Activation of target signal transducers utilizing chimeric receptors with signaling-molecule binding motifs.
Cellular fates such as proliferation, differentiation, and death are controlled by a variety of cytokine receptors, which are crucial in initiating downstream signaling cascades. To initiate signaling, the cytokine receptor cytoplasmic domain recruits specific signaling molecules with a range of tyrosine-containing motifs. Thus, we postulate that it is possible to regulate signal transduction artificially by locating the tyrosine motif of interest into the intracellular domain of specific receptors. Construction of such artificial receptors was based on an anti-fluorescein ScFv/c-Mpl chimera (S-Mpl). We selected several known tyrosine motifs from native cytokine receptors that strongly bind to their target molecule, and located them downstream of the Janus kinase (JAK) binding domain of S-Mpl, which would be necessary for phosphorylation of the receptor. Next, we used retroviral transduction to express chimeric receptors in a murine IL-3-dependent pro-B cell line, Ba/F3, which was stimulated with BSA-fluorescein. The results indicated that each chimeric receptor preferentially activated the corresponding signaling molecule. We also examined whether the position of the tyrosine motif in the receptor could influence the activation levels of the signal transducer, and found that the chimeric receptors could activate the corresponding signaling molecule even when the tyrosine motif was distant from the JAK binding domain.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Koichiro
K
Saka
Masahiro
M
Kawahara
Hiroshi
H
Ueda
Teruyuki
T
Nagamune
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology; D001665:Binding Sites; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D051379:Mice; D018121:Receptors, Cytokine / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D016207:Cytokines; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins