Abstract
Although cytokine receptors regulate many cellular functions, the contribution of receptor’s domains and their conformation to signal transduction is still not clear. In this study, we designed a series of chimeric erythropoietin receptor (EpoR) variants encoding a hemagglutinin (HA) epitope-tagged anti-fluorescein single-chain Fv (ScFv) and different combinations of extracellular D1/D2 domain(s) of EpoR as the extracellular domain. Furthermore, 1–4 Ala residue(s) were inserted at the intracellular juxtamembrane region of each chimeric receptor to modulate the conformation of the intracellular domain. When the chimeric receptors were expressed in mouse IL-3-dependent Ba/F3 cells, the cell-surface expression of the chimeric receptors without the D2 domain was markedly lowered, suggesting a role of the D2 domain for stabilizing the receptor. A cell growth assay would be one of the methods to characterize the contribution to signal transduction of extracellular and intracellular domains in the chimeric receptors. It was performed to examine whether the cells expressing the chimeric receptors could grow in response to fluorescein-conjugated BSA (BSA-FL), which could bind to the ScFv domain of the chimeric receptors. To confirm ligand-specificity of the BSA-FL-responsive chimeric receptors, we used a different set of ligands for the chimeric receptors including fluorescein-conjugated OVA (OVA-FL) as a specific ligand, whereas unconjugated ligands (BSA, OVA and free FL) or specific ligands in the presence of excess free FL (BSA-FL + free FL, OVA-FL + free FL) were also tested to investigate the specificity of the chimeric receptors. These results clearly indicate that signal transduction of the characteristic of chimeric receptors based on EpoR is strongly affected by the conformation of both extracellular and intracellular domains.
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Liu, W., Kawahara, M., Ueda, H., Nagamune, T. (2010). The Characteristic of Chimeric Receptors Based on Erythropoietin Receptor. In: Kamihira, M., Katakura, Y., Ito, A. (eds) Animal Cell Technology: Basic & Applied Aspects. Animal Cell Technology: Basic & Applied Aspects, vol 16. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-3892-0_43
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DOI: https://doi.org/10.1007/978-90-481-3892-0_43
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