Jin L W, Ninomiya H, Roch J M, Schubert D, Masliah E, Otero D A, Saitoh T
Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla 92093-0624.
J Neurosci. 1994 Sep;14(9):5461-70. doi: 10.1523/JNEUROSCI.14-09-05461.1994.
Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.
淀粉样β蛋白/A4蛋白前体（APP）可被大多数培养细胞分泌到培养基中，并可作为一种自分泌因子发挥作用。为了研究分泌型APP（sAPP）对神经元的生物学功能，我们使用了一种克隆的中枢神经系统神经元系B103，该细胞系不合成可检测水平的APP。当接种在无血清限定培养基中时，用APP构建体转染的B103细胞比亲代B103细胞更快地长出神经突。APP - 695高表达细胞的条件培养基或细菌产生的sAPP - 695（命名为KB75）可促进B103细胞的神经突生长。一系列具有APP - 695的Ala - 319至Met - 335之间序列的肽也刺激了B103细胞的神经突生长。在这段序列中的五个氨基酸序列RERMS（APP 328 - 332）是最短的活性肽，尽管其促神经突生长活性所需的浓度高于KB75。使用对应于APP - 695的Ala - 319至Met - 335的125I标记的APP 17肽作为配体的结合试验表明存在特异性和可饱和的细胞表面结合位点。预测的KD值为20±5 nM，Bmax值为80±8 fmol/10(6)细胞。该结合可被KB75取代。具有反向序列的17肽既不诱导神经突生长也不竞争结合。一种缺乏活性17肽序列的细菌产生的sAPP片段（命名为KB75δ）不与125I标记的17肽竞争结合或刺激神经突延伸。序列为RMSQ（APP 330 - 333）的肽部分与活性序列RERMS重叠，在较高浓度下可阻断KB75和17肽的促神经突生长作用。还发现APP 17肽可诱导肌醇多磷酸的积累，这表明APP 17肽的作用涉及肌醇磷脂信号转导系统的激活。这些数据表明，sAPP通过细胞表面结合诱导神经突延伸，并且包含RERMS序列（APP 328 -
