Kolkova K, Pedersen N, Berezin V, Bock E
Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Denmark.
J Neurochem. 2000 Sep;75(3):1274-82. doi: 10.1046/j.1471-4159.2000.751274.x.
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.
神经细胞黏附分子（NCAM）胞外结构域的功能已得到广泛研究，而NCAM跨膜形式的胞质结构域的作用则较少阐明。我们通过评估与NCAM阴性或NCAM阳性成纤维细胞共培养的PC12-E2细胞中NCAM依赖性神经突生长，研究了140-kDa NCAM亚型（cytNCAM-140）和180-kDa NCAM亚型（cytNCAM-180）的胞质结构域在NCAM诱导的神经突延伸中的重要性。用编码cytNCAM-140、cytNCAM-180、丝裂原活化蛋白（MAP）激酶/细胞外信号调节激酶激酶（MEK2）的组成型活性形式以及绿色荧光蛋白（EGFP）增强变体的表达质粒瞬时转染PC12-E2细胞。EGFP表达用于鉴定转染细胞。我们发现cytNCAM-180的表达对NCAM刺激的神经突生成没有影响，而cytNCAM-140的表达强烈抑制这一过程。然而，如果MEK2与cytNCAM-140同时表达，神经突生长得以恢复，表明cytNCAM-140通过Ras-MAP激酶途径参与信号传导。随后用编码cytNCAM-140一系列片段和各种全长cytNCAM-140突变体的构建体瞬时转染PC12-E2细胞，并且鉴定出苏氨酸-谷氨酸-缬氨酸-赖氨酸-苏氨酸（839-843）残基在NCAM刺激的神经突生成中是必需的。用丙氨酸联合取代谷氨酸（840）和赖氨酸（842）消除了构建体的作用，赋予这两个残基关键作用。
