Meiri K F, Willard M, Johnson M I
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Neurosci. 1988 Jul;8(7):2571-81. doi: 10.1523/JNEUROSCI.08-07-02571.1988.
Sympathetic neurons regenerating in culture were studied in order to gain further insight into the intracellular distribution and phosphorylation of GAP-43, a protein that has been suggested to have a role in axonal outgrowth and neuronal plasticity (Willard et al., 1987). Superior cervical ganglion neurons from embryonic rats were highly reactive with a polyclonal antibody against the growth-associated protein GAP-43 soon after they were placed in culture on a laminin substrate. As these neurons extended neurites, the distribution of GAP-43 reactivity changed. The cell body became progressively less reactive, whereas the growth cone at the tip of the growing neurite reacted strongly. The pattern of immunofluorescence was punctate both in the growth cone and the adjacent neurite, but appeared more diffusely distributed in the cell body. The antibody reacted only with cells that had been subjected to treatment that permeabilized the plasma membrane. When antibody was supplied in the medium of growing neurons, it neither bound to the cells nor altered normal neurite initiation or elongation. Of the different types of cells in these cultures, the antibody reacted only with neurons; it did not react with Schwann cells or fibroblasts. The stimulation of protein kinase C in these cultures resulted in a 7-fold stimulation of the phosphorylation of a protein of similar electrophoretic mobility to GAP-43. These observations demonstrate that GAP-43 is neuron-specific, is present throughout the neuron but at higher levels in the growth cone, and is a major substrate of protein kinase C. The high concentration of GAP-43 in the growth cones may necessitate its increased synthesis in neurons with elongating axons. Its location and phosphorylation by kinase C suggest that it could perform a function in the growth cone that is modulated by extracellular signals, such as those used in pathfinding or in the control of axonal elongation.
为了更深入了解生长相关蛋白43（GAP - 43）的细胞内分布和磷酸化情况，对在培养中再生的交感神经元进行了研究。GAP - 43是一种被认为在轴突生长和神经元可塑性中起作用的蛋白质（威拉德等人，1987年）。将来自胚胎大鼠的颈上神经节神经元置于层粘连蛋白底物上培养后不久，它们就与抗生长相关蛋白GAP - 43的多克隆抗体发生强烈反应。随着这些神经元长出神经突，GAP - 43反应性的分布发生了变化。细胞体的反应性逐渐降低，而生长中的神经突顶端的生长锥反应强烈。免疫荧光模式在生长锥和相邻神经突中呈点状，但在细胞体中分布更弥散。该抗体仅与经过使质膜通透化处理的细胞发生反应。当在生长中的神经元培养基中提供抗体时，它既不与细胞结合，也不改变正常的神经突起始或伸长。在这些培养物的不同类型细胞中，该抗体仅与神经元发生反应；它不与施万细胞或成纤维细胞发生反应。在这些培养物中刺激蛋白激酶C导致一种电泳迁移率与GAP - 43相似的蛋白质的磷酸化增加了7倍。这些观察结果表明，GAP - 43是神经元特异性的，在整个神经元中都存在，但在生长锥中含量更高，并且是蛋白激酶C的主要底物。生长锥中高浓度的GAP - 43可能需要在轴突伸长的神经元中增加其合成。它的位置以及被激酶C磷酸化表明它可能在生长锥中执行一种由细胞外信号调节的功能，例如在路径寻找或轴突伸长控制中使用的信号。
