Identification of an amino acid sequence motif in the cytoplasmic domain of the NCAM-140 kDa isoform essential for its neuritogenic activity
Identification of an amino acid sequence motif in the cytoplasmic domain of the NCAM-140 kDa isoform essential for its neuritogenic activity
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DOI：
10.1046/j.1471-4159.2000.751274.x
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发表时间：
2000-09-01
影响因子：
4.7
通讯作者：
Bock, E
中科院分区：
文献类型：
--
作者：
Kolkova, K;Pedersen, N;Bock, E
神经细胞粘附分子（NCAM）胞外结构域的功能已经被广泛研究，而NCAM跨膜形式的细胞质结构域的作用却很少被阐明。我们研究了140 kda NCAM异构体（cytNCAM-140）和180 kda NCAM异构体（cytNCAM-180）的细胞质结构域在NCAM诱导的神经突延伸中的重要性，通过估计与NCAM阴性或NCAM阳性成纤维细胞共培养的PC12-E2细胞的NCAM依赖性神经突生长。用编码有丝分裂原活化蛋白（MAP）激酶/细胞外信号调节激酶（MEK2）的组成活性形式cytNCAM-140、cytNCAM-180的表达质粒和绿色荧光蛋白（EGFP）的增强变体瞬时转染PC12-E2细胞，EGFP表达用于鉴定转染细胞。我们发现，表达cytNCAM-180对ncam刺激的神经细胞发生没有影响，而表达cytNCAM-140则强烈抑制这一过程。然而，如果MEK2与cytNCAM-140同时表达，则神经突的生长被挽救，这表明cytNCAM-140通过Ras-MAP激酶途径参与信号传导。随后，将编码一系列cytNCAM-140片段和各种全长cytNCAM-140突变体的构建物短暂转染PC12-E2细胞，并鉴定残基Thr-Glu-Val-Lys-Thr（839-843）在ncam刺激的神经细胞发生中是必需的。Glu（840）和Lys（842）与Ala的联合取代消除了该结构的作用，赋予这两个残基一个关键的作用。
The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated, We investigated the importance of the cytoplasmic domain of the 140-kDa NCAM isoform (cytNCAM-140) and of the 180-kDa NCAM isoform (cytNCAM-180) in NCAM-induced neurite extension by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding cytNCAM-140, cytNCAM-180, the constitutively active form of the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP), EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM-180 had no effect on NCAM-stimulated neuritogenesis, whereas expression of cytNCAM-140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM-140, neurite outgrowth was rescued, indicating that cytNCAM-140 is involved in signaling via the Ras-MAP kinase pathway. PC12-E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM-140 and various full-length cytNCAM-140 mutants, and the residues Thr-Glu-Val-Lys-Thr (839-843) were identified as essential in NCAM-stimulated neuritogenesis. The combined substitution of Glu(840) and Lys(842) with Ala abrogated the effect of the construct, assigning a critical role to these two residues.