Modulation of MUC1 Mucin Trafficking

描述（由申请人提供）：人MUC1是一种膜糖蛋白，在许多分泌性上皮细胞的顶端表面表达。累积数据表明，MUC1具有高度保守的胞质结构域和高度糖基化的外结构域，可作为细胞表面受体/传感器和表皮生长因子（EGF）信号的调节剂。肿瘤中MUC1的过表达通过与EGF受体（EGFR/ErbB1）相关的异常信号转导明显诱导细胞转化。相反，EGFR磷酸化MUC1可增强MUC1与-catenin的结合、稳定性和核靶向性。虽然EGFR主要位于极化上皮细胞的基底外侧，但有一部分EGFR与MUC1共同定位于顶端细胞表面。MUC1和EGFR在信号转导、内化和降解所需的细胞质结合伙伴对接中具有共同的位点。因此，MUC1和EGFR的相互作用在稳定极化上皮的正常完整性和MUC1过表达时破坏肿瘤单层中都起着重要作用。在上皮起源的肿瘤中，细胞极性受损，在所有细胞表面和细胞内都发现过表达MUC1。最近的数据表明，MUC1的错误定位与侵袭性肿瘤表型、转移增加和癌症患者预后不良相关。MUC1异常定位的基础尚不清楚。我们之前发现，通过网格蛋白介导的内吞作用，MUC1的内化是缓慢的，并由其糖基化调节，而循环是快速的，并依赖于其s -棕榈酰化。现在清楚的是，MUC1通过与凝集素结合而保留在MDCK细胞表面，而EGFR则通过与通常在上皮细胞顶端表面分泌的凝集素-3结合而保留在非极化肿瘤细胞表面。半乳糖凝集素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的细胞外糖缀合物。因此，我们将验证MUC1的顶端靶向和EGFR-MUC1复合物的形成依赖于凝集素与末端加工聚糖上的聚n -乙酰乳胺的结合这一假设。我们将在正常和糖基化缺陷的MDCK细胞中结合使用内源性EGFR、MUC1和重组突变体的代谢标记、表面生物素化和免疫印迹，以确定1)极化上皮细胞中MUC1的顶端靶向机制，2)极化上皮细胞中MUC1的内吞噬和再循环调节机制。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依赖性信号。我们在这些研究中的成功将为肿瘤中MUC1依赖的信号传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.