Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells.
Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells.
复制标题
受体结合的凝血酶不会通过小鼠胚胎细胞中的包被凹坑内化。
DOI：
10.1002/jcb.240200305
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发表时间：
1982
影响因子：
4
通讯作者：
Carney,DH
中科院分区：
文献类型：
--
作者：
Bergmann,JS;Carney,DH
使用电子显微镜（EM）免疫细胞学技术检查小鼠胚胎（ME）细胞上凝血酶受体的定位。 ME 细胞在凝血酶结合之前用甲醛固定，并使用亲和纯化的抗凝血酶兔抗体和胶体金标记的抗兔 IgG 在细胞表面观察凝血酶。在与凝血酶一起孵育的细胞表面上发现了成簇的胶体金颗粒。在薄片中观察到每个簇大约有 7 个颗粒，簇直径范围为 70 至 200 nm。在没有凝血酶的情况下孵育的细胞上没有观察到这些簇。使用和不使用凝血酶孵育的细胞上存在的颗粒总数表明，胶体金标记对凝血酶的特异性约为 98%。大约 1,200 个胶体金颗粒中只有 4 个与涂层凹坑相关。因此，凝血酶受体簇似乎不与包被膜区域相关。为了确定受体结合的凝血酶是否通过受体介导的内吞作用内化，将ME细胞与125I-凝血酶一起孵育，并使用EM放射自显影和与细胞相关的125I-凝血酶的胰蛋白酶敏感性进行检查。在两种类型的实验中，凝血酶与细胞在 4°C 下孵育，温度升至 37°C，初始孵育温度为 37°C，受体导向的特异性内化的进行速度与非特异性内化的速度大致相同。这些研究表明，与 ME 细胞上的受体结合的凝血酶不会通过受体介导的内吞作用快速内化。
The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity‐purified antithrombin rabbit antibody and colloidal gold labeled anti‐rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor‐bound thrombin was internalized by receptor‐mediated endocytosis, ME cells were incubated with125I‐thrombin and examined using EM autoradiography and the trypsin sensitivity of125I‐thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4°C and the temperature increased to 37°C and where initial incubation was at 37°C, the receptor‐directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor‐mediated endocytosis.