THROMBOMODULIN LACKING THE CYTOPLASMIC DOMAIN EFFICIENTLY INTERNALIZES THROMBIN VIA NONCLATHRIN-COATED, PIT-MEDIATED ENDOCYTOSIS
THROMBOMODULIN LACKING THE CYTOPLASMIC DOMAIN EFFICIENTLY INTERNALIZES THROMBIN VIA NONCLATHRIN-COATED, PIT-MEDIATED ENDOCYTOSIS
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DOI：
10.1002/jcp.1041580211
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发表时间：
1994-02-01
影响因子：
5.6
通讯作者：
STEINERMOSONYI, M
中科院分区：
文献类型：
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作者：
CONWAY, EM;NOWAKOWSKI, B;STEINERMOSONYI, M
血栓调节蛋白(TM)是一种跨膜血管内皮细胞受体，是主要的生理相关天然抗凝剂系统的辅因子。我们最近开发了一个细胞模型来研究TM细胞表面表达的一种调节机制，并直观地证明了受体主要通过非涂层凹坑进行内化(Conway等人，1992，J.Cell)。体育，151：604-612)。我们将这些研究扩展到通过在COS细胞(COS.Cyto.Del细胞)中删除该区域并表达该分子的截短版本来检查TM的细胞质结构域的作用。电子显微镜显示金标记的抗TM抗体或凝血酶以时间和温度依赖的方式内化，类似于野生型转基因细胞(COS.TM-CR)。内吞作用的特征是金颗粒最初在表面聚集，然后聚集到未包被的凹坑中，早期内吞形成，最后进入多囊体和溶酶体。网状蛋白包被的小凹和囊泡中没有明显的金颗粒。比较了I-125标记配体在COS.Cyto.Del细胞和COS.TM-CR细胞中的结合和内化动力学，两者无显著差异。这些研究提供了超微结构和定量数据，表明当受体缺乏细胞质结构域时，TM通过非分子筛蛋白包裹的凹坑有效地进行内吞作用。这一发现表明，分子中可能存在替代区域，负责调节内化所需的那些信号。
Thrombomodulin (TM) is a transmembrane vascular endothelial cell receptor that is a cofactor in a major physiologically relevant natural anticoagulant system. We recently developed a cell model to examine one mechanism of regulation of TM cell surface expression and visually demonstrated that the receptor undergoes internalization predominantly via noncoated pits (Conway et al., 1992, J. Cell. Phys., 151:604-612). We have extended these studies to examine the role of the cytoplasmic domain of TM by deleting this region and expressing the truncated version of the molecule in COS cells (COS.Cyto.Del cells). Electron microscopy demonstrated internalization of gold-labeled anti-TM antibody or thrombin in a time- and temperature-dependent manner, similar to that seen with the wild-type transfected cells (COS.TM-CR). Endocytosis was characterized by initial surface clustering of gold particles, followed by aggregation into noncoated pits, early endosome formation, and, finally, entry into multivesicular bodies and lysosomes. There was a notable absence of gold particles in clathrin-coated pits and vesicles. The kinetics of binding and internalization of I-125-labeled ligand in COS.Cyto.Del cells was compared with that of COS.TM-CR cells and was not significantly different. These studies provide ultrastructural and quantitative data to indicate that TM efficiently undergoes endocytosis via nonclathrin-coated pits when the receptor is lacking the cytoplasmic domain. This finding suggests that there may be alternative regions of the molecule that mediate those signals necessary for internalization.