MUC1 membrane trafficking is modulated by multiple interactions
MUC1 membrane trafficking is modulated by multiple interactions
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DOI：
10.1074/jbc.m409360200
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发表时间：
2004-12-17
影响因子：
4.8
通讯作者：
Hughey, RP
中科院分区：
文献类型：
--
作者：
Kinlough, CL;Poland, PA;Hughey, RP
MUC 1是在许多上皮细胞的顶端表面上发现的粘蛋白样跨膜蛋白。由于肿瘤细胞中MUC 1的异常细胞内定位与侵袭性肿瘤和患者的不良预后相关，因此设计实验以表征调节MUC 1膜运输的特征。通过在糖基化缺陷的中国仓鼠卵巢细胞中跟踪[S-35]Met/Cys标记的MUC 1，我们先前发现MUC 1上的O-聚糖的截短抑制其表面表达并通过网格蛋白介导的内吞作用刺激其内化。为了鉴定与其糖基化状态无关的MUC 1内化信号，用Tac的胞外域替换MUC 1的胞外域，并通过相同的方案测量嵌合体内吞作用。嵌合体的内吞作用显著快于MUC 1，表明高度延伸的胞外域的特征抑制MUC 1内化。对截短突变体和酪氨酸突变体的分析表明，Tyr（20）和Tyr（60）都是有效内吞所必需的。Tyr（20）的突变显著阻断了嵌合体与AP-2的免疫共沉淀，表明（YHPM）-H-20被mu 2亚基识别为YXXphi基序。酪氨酸磷酸化（YTNP）-T-60先前被鉴定为Grb 2结合的SH 2位点，并且我们发现Tyr（60）的突变阻断了嵌合体与Grb 2的免疫共沉淀。这是Grb 2在MUC 1的内吞作用中起重要作用的第一个迹象。
MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [S-35]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr(20) and Tyr(60) were both required for efficient endocytosis. Mutation of Tyr(20) significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that (YHPM)-H-20 is recognized as a YXXphi motif by the mu2 subunit. The tyrosine-phosphorylated (YTNP)-T-60 was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr(60) blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1.