Benlimame N, Le P U, Nabi I R
Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada H3C 3J7.
Mol Biol Cell. 1998 Jul;9(7):1773-86. doi: 10.1091/mbc.9.7.1773.
Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4 degreesC exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1-2 h at 37 degreesC, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.
自分泌运动因子受体（AMF-R）是一种细胞表面受体，也定位于内质网的一个光滑亚结构域，即AMF-R小管。通过包埋后免疫电子显微镜观察，在NIH-3T3成纤维细胞和HeLa细胞中，AMF-R集中在光滑的质膜囊泡或小窝中。通过共聚焦显微镜观察，在4℃下向活细胞中添加抗AMF-R抗体标记的细胞表面AMF-R与小窝蛋白部分共定位，证实细胞表面AMF-R定位于小窝。用抗AMF-R抗体或生物素化的AMF（bAMF）标记细胞表面AMF-R显示出广泛的共定位，在37℃下脉冲1-2小时后，bAMF积聚在密集标记的核周结构以及与AMF-R小管共定位的较淡的管状结构中。在随后的2至4小时追踪后，bAMF主要定位于AMF-R小管。细胞质酸化，阻断网格蛋白介导的内吞作用，导致内化的bAMF基本上排他性地分布到AMF-R小管中。通过共聚焦显微镜观察，内化的bAMF标记的管状结构与AMF-R小管完全共定位。在存在10倍过量未标记AMF的情况下内化的bAMF标记核周点状结构，因此这些是液相内吞作用的产物，但不标记AMF-R小管，表明bAMF靶向AMF-R小管是通过受体介导的途径发生的。通过电子显微镜观察，内化10分钟的bAMF位于细胞表面小窝中，30分钟后存在于光滑和粗糙的内质网小管中。因此，AMF-R通过受体介导的非网格蛋白依赖性途径内化到光滑内质网中。AMF-R在小窝中的稳态定位表明这些细胞表面内陷参与了AMF-R的内吞作用。
