Modulation of MUC1 Mucin Trafficking

MUC1 粘蛋白运输的调节

• 批准号: 7385940
• 负责人: Rebecca P Hughey
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7385940
• 关键词: Apical; Binding; Binding Proteins; Biology; Biotinylation; Cancer Patient; Cell Nucleus; Cell Polarity; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Clathrin; Complex; Cyclin D1; Cytoplasm; Cytoplasmic Tail; Data; Docking; Endocytosis; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Epithelium; Exhibits; Galactose Binding Lectin; Galactosides; Galectin 3; Genetic Transcription; Glycoconjugates; Human; Immunoblotting; Individual; Label; Link; Localized; MDCK cell; Malignant Neoplasms; Measures; Mediating; Membrane Glycoproteins; Membrane Protein Traffic; Metabolic; Mucin-1 Staining Method; Mucins; Mutation; N-Glycosylation Site; Neoplasm Metastasis; Neoplasms; Nuclear; Pathway interactions; Phenotype; Phosphorylation; Play; Polysaccharides; Process; Protein Analysis; Protein Overexpression; Protocols documentation; Publishing; Rate; Recombinants; Recycling; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Site; Structure; Surface; Testing; Ubiquitination; base; crosslink; extracellular; glycosylation; insight; metaplastic cell transformation; monolayer; mutant; neoplastic cell; novel; outcome forecast; palmitoylation; poly-N-acetyllactosamine; programs; research study; sensor; success; trafficking; tumor

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.

描述（由申请方提供）：人MUC 1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，MUC 1，其高度保守的胞质结构域和高度糖基化的胞外域，作为细胞表面受体/传感器和表皮生长因子（EGF）信号转导的修饰剂。肿瘤中MUC 1的过表达通过与EGF受体（EGFR/ErbB 1）相关的异常信号转导明显诱导细胞转化。相反，MUC 1的EGFR磷酸化增强MUC 1结合、稳定和β-连环蛋白的核靶向。尽管EGFR主要在极化的上皮细胞中位于基底外侧，但EGFR的一部分与MUC 1共定位于顶端细胞表面。MUC 1和EGFR表现出信号传导、内化和降解所需的细胞质结合配偶体对接的共同位点。因此，MUC 1和EGFR的相互作用在稳定极化上皮细胞的正常完整性和当MUC 1过表达时在破坏肿瘤形成中的单层中起重要作用。在上皮来源的肿瘤中，细胞极性受损，并且在所有细胞表面和细胞内发现过表达的MUC 1。最近的数据表明，MUC 1的错误定位与癌症患者的侵袭性肿瘤表型、增加的转移和不良预后相关。MUC 1异常定位的基础尚不清楚。我们以前发现，MUC 1内化网格蛋白介导的内吞作用是缓慢的，其糖基化调制，而回收是快速的，并依赖于其S-棕榈酰化。现在清楚的是，MUC 1通过半乳糖凝集素结合保留在MDCK细胞的表面上，而EGFR通过结合通常在上皮细胞的顶端表面分泌的半乳糖凝集素-3保留在非极化肿瘤细胞的表面上。半乳糖凝集素是可溶性的？通过非经典途径从细胞质分泌的半乳糖苷结合蛋白，以交联细胞外糖缀合物。因此，我们将检验MUC 1的顶端靶向和EGFR-MUC 1复合物的形成依赖于半乳糖凝集素与末端加工聚糖上的聚-N-乙酰乳糖胺的结合的假设。我们将在正常和糖基化缺陷的MDCK细胞中使用内源性EGFR和MUC 1以及重组突变体的代谢标记、表面生物素化和免疫印迹的组合来确定1）MUC 1在极化上皮细胞中顶端靶向的机制，2）调节MUC 1在极化上皮细胞中的内吞和再循环的机制，以及3）MUC 1和EGFR的相互作用将如何影响极化上皮细胞顶端表面的EGF依赖性信号传导。我们在这些研究中的成功将为MUC 1依赖性信号传导和肿瘤中的错误定位提供新的和基本的见解，这将有助于对MUC 1生物学在人类癌症中的理解达到新的水平。