Analysis of Signal Transduction Machineries Involving Two Types of NELL Proteins in the Differentiation Process of Neural Crest-derived Cells

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NELL proteins (NELL1,NELL2), found in 1999 by us, possess a unique molecular organization. From N- to C-terminal, a signal peptide, thrombospondin 1-N terminal domain, EGF-like motifs, and vWF-derived motifs are found in both proteins. These Two 140-kDa proteins show about 55% similarity in the amino acid sequence level, are synthesized as a secreted protein, and form a about 400-kDa homotrimeric proteins. By our previous studies, since NELL1 mRNA was significantly expressed at the suture of craniocynostosis patients' skull, the NELL1 was finally demonstrated as a novel bone morphogenic protein(BMP). Unlike the conventional BMPs (e.x., BMP2,BMP4,BMP7), the NELL1 protein showed exclusively the induction of bone formation in osteoblast cells and not affected the determination of dorsal-ventral axes in the embryos.In this study, we established the continuous synthesis of NELL proteins using insect cell system (continuous, stable, and serum-free) and succeeded in the preparation of large amount of pure NELL proteins. These NELL proteins facilitated us to identify the novel molecules as candidates of NELL-specific receptors by using expression cloning techniques and analyze the 3D-structure by using an X-ray crystallographic technique. Furthermore, we succeeded in the establishing NELL1-overexpressing transgenic mice and NELL2-deflcieat mice for analyzing the physiological roles of NELL proteins.