Xie Jiansong, Qian Limin, Wang Yanru, Rose Chadron M, Yang Tao, Nakamura Tamako, Hamm-Alvarez Sarah F, Mircheff Austin K
Department of Physiology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
J Cell Physiol. 2004 Apr;199(1):108-25. doi: 10.1002/jcp.10458.
The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [(125)I]-EGF exhibited saturable and non-saturable, temperature-dependent components, suggesting both receptor-mediated and fluid phase endocytosis. Accumulation of [(125)I] was time-dependent over a 120-min period, but the content of intact [(125)I]-EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37 degrees C [(125)I] reached an early endosome, basal-lateral recycling endosome, pre-lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans-Golgi network. Intact [(125)I]-EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [(125)I]-labeled degradation products appeared in the pre-lysosomes and lysosomes. Confocal microscopy imaging of FITC-EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non-acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane-expressed pool at all times. Cells loaded with [(125)I]-EGF released a mixture of intact EGF and [(125)I]-labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF-EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF-stimulation generates time-dependent signals that initially decrease, then increase, EGF-EGFR traffic to degradative compartments.
本研究的目的是描绘表皮生长因子（EGF）和表皮生长因子受体（EGFR）在兔泪腺原代培养腺泡上皮细胞中的运输模式。[（125）I]-EGF的摄取表现出可饱和和不可饱和的、温度依赖性成分，提示存在受体介导的内吞作用和液相内吞作用。[（125）I]的积累在120分钟内呈时间依赖性，但完整的[（125）I]-EGF含量在20分钟达到最大值后下降。通过山梨醇密度梯度离心和相分配进行的分析分级表明，在37℃下20分钟内，[（125）I]到达早期内体、基底外侧循环内体、前溶酶体和溶酶体。标记的小部分似乎也到达了高尔基体复合体和反式高尔基体网络。完整的[（125）I]-EGF最初在循环内体中积累；循环内体中的含量随后下降，到120分钟时，前溶酶体和溶酶体中出现了越来越多的[（125）I]标记的降解产物。FITC-EGF和LysoTrackerRed的共聚焦显微镜成像显示，FITC在20分钟时富集在非酸性区室的分散系统中，在120分钟时富集在酸性区室中。共聚焦免疫荧光显微镜和分析分级均表明，细胞内EGFR库在所有时间都比质膜表达的库大得多。加载了[（125）I]-EGF的细胞释放出完整EGF和[（125）I]标记的降解产物的混合物。这些观察结果表明，在泪腺腺泡细胞中，EGFR和EGF-EGFR复合物在质膜和内膜区室系统之间持续运输；EGF刺激产生时间依赖性信号，最初减少，然后增加，EGF-EGFR向降解区室的运输。
