| Summary: | 碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 101 === FMS-like tyrosine kinase receptor 3 (FLT3) belongs to the type-III RTK (Receptor Tyrosine Kinase) family to which c-KIT and the PDGFR (Platelet Derived Growth Factor Receptor) also belong. They share the same structure: 1) Extracellular domain with five immunoglobulin-like domains, 2) Transmembrane domain, 3) Juxtamembrane domain, 4) Cytoplasmic domain with tyrosine-kinase domain with a kinase insert loop. FLT3 ligand (FLT3L) is biologically active both as a transmembrane form and as a soluble form. Soluble FLT3L can be generated by proteolytic cleavage of the transmembrane ligands and/or alternative splicing. The crystal structure of FLT3L was published in 2000. The binding of FLT3L with its cognate receptor (FLT3) will induce the ligand-mediated receptor dimerization which will activate intracellular tyrosine kinase of receptors undergoing auto-phosphorylation and propagate signal-transduction cascade. FLT3 and its cytokine ligand (FLT3L) play a critical role for hematopoiesis and the immune system.
Although the physiologic role of the FLT3 ligand-receptor interaction has been characterized, the FLT3 signaling complex has remained unclear at the molecular and structural levels. Until 2011, Verstraete et al. reported the low-resolution crystal structures of FLT3L in complex with FLT3-D1-D4 and FLT3-ECD, which resolution is 4.3A and 7.8A, respectively. Further, the results are not consistent with previous homology models and the structural features of RTKIII complex by the following two findings. First, ligand-receptor interactions of FLT3 complex are very different from its homologous counterparts. For the FLT3 complex, the FLT3 ligand only interacts with the D3 (the third Ig-like domain) of FLT3. For c-KIT/SCF complex and CSFR-1/CSF-1 complex, the dimer ligand is embraced by two receptor molecules through D2 and D3 domains. Second, no homotypic interaction between two receptors has been observed in the FLT3 complex. In the c-KIT/SCF 2:2 complex, the binding with dimer of ligand induces the conformational change of receptor through the interactions between D4-D4 and D5-D5 interfaces. Therefore, we constructed Fc fusion protein in order to improve the ligand binding affinity and aid for crystallization.
In this study, recombinant proteins of FLT3 ligand, FLT3-D2-D5 with/without Fc region and FLT3-ECD with/without Fc region were expressed by Drosophila Schneider 2 (S2) cells for functional and structural studies of ligand-receptor interaction After purification procedures (Ni-affinity and gel filtration chromatography), we used mass spectrometry to characterize recombinant proteins and conducted ELISA-based binding assays. Our data revealed an increased binding affinity for FLT3 with Fc region. For the formation of FLT3L/FLT3-Fc complex, we co-culture by mixing the stable line of FLT3L and FLT3-Fc together in an attempt that both of them can form complex during secretion in culture medium. With this new expression approach, we obtain FLT3L/FLT3-Fc complex with one–step purification procedure using Protein A affinity gel. The preliminary shape of FLT3L/FLT3-Fc complex was determined by SAXS with the programs implant in ATSAS. To acquire the more detail information of FLT3 signaling complex, we had tried crystal growing for further study by X-ray crystallography.
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