Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex
Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex
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DOI：
10.1681/asn.2004110925
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发表时间：
2005-08-01
影响因子：
13.6
通讯作者：
Maurice, M
中科院分区：
文献类型：
--
作者：
Coudroy, G;Gburek, J;Maurice, M
Cubilin是多种配体的外周顶膜受体，这些配体在几种吸收性上皮细胞中被摄取。最近，无糖蛋白（AMN）被鉴定为与cubilin形成功能性受体复合物。通过在转染的极化MDCK细胞中表达AMN和几个cubilin片段，包括一个功能性的“迷你”版本的cubilin，复合物的处理，分选和膜锚定到顶端膜进行了研究。结果表明，截短突变体，包括cubilin的N-末端结构域，没有出现在质膜上，而是保留在内质网或部分分泌到培养基中。与AMN的共表达导致cubilin构建体（其中包括EGF结构域）有效转运至顶端细胞表面，并防止释放至培养基中。AMN与cubilin共沉淀，并与cubilin共定位在顶端细胞表面。在没有AMN的情况下，观察到一组广泛的不重叠的cubilin片段，没有N-末端区域的顶端分选。当衣霉素抑制糖基化时，顶端分选的偏好消失。总之，它表明，这两个单位有助于处理cubilin-AMN复合物的顶端膜：AMN与EGF结构域的cubilin相互作用，并负责膜附着和出口的复合物从内质网，而细胞外cubilin分子是负责顶端排序的复合物在碳水化合物依赖的方式。
Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with cubilin. By expression in transfected polarized MDCK cells of AMN and several cubilin fragments, including a functional "mini" version of cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with cubilin and co-localized with cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the cubilin-AMN complex to the apical membrane: AMN interacts with the EGF domains of cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.