Construction of a Fluorescein-Responsive Chimeric Receptor with Strict Ligand Dependency and Analysis of the Role of Erythropoietin Receptor Domains in Signal Transduction

摘要

In our previous study, we designed anti-fluorescein (FL) antibody/ receptor chimeras in response to FL-conjugated BSA (BSA-FL). However, considerable background cell proliferation was observed without antigen. Therefore, we tried to redesign chimericreceptor constructs with different combinations of the domains containing anti-FL single chain Fv (ScFv), extracellular D1/D2 domains, transmembrane/intracellular domains of erythropoietin receptor (EpoR) or glycoprotein 130 (gpl30), for construction ofa strictly fluorescein-dependent chimeric receptor. We also tried to analyze the role of erythropoietin receptor domains in signal transduction. We designed a series of chimeric receptors. Firstly an anti-FL ScFv was fused to full-length EpoR. Next we tried to delete extracellular Dl or D2 domain of EpoR, to mutate transmembrane (TM) domain, to exchange the intracellular domain (ID) into that of gpl30, and/or to insert several Ala residues into juxtamembrane domain to modulate the conformation of intracellular domain. Chimeric receptors were expressed in IL-3-dependent Ba/F3 cells to compare their growth characteristics. We found that BSA-FL acted as an inverse agonist at some chimeric receptors, whereas it also acted as an agonist at other chimeric receptors. We also found the effect on cell growth induced by the TM domain mutation and the insertion of Ala residues between TM and intracellular domains of chimeric receptors. Notably, one chimeric receptor, ScFv-EpoRTM-gpl30ID, transduced a strict BSA-FL dependent growth signal without any background cell growth. Therefore, this chimera might be promising as a basis for cell growth-based screening of high-affinity ScFvs derived from a randomized antibody library, where simple culture in antigen-containing medium results in growing cells with a high-affinity antibody gene, leading to antibody selection.