Saffell J L, Doherty P, Tiveron M C, Morris R J, Walsh F S
Department of Experimental Pathology, UMDS, Guy's Hospital, London, United Kingdom.
Mol Cell Neurosci. 1995 Dec;6(6):521-31. doi: 10.1006/mcne.1995.0004.
The neural cell adhesion molecule (NCAM) promotes axonal growth via a homophilic binding mechanism by acting both as a neuronal receptor and a substratum ligand. We have previously shown that the GPI-linked 120-kDa isoform of NCAM, which lacks a cytoplasmic domain, is effective at promoting neurite outgrowth as a cellular ligand. To test its ability to function as a neuronal receptor, we have transfected PC12 cells with a cDNA encoding human GPI-linked NCAM and tested clones displaying stable cell surface expression of this isoform for their ability to respond to NCAM in a cellular substratum. Although they continued to express endogenous transmembrane rat isoforms of NCAM (140 and 180 kDa), PC12 cells expressing the GPI-linked NCAM lost their ability to extend neurites in response to substratum associated NCAM. However, their outgrowth response to N-cadherin and other activators of axonal growth was undiminished. Removal of GPI-linked NCAM from the surface of these clones using phosphatidylinositol-specific phospholipase C (PIPLC) fully restored their responsiveness to NCAM, indicating that the inhibition was a direct consequence of cell surface expression of this "dominant negative" isoform of NCAM. We have previously shown that expression of transfected 140- and 180-kDa isoforms of human NCAM in PC12 cells does not result in a loss of the neurite outgrowth response to NCAM. However, we show that deletion of the cytoplasmic domain of the 140-kDa isoform has the same effect as expression of GPI-linked NCAM. We conclude that the cytoplasmic domain of NCAM is required for an appropriate neurite outgrowth response.
神经细胞黏附分子（NCAM）通过同源结合机制促进轴突生长，它既作为神经元受体，又作为底物配体发挥作用。我们之前已经表明，缺乏胞质结构域的GPI连接的120 kDa NCAM同工型作为细胞配体，在促进神经突生长方面是有效的。为了测试其作为神经元受体的功能能力，我们用编码人GPI连接的NCAM的cDNA转染PC12细胞，并测试显示该同工型稳定细胞表面表达的克隆对细胞底物中NCAM的反应能力。尽管它们继续表达内源性跨膜大鼠NCAM同工型（140 kDa和180 kDa），但表达GPI连接的NCAM的PC12细胞失去了对底物相关NCAM作出反应而延伸神经突的能力。然而，它们对N-钙黏蛋白和其他轴突生长激活剂的生长反应并未减弱。使用磷脂酰肌醇特异性磷脂酶C（PIPLC）从这些克隆的表面去除GPI连接的NCAM，完全恢复了它们对NCAM的反应性，表明这种抑制是NCAM这种“显性负性”同工型细胞表面表达的直接结果。我们之前已经表明，在PC12细胞中表达转染的人NCAM 140 kDa和180 kDa同工型不会导致对NCAM的神经突生长反应丧失。然而，我们表明，140 kDa同工型的胞质结构域缺失具有与表达GPI连接的NCAM相同的效果。我们得出结论，NCAM的胞质结构域是适当的神经突生长反应所必需的。
