Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cells
Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cells
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DOI：
10.1113/jphysiol.2007.129171
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发表时间：
2007-06-01
影响因子：
5.5
通讯作者：
Seetharam, Bellur
中科院分区：
文献类型：
--
作者：
Bose, Santanu;Kalra, Seema;Seetharam, Bellur
具有代谢活性并处于高度分化和增殖状态的细胞需要钴胺素（Cbl：维生素B-12），并且它们通过转钴胺素受体（TC-R）从与转钴胺素（TC）结合的循环中获得钴胺素。本研究采用脉冲追踪标记、结构域特异性生物素化和细胞分级分离技术研究了极化的人肠上皮Caco-2细胞中TC-R表达的质膜动力学。内源性合成的TC-R在递送至基底外侧质膜（BLM）后以8小时的半衰期（T-1/2）翻转。BLM递送的T-1/2为15 min，递送至BLM的TC-R被内吞，随后被亮抑酶肽敏感蛋白酶降解。然而，从BLM内吞的约15%的TC-R被转胞吞（T-1/2，45分钟）到顶端膜（BBM），在那里它经历内吞作用并被降解。布雷菲德菌素A和衣霉素抑制TC-R向BLM和BBM的递送，但不抑制渥曼青霉素或亮抑酶肽。秋水仙素抑制TC-R向BBM的递送，但不抑制BLM。在稳态下，顶端TC-R与巨蛋白相关，并且这两种蛋白质都富集在还含有Rab 5和转铁蛋白受体的细胞内隔室中。这些结果表明，在快速递送至Caco-2细胞的两个质膜结构域后，TC-R经历组成性内吞作用和由亮抑酶肽敏感性蛋白酶降解。TC-R在其从BLM转胞吞期间在顶端BBM与megalin复合物中表达。
Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B-12) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T-1/2) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T-1/2 of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T-1/2, 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM.