Katsuma Susumu, Daimon Takaaki, Mita Kazuei, Shimada Toru
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
J Virol. 2006 Jun;80(11):5474-81. doi: 10.1128/JVI.00248-06.
The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an approximately 110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis.
家蚕核型多角体病毒（BmNPV）编码一个与哺乳动物成纤维细胞生长因子（FGF）家族同源的基因。我们报道了果蝇无呼吸基因（btl）在家蚕和草地贪夜蛾中的直系同源基因（分别为Bmbtl和Sfbtl）的克隆，该基因编码无分支/FGF的受体，并表明这些基因编码杆状病毒编码的FGF（vFGF）的受体。序列分析表明，BmBtl由856个氨基酸残基组成，可能编码一个97.3 kDa的多肽，与FGF受体家族具有结构特征和序列相似性。逆转录PCR实验表明，Bmbtl在家蚕幼虫的气管和中肠中大量表达，在血细胞和家蚕培养细胞系BmN中表达适中。我们构建了稳定表达His标签的BmBtl的Sf-9细胞。蛋白质免疫印迹分析显示，BmBtl是一种约110 kDa的蛋白质。免疫沉淀实验表明，BmNPV vFGF在Sf-9细胞中显著磷酸化BmBtl。此外，我们发现BmBtl的过表达增强了对BmNPV vFGF的迁移活性。此外，我们通过转染表达双链RNA的质粒构建了Sfbtl被敲低的Sf-9细胞。在这些细胞中，vFGF触发的细胞运动性显著降低。这些结果强烈表明，Btl直系同源物BmBtl和SfBtl是vFGF的受体，介导vFGF诱导的宿主细胞趋化作用。
