Hobert M, Carlin C
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970.
J Cell Physiol. 1995 Mar;162(3):434-46. doi: 10.1002/jcp.1041620316.
Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin-Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans-Golgi network with a half-time of approximately 45 min and have an approximate t1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg-651 were localized predominantly at the apical surface of filter-grown cells, whereas receptors truncated at Leu-723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise location of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states.
尽管表皮生长因子受体（EGFRs）在极化上皮细胞的基底外侧质膜上不对称表达已得到充分证实，但该过程的调控机制尚不清楚。本研究的目的是利用Madin-Darby犬肾（MDCK）细胞模型探讨EGFR定向基底外侧分选的机制。第一组实验确定了内源性犬EGFR的分选模式。MDCK I和MDCK II细胞系所表现出的电阻差异对犬EGFR的极性没有定量影响。在这两种情况下，超过90%的总表面EGFR定位于基底外侧表面。犬EGFR从反式高尔基体网络直接分选到基底外侧膜，半衰期约为45分钟，一旦到达基底外侧表面，其半衰期约为12.5小时。在稳定转染的MDCK细胞中表达的人全受体也以相似的效率定位于基底外侧膜。为了确定基底外侧分选所需的EGFR序列，用编码细胞质截短的人受体蛋白的cDNA转染MDCK细胞。在Arg-651处截短的人EGFR主要定位于滤器培养细胞的顶端表面，而在Leu-723处截短的受体主要位于基底外侧。这些结果表明，细胞质近膜结构域包含一个正向基底外侧分选决定簇。此外，一旦主要的基底外侧分选信号被去除，EGFR胞外结构域或跨膜结构域可能具有一个隐蔽序列，该序列与顶端分选机制特异性相互作用。进一步阐明这些信号的精确位置将增进我们对调控质膜分选的基本理解，以及与某些病理生理和恶性状态相关的EGFR表达不当的功能后果。
