a short hydrophobic stretch of amino acids, termed the signal sequence. We have developed a method that detects signal sequences in cDNA fragments based on their ability to redirect a constitutively active mutant of a cytokine receptor to the cell surface, thereby permitting interleukin-3 (IL-3)-independent growth of Ba/F3 cells. Retrovirus-mediated expression of the fusions in IL-3-dependent cells was followed by selection of clones for growth in the absence of IL-3. Infection of cells with 5x10(6) viral particles in a pilot experiment led to the isolation of 150 known and 48 novel cDNA clones, and all the known cDNA clones were found to encode secreted and cell-surface proteins. In addition, we isolated type II membrane proteins, which have not been detected by existing signal sequence trap strategies.
Kojima
T
T
Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Japan.
Kitamura
T
T
D000818:Animals; D002478:Cells, Cultured; D018076:DNA, Complementary; D015723:Gene Library; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000235:genetics* / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013926:Thrombopoietin; D014162:Transfection
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D018121:Receptors, Cytokine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013926:Thrombopoietin