Journal of Biochemistry Advance Access originally published online on January 20, 2009
Journal of Biochemistry 2009 145(5):575-584; doi:10.1093/jb/mvp013
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The Influence of Domain Structures on the Signal Transduction of Chimeric Receptors Derived from the Erythropoietin Receptor
1Department of Chemistry and Biotechnology, School of Engineering; 2Center for NanoBio Integration (CNBI); and 3Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, 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 8, 2008; Accepted January 10, 2009
 |
Abstract |
|---|
Although cytokine receptors regulate many cellular functions, contribution of receptor's domains and their conformation to signal transduction remains unclear. In this study, we designed a series of chimeric erythropoietin receptor (EpoR) variants encoding a haemagglutinin epitope-tagged anti-fluorescein single-chain Fv and different combinations of extracellular D1/D2 domain(s) of EpoR as the extracellular domain to allow the receptor to be activated by multiple ligands. Furthermore, one to four Ala residues 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 Ba/F3 cells, cell-surface expression levels of chimeric receptors without D2 domain were markedly lowered, suggesting a role of D2 domain for stabilizing the receptor. Furthermore, the ligand-dependent cell proliferation was strongly affected by extracellular domain structures and the number of inserted Ala residues. Moreover, the conformational change of chimeric receptors was induced by various ligands to detect the phosphorylation of JAK2, STAT5 and ERK2, whose activations are characteristics of EpoR signalling. Consequently, the phosphorylation pattern of these signal transducers was significantly influenced by ligands and receptor variants. These results indicate that signal transduction of EpoR is strongly affected by conformation of both extracellular and intracellular domains.
Key Words: chimeric receptor, conformational change, erythropoietin receptor, ligand, signal transduction
Abbreviations: BSA-FL, fluorescein-conjugated BSA; EGFP, enhanced green fluorescent protein; EMP, Epo-mimetic peptides; EpoR, erythropoietin receptor; ERK2, the extracellular signal-regulated kinase 2; HA, haemagglutinin; IL-3, interleukin-3; JAK2, Janus kinase 2; STAT5, signal transducer and activator of transcription 5; TM, transmembrane