Construction of a fluorescein-responsive chimeric receptor with strict ligand dependency.
Since many cell functions are regulated by members of the cytokine receptor superfamily, the artificial mimicry of the cytokine receptor system would be attractive for cellular engineering. We previously showed that an antibody/cytokine receptor chimera can transduce a growth signal in response to non-natural ligands, such as fluorescein-conjugated BSA. However, considerable background of cell proliferation was observed without antigen. Therefore, we redesigned chimeric receptor constructs with different combinations of domains containing anti-fluorescein single chain Fv (ScFv), extracellular D1/D2 as well as transmembrane domains of erythropoietin receptor (EpoR), and the intracellular domain of glycoprotein 130 (gp130), to obtain strictly fluorescein-dependent chimeric receptors. When interleukin-3-dependent Ba/F3 cells were transduced with retroviral vectors encoding individual chimeric receptors, the chimeras either with both D1 and D2 domains or without any EpoR extracellular domain attained a strict ligand-dependent ON/OFF regulation.
Liu
Wenhai
W
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Kawahara
Masahiro
M
Ueda
Hiroshi
H
Nagamune
Teruyuki
T
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D000941:Antigens / Q000235:genetics / Q000378:metabolism; D048708:Cell Growth Processes / Q000235:genetics; D002460:Cell Line; D050822:Cytokine Receptor gp130 / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D019793:Fluorescein / Q000378:metabolism; D005822:Genetic Vectors; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D051379:Mice; D015202:Protein Engineering / Q000379:methods; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D013379:Substrate Specificity
D000906:Antibodies; D000941:Antigens; D007135:Immunoglobulin Variable Region; D008024:Ligands; D017467:Receptors, Erythropoietin; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130; D019793:Fluorescein