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Journal of Biochemistry 2007 141(4):563-571; doi:10.1093/jb/mvm056
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© 2007 The Japanese Biochemical Society.

Mimicry of Erythropoietin and Interleukin-6 Signalling by an Antibody/Cytokine Receptor Chimera in Murine Myeloid 32D Cells

Masahiro Kawahara1,*, Hiroshi Ueda1, Kouhei Tsumoto2, Izumi Kumagai3 and Teruyuki Nagamune1

1Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656; 2Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 301, Bioscience Building, 5-1-5, Kashiwa-no-ha, Kashiwa 277-8562; and 3Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, 980-8579, Japan

*To whom correspondence should be addressed. Tel: +81-3-5841-7356, Fax: +81-3-5841-8657, E-mail: kawahara{at}bio.t.u-tokyo.ac.jp

Received December 11, 2006; Accepted February 5, 2007


  Abstract

We have previously designed antibody-cytokine receptor chimeras that could respond to a cognate antigen. While these chimeric receptors were functional, it has not been investigated exactly how they mimic signal transduction through corresponding wild-type receptors. In this study, we compared the growth properties and the phosphorylation status of intracellular signal transducers between the erythropoietin receptor (EpoR)- or gp130-based chimeric receptors and wild-type EpoR or EpoR-gp130 chimera, respectively. Expression plasmids, encoding VH or VL region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domain of EpoR and transmembrane/cytoplasmic domains of either EpoR or gp130, were constructed, and pairs of chimeric receptor combinations (VH-EpoR and VL-EpoR, VH-gp130 and VL-gp130, VH-EpoR and VL-gp130, VH-gp130 and VL-EpoR) were expressed in an IL-3-dependent myeloid cell line, 32D. Growth assay revealed that the transfectants all grew in a HEL-dependent manner. As for phosphorylation of Stat3, Stat5, ERK and Akt, the chimeric receptors showed similar activation pattern of signalling molecules with wild-type receptors, although the chimeric receptors showed ligand-independency and a little lower maximal phosphorylation than the corresponding wild-type receptors. The results demonstrate that antibody-receptor chimeras could substantially mimic wild-type receptors.

Key Words: antibody, chimeric receptor, erythropoietin receptor, gp130, haematopoietic cell

Abbreviations: Epo, erythropoietin; EpoR, erythropoietin receptor; HEL, hen egg lysozyme; Stat, signal transducers and activators of transcriptions; VH, antibody variable domain of heavy chain; VL, antibody variable domain of light chain


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