Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability
Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability
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DOI：
10.1042/bj20070919
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发表时间：
2008-03-01
影响因子：
4.1
通讯作者：
Seetharam, Bellur
中科院分区：
文献类型：
--
作者：
Ahuja, Rajiv;Yamman, Raghunatha;Seetharam, Bellur
Cubilin是一种456 kDa的多功能受体，缺乏跨膜和胞质结构域，在极化上皮细胞的顶端BBM（刷状缘膜）中表达。Cubilin与两种跨膜蛋白AMN和megalin相互作用，AMN是一种45-50 kDa的无糖蛋白基因蛋白产物，megalin是一种600 kDa的巨内吞受体。在体外，cubilin的三个片段，113个残基的N-末端和CUB结构域12-17和22-27，表现出Ca 2+依赖性结合巨蛋白。使用大鼠肾脏BBM的洗涤剂提取物进行的免疫沉淀和免疫印迹研究表明，cubilin与megalin和AMN相互作用。配体（内在因子-钴胺素）亲和层析显示，在肾BBN 1中，功能性cubilin作为与AMN和megalin两者的复合物存在。在从产生megalin抗体的兔的肾脏获得的总膜和BBM中，Cubilin和AMN水平分别降低80%和55-60%。巨蛋白或AMN基因沉默的负鼠肾细胞中cubilin的免疫组织化学分析和周转研究显示cubilin染色显著减少（85-90%），半衰期减少2倍。总之，这些结果表明，三个不同的区域cubilin结合megalin和它的相互作用与megalin和AMN是必不可少的细胞内稳定性。
Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)affinity chromatography showed that in renal BBNls, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.