Endocytic trafficking of megalin/RAP complexes: Dissociation of the complexes in late endosomes
Endocytic trafficking of megalin/RAP complexes: Dissociation of the complexes in late endosomes
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DOI：
10.1091/mbc.8.3.517
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发表时间：
1997-03-01
影响因子：
3.3
通讯作者：
Farquhar, MG
中科院分区：
文献类型：
--
作者：
Czekay, RP;Orlando, RA;Farquhar, MG
Megalin (gp330) 是低密度脂蛋白受体基因家族的成员。与该家族的其他成员一样，它是一种结合许多特定配体的内吞受体。巨蛋白还结合受体相关蛋白 (RAP)，作为胞吐交通伴侣并抑制配体与受体的结合。为了研究巨蛋白/RAP复合物的命运，我们将RAP谷胱甘肽-S-转移酶融合蛋白（RAP-GST）与L2卵黄囊癌细胞表面的巨蛋白结合，并通过免疫荧光和免疫金标记及其在Percoll梯度上的分布来跟踪复合物的运输。我们发现巨蛋白/RAP-GST 复合物通过网格蛋白包被的凹坑内化，被递送到早期内体，在那里它们在 18 摄氏度的温度阻断期间积累并与转铁蛋白和转铁蛋白受体共定位。从温度块释放后，复合物行进至晚期内体，在那里它们与 rab7 共定位，并且可以与抗 RAP-GST 抗体共沉淀。复合物的解离发生在晚期内体中，并且很可能是由该区室的低 pH（类似于 5.5）触发的。然后 RAP 被快速递送至溶酶体并被降解，而巨蛋白则被循环至细胞表面。当配体脂蛋白脂肪酶与巨蛋白结合时，发现受体通过早期内体循环。我们得出的结论是，与受体/配体复合物相比，巨蛋白/RAP复合物通过晚期内体进行运输，这对于低密度脂蛋白受体基因家族的成员来说是一个新事物。
Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (similar to 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.