Characterization of the Intracellular Localization, Processing, and Secretion of Two Glial Cell Line-Derived Neurotrophic Factor Splice Isoforms
Characterization of the Intracellular Localization, Processing, and Secretion of Two Glial Cell Line-Derived Neurotrophic Factor Splice Isoforms
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DOI：
10.1523/jneurosci.5888-09.2010
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发表时间：
2010-08-25
影响因子：
5.3
通讯作者：
Saarma, Mart
中科院分区：
文献类型：
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作者：
Lonka-Nevalaita, Liina;Lume, Maria;Saarma, Mart
内分泌和神经细胞具有高度发达的分泌机制，并且分泌可以是组成性的或受生理刺激调节。在组成性途径中，细胞内运输囊泡在到达靶标后立即发生融合反应。在调节分泌中，囊泡在靶膜附近积聚，直到触发融合，通常是通过游离Ca 2+的局部升高。在本研究中，我们的特点的加工和分泌机制的胶质细胞源性神经营养因子（GDNF）。虽然GDNF的功能已被广泛研究，但对GDNF及其具有不同前区的前体形式（α）pro-GDNF和（β）pro-GDNF的基本细胞生物学知之甚少。我们的研究结果表明，（α）pro-GDNF和（β）pro-GDNF分泌。我们证明KCl诱导的去极化以Ca 2+依赖的方式增加（β）pro-GDNF和相应成熟GDNF向细胞培养基的分泌，但不增加（α）pro-GDNF和相应成熟GDNF向细胞培养基的分泌。与此同时，细胞的免疫荧光分析表明，（α）pro-GDNF/GDNF主要定位于高尔基复合体，而（β）pro-GDNF/GDNF主要定位于分泌颗粒蛋白II和Rab 3A阳性囊泡的调节分泌途径。此外，我们发现切割pro-BDNF和pro-NGF的基质金属蛋白酶和纤溶酶不负责pro-GDNF的切割，而弗林蛋白酶内切蛋白酶、PACE 4和前蛋白转化酶PC 5A、PC 5 B和PC 7可以将pro-GDNF切割成成熟的GDNF。因此，GDNF的加工和分泌机制不同于BDNF和NGF。
Endocrine and neuronal cells have highly developed secretion mechanisms, and the secretion can be either constitutive or regulated by physiological stimuli. In the constitutive pathway, intracellular transport vesicles undergo immediate fusion reactions after arrival at the target. In regulated secretion, vesicles accumulate near the target membrane until triggered to fuse, typically by a local rise in free Ca2+. In the present study, we characterize the processing and secretion mechanisms of the glial cell line-derived neurotrophic factor (GDNF). Although the function of GDNF has been extensively studied, very little is known about the basic cell biology of GDNF and its precursor forms (alpha)pro-GDNF and (beta)pro-GDNF that have different pro-regions. Our results show that both (alpha)pro-GDNF and (beta)pro-GDNF are secreted. We demonstrate that KCl-induced depolarization increases the secretion of (beta)pro-GDNF and corresponding mature GDNF, but not (alpha)pro-GDNF and corresponding mature GDNF, to the cell medium in a Ca2+ -dependent manner. In parallel with this, immunofluorescence analysis of cells show that (alpha)pro-GDNF/GDNF is localized mostly in the Golgi complex, whereas (beta)pro-GDNF/GDNF is localized primarily in secretogranin II and Rab3A-positive vesicles of the regulated secretory pathway. In addition, we find that matrix metalloproteinases and plasmin that cleave pro-BDNF and pro-NGF are not responsible for the cleavage of pro-GDNF, whereas furin endoproteinase, PACE4, and proprotein convertases PC5A, PC5B, and PC7 can cleave pro-GDNF into mature GDNF. Thus, the processing and secretion mechanisms of GDNF are different from those of BDNF and NGF.