Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.
Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.
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DOI：
10.1083/jcb.109.6.2741
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发表时间：
1989-12
影响因子：
7.8
通讯作者：
Bergeron, J J
中科院分区：
文献类型：
--
作者：
Lai, W H;Cameron, P H;Wada, I;Doherty, J J 2nd;Kay, D G;Posner, B I;Bergeron, J J
EGF受体内化，再循环，和下调作为一个函数的肝实质中注射EGF剂量增加进行了评价。研究了体内配体占有率对内化动力学和程度的影响，并通过直接结合、Scatchard分析和Western印迹评价了分离的质膜和内体组分的受体含量的变化。对于所有剂量的注射EGF，受体从质膜中丢失，并以时间和剂量依赖性的方式积累在内体中。然而，在注射的EGF剂量等于小于或等于50%的表面受体占有率（即，小于或等于1微克/100克体重），受体水平在120分钟后恢复到初始值。这种回归对放线菌酮具有抗性，因此不代表新合成的受体。也不是由于低亲和力受体的细胞内池的补充而导致的返回，因为这样的池不能通过Scatchard分析或Western印迹检测。因此，受体返回是由于先前内化的受体的再循环。在注射的EGF剂量大于50%受体占有率时，净受体损失-即，下调-通过评估肝匀浆的总颗粒级分的受体含量来观察。在较高的饱和剂量注射EGF（5和10微克/100克体重），大部分表面受体含量在15分钟时丢失，并在至少另外105分钟内保持较低水平。由于所有剂量的EGF注射的配体从循环和肝实质中清除的动力学相似，那么配体介导的表面受体含量的调节和下调不是配体与受体延长的时间相互作用的结果。相反，该现象必须是EGF与细胞表面和/或内体中的受体相互作用的绝对浓度的结果。
EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.