Brändli A W, Adamson E D, Simons K
European Molecular Biology Laboratory, Cell Biology Programme, Heidelberg, Germany.
J Biol Chem. 1991 May 5;266(13):8560-6.
Madin-Darby canine kidney (MDCK) cells polarize and generate distinct apical and basolateral membrane domains when grown on permeable filter supports. Under these conditions, they transcytose fluid-phase markers. Recently, receptor-mediated transcytosis of epidermal growth factor (EGF) across MDCK cells has been reported (Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601). We examined the role of the EGF receptor in this process. Transcytosis of EGF occurred only in the basolateral-to-apical direction, was time-dependent, and inhibited by the addition of unlabeled EGF in a concentration-dependent manner. In contrast to previous work, we found that only about 5% of basolaterally bound EGF was transported to the apical chamber. The half-time of transport was 90 min. A mutant cell line of MDCK, MDCKII-RCAr, was used to study the expression of the EGF receptor. Cell surface glycoproteins of these mutant cells can be efficiently labeled with [3H]galactose by exogalactosylation. The EGF receptor was found to be expressed only on the basolateral surface. Addition of EGF to the basolateral medium resulted in rapid internalization and degradation of the receptor. Testing directly for transcytosis of basolateral glycoproteins, we detected several proteins transported across the cell. The EGF receptor, however, was not among this group of proteins. Taking these results together, we suggest the following model. Internalization of EGF on the basolateral surface is mediated by the EGF receptor. EGF dissociates from the receptor in an endocytic compartment. A fraction of the EGF is then diverted nonselectively to the transcytotic pathway, as found for other fluid-phase markers previously (Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243-3258.
当在可渗透滤膜支持物上生长时，犬肾传代细胞（MDCK）会发生极化，并产生不同的顶端和基底外侧膜结构域。在这些条件下，它们会进行液相标记物的转胞吞作用。最近，有报道称表皮生长因子（EGF）可通过MDCK细胞进行受体介导的转胞吞作用（Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595 - 1601）。我们研究了EGF受体在这一过程中的作用。EGF的转胞吞作用仅发生在基底外侧到顶端的方向，具有时间依赖性，并且会被未标记的EGF以浓度依赖性方式抑制。与之前的研究不同，我们发现只有约5%的基底外侧结合的EGF被转运到顶端腔室。转运的半衰期为90分钟。使用MDCK的突变细胞系MDCKII-RCAr来研究EGF受体的表达。这些突变细胞的细胞表面糖蛋白可通过外源性半乳糖基化用[3H]半乳糖有效地进行标记。发现EGF受体仅在基底外侧表面表达。向基底外侧培养基中添加EGF会导致受体迅速内化和降解。直接检测基底外侧糖蛋白的转胞吞作用时，我们检测到几种蛋白质穿过细胞。然而，EGF受体并不在这组蛋白质之中。综合这些结果，我们提出以下模型。基底外侧表面的EGF内化是由EGF受体介导的。EGF在内吞小室中与受体解离。然后，一部分EGF会像之前对其他液相标记物所发现的那样，非选择性地转向转胞吞途径（Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243 - 3258）。
