Modulation of MUC1 Mucin Trafficking

描述(申请人提供)：人MUC1是一种膜糖蛋白，表达在许多分泌上皮细胞的顶端表面。研究结果表明，MUC1具有高度保守的胞质结构域和高度糖基化的胞外结构域，是细胞表面受体/感受器和表皮生长因子(EGF)信号的修饰者。MUC1在肿瘤中的过表达通过与EGF受体(EGFR/ErbB1)相关的异常信号转导明显地诱导细胞转化。相反，MUC1的EGFR磷酸化增强了MUC1的结合、稳定性和核靶向性。尽管EGFR在极化的上皮细胞中主要是基侧的，但一小部分EGFR与MUC1共同定位于顶端细胞表面。MUC1和EGFR展示了信号传递、内化和降解所需的细胞质结合伙伴对接的共同位置。因此，MUC1和EGFR的相互作用在稳定极化上皮细胞的正常完整性和当MUC1过表达时破坏肿瘤的单层方面都起着重要作用。在上皮性肿瘤中，细胞极性受损，在所有细胞表面和细胞内均可发现MUC1的过度表达。最近的数据表明，MUC1的错误定位与肿瘤的侵袭性表型、转移增加以及癌症患者的不良预后有关。MUC1基因异常定位的基础尚不清楚。我们先前发现，MUC1的内吞是缓慢的，受其糖基化的调节，而循环是快速的，依赖于它的S棕榈酰化。现已清楚的是，MUC1通过Galectin结合保留在MDCK细胞表面，而EGFR通过结合Galectin-3保留在未极化的肿瘤细胞表面，Galectin-3通常分泌在上皮细胞的顶端表面。Galectins是一种可溶性的半乳糖苷结合蛋白，通过非经典途径从细胞质分泌到交联胞外糖结合物。因此，我们将检验这样的假设，即MUC1的顶端靶向和EGFR-MUC1复合体的形成依赖于Galectin与末端加工的多糖上的多-N-乙酰乳糖胺的结合。我们将使用代谢标记、表面生物素化和免疫印迹相结合的方法，在正常和糖基化缺陷的MDCK细胞中使用内源性EGFR和MUC1及其重组突变体来确定1)极化上皮细胞中MUC1顶端靶向的机制，2)调控极化上皮细胞内吞和再循环的机制，以及3)MUC1和EGFR的相互作用如何影响极化上皮细胞顶端表面的EGF依赖信号。我们在这些研究中的成功将为肿瘤中MUC1依赖的信号转导和错误定位提供新的和基本的见解，这将有助于对人类癌症中MUC1生物学的新水平的理解。

DESCRIPTION (provided by applicant): Human MUC1 is a membrane glycoprotein that is expressed on the apical surface of many secretory epithelial cells. The cumulative data indicate that MUC1, with its highly conserved cytoplasmic domain and heavily glycosylated ectodomain, functions as a cell surface receptor/sensor and a modifier of epidermal growth factor (EGF) signaling. Overexpression of MUC1 in tumors clearly induces cellular transformation through aberrant signal transduction linked to the EGF receptor (EGFR/ErbB1). Conversely, EGFR phosphorylation of MUC1 enhances MUC1 binding, stabilization and nuclear targeting of ¿-catenin. Although the EGFR is primarily basolateral in polarized epithelial cells, a fraction of the EGFR co-localizes with MUC1 at the apical cell surface. MUC1 and EGFR exhibit common sites for docking of cytoplasmic binding partners required for signaling, internalization and degradation. Thus, the interaction of MUC1 and EGFR plays a significant role both in stabilizing the normal integrity of polarized epithelia, and in disrupting the monolayer in neoplasia when MUC1 is overexpressed. In tumors of epithelial origin, cell polarity is compromised and overexpressed MUC1 is found on all cell surfaces and inside the cell. Recent data indicate that mis- localization of MUC1 correlates with an aggressive tumor phenotype, increased metastasis, and a poor prognosis for the cancer patient. The basis for the aberrant localization of MUC1 is unknown. We previously found that MUC1 internalization by clathrin-mediated endocytosis is slow and modulated by its glycosylation, while recycling is rapid and dependent on its S-palmitoylation. It is now clear that MUC1 is retained on the surface of MDCK cells by galectin binding, whereas the EGFR is retained on the surface of non-polarized tumor cells by binding galectin-3 that is normally secreted at the apical surface of epithelial cells. Galectins are soluble ¿ -galactoside-binding proteins secreted by a non-classical pathway from the cytoplasm to cross-link extracellular glycoconjugates. Therefore, we will test the hypothesis that apical targeting of MUC1 and formation of an EGFR-MUC1 complex is dependent on galectin binding to poly-N- acetyllactosamine on terminally processed glycans. We will use a combination of metabolic labeling, surface biotinylation and immunoblotting of endogenous EGFR and MUC1 and recombinant mutants in normal and glycosylation-defective MDCK cells to determine 1) the mechanism of MUC1 apical targeting in polarized epithelial cells, 2) the mechanisms that regulate MUC1 endocytosis and recycling in polarized epithelial cells, and 3) how interaction of MUC1 and the EGFR will influence EGF-dependent signaling at the apical surface of polarized epithelial cells. Our success in these studies will provide novel and fundamental insights into both MUC1-dependent signaling and mislocalization in tumors, which will contribute to a new level of understanding of MUC1 biology in human cancer.