Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.
Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.
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DOI：
10.1083/jcb.97.2.508
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发表时间：
1983-08
期刊：
影响因子：
--
通讯作者：
Trowbridge IS
中科院分区：
文献类型：
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作者：
Hopkins CR;Trowbridge IS
用~(125)I-转铁蛋白和~(125)I标记的转铁蛋白受体(ATR)单抗，在A431细胞中研究了转铁蛋白和转铁蛋白受体的结合及随后的细胞内处理。使用125I-转铁蛋白，我们已经证明，在37℃下线性摄取长达60分钟，大多数结合的配体被内化，然后迅速回到孵育介质中而不被降解。在37℃下，循环最快的转铁蛋白在细胞内的半衰期为7.5分钟。125I-ATR表现出相同的摄取动力学，但在37℃内化后，它被部分降解。在22℃及以下，125I-ATR在细胞内的降解被选择性地抑制，结果是它在细胞内积累。用常规薄片和全细胞悬架的电子显微镜观察ATR-金胶体络合物标记的转铁蛋白受体的摄取和加工过程。利用脉冲追逐方案，详细描述了内化ATR金受体复合体所遵循的细胞内途径。在22℃的5分钟内，内化的复合体从细胞表面的涂层凹坑转移到外围细胞质内的狭窄、分叉的脑池系统中。到15分钟时，它们达到了更大、更扩张的成分，在薄片中，这些成分看起来像是含有小(30-50 nm直径)小泡的不规则轮廓。30min时，金复合体主要位于胞浆周围典型的球形多泡小体内；40-60min时，金复合体在核旁区域形成一个囊状和多泡小体系统。在22摄氏度时，没有其他隔室被标记，但如果它们加热到37摄氏度，金络合物就会转移到溶酶体样元素上。用Triton X在22℃下追逐30分钟后提取ATR-金复合体，并用琼脂糖转铁蛋白对其进行纯化，结果表明内化的复合体在细胞内加工过程中仍与转铁蛋白受体结合。
The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half- life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR- gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome- like elements. Extracting ATR-gold complexes with Triton X after a 30- min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.