Lai W H, Cameron P H, Wada I, Doherty J J, Kay D G, Posner B I, Bergeron J J
Department of Anatomy, McGill University, Montreal, Quebec, Canada.
J Cell Biol. 1989 Dec;109(6 Pt 1):2741-9. doi: 10.1083/jcb.109.6.2741.
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.
在肝实质中，评估了表皮生长因子（EGF）受体的内化、再循环和下调情况，作为注射EGF剂量增加的函数。通过直接结合、Scatchard分析和蛋白质印迹法评估分离的质膜和内体组分中受体含量的变化，研究了体内配体占据对内化动力学和程度的影响。对于所有注射剂量的EGF，受体从质膜丢失并以时间和剂量依赖性方式在内体中积累。然而，当注射的EGF剂量相当于小于或等于50%的表面受体占据率（即小于或等于1微克/100克体重）时，受体水平在120分钟时恢复到初始值。这种恢复对放线菌酮有抗性，因此不代表新合成的受体。这种恢复也不是由于低亲和力受体内池的补充，因为通过Scatchard分析或蛋白质印迹法无法检测到这样一个池。因此，受体的恢复是由于先前内化的受体的再循环。当注射的EGF剂量大于50%受体占据率时，通过评估肝匀浆总颗粒组分中的受体含量，观察到净受体丢失，即下调。在较高的饱和剂量注射EGF（5和10微克/100克体重）时，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。由于所有注射剂量的EGF从循环和肝实质中清除配体的动力学相似，那么配体介导的表面受体含量调节和下调不是配体与受体长时间相互作用的结果。相反，这些现象一定是细胞表面和/或内体中与受体相互作用的EGF绝对浓度的结果。
