TRKA CROSS-LINKING MIMICS NEURONAL RESPONSES TO NERVE GROWTH-FACTOR
TRKA CROSS-LINKING MIMICS NEURONAL RESPONSES TO NERVE GROWTH-FACTOR
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DOI：
10.1091/mbc.5.5.549
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发表时间：
1994-05-01
影响因子：
3.3
通讯作者：
REICHARDT, LF
中科院分区：
文献类型：
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作者：
CLARY, DO;WESKAMP, G;REICHARDT, LF
TrkA 是一种酪氨酸激酶受体，是神经生长因子 (NGF) 反应途径的重要组成部分。 NGF与受体的结合诱导受体自身磷酸化并激活细胞内信号通路，从而产生多种生物学效应。我们制备了针对使用杆状病毒表达系统产生的大鼠 trkA 整个胞外域的多克隆抗体。这些抗体在抗原印迹和免疫沉淀中特异性识别大鼠 trkA。 IgG 和 Fab 片段均可阻断 NGF 与 PC12 细胞系表达的 trkA 的结合。在使用抗 trkA 和抗低亲和力 NGF 受体 (LNGFR) 免疫球蛋白 (Ig) G 的 NGF 结合研究中，基本上所有 NGF 的结合都可以被抑制。结果表明，PC12 细胞上大于或等于 97% 的 NGF 结合位点由 trkA 和 LNGFR 占据。结合数据还表明，PC12 细胞上的所有低亲和力 NGF 结合位点都反映了与 LNGFR 的相互作用，而所有高亲和力位点均依赖于 trkA。一小部分高亲和力（或慢速）结合位点似乎同时需要 trkA 和 LNGFR。尽管单价抗 trkA Fab 片段抑制 NGF 的生物效应，例如诱导酪氨酸磷酸化以及交感神经元的存活和神经突生长，但 IgG 制剂作为抑制剂并不有效。相反，IgG 组分本身在刺激受体激活、细胞存活和神经突生长方面几乎与 NGF 一样有效。因此，抗体诱导的交联引起的 trkA 寡聚化似乎足以产生 NGF 的已知细胞效应。
TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that greater than or equal to 97% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent anti-trkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.