Molecular Mechanism of Polarized Cubilin Expression

极化Cubilin表达的分子机制

• 批准号: 6732074
• 负责人: JOHN C FYFE
• 金额: $ 14.95万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6732074
• 关键词: apical membrane; biotechnology; cell surface receptors; cobalamin; disease /disorder classification; disease /disorder etiology; disease /disorder model; dogs; functional /structural genomics; gastrointestinal nutrient absorption; gene mutation; genetic disorder; genetic markers; linkage mapping; malabsorption; molecular cloning; molecular genetics; nutrition related tag; protein transport; proteinuria; receptor expression; single nucleotide polymorphism; small intestines; southern blotting; syndrome

DESCRIPTION (provided by applicant): Cobalamin (vitamin B 12) is an essential micronutrient for the maintenance of normal metabolism. Humans and other animals have evolved a highly specific mechanism for concentrating dietary cobalamin on the absorptive surface of distal small intestinal enterocytes. Cobalamin endocytosis is mediated by cubilin, a large multiligand receptor of the apical membrane. Imerslund-Grasbeck syndrome (I-GS) is an autosomal recessive trait of selective cobalamin malabsorption and proteinuria due to failure to express functional cubilin in the apical membrane of ileal enterocytes and renal proximal tubule cells. Cubilin mutations cause human I-GS in one population, but other causes remain to be determined. An inherited defect of cubilin trafficking to the apical membrane has been described in a canine I-GS model, but cubilin was excluded as the disease gene, indicating that an unknown accessory activity is essential for polarized cubilin expression in intestinal and renal epithelial cells. Our goal is to take advantage of this unique, naturally-occurring animal model to better understand the cell biology of receptor expression in polarized epithelia. Lacking functional gene candidates, we initiated a comparative positional-candidate gene approach to determine the I-GS disease gene. Significant linkage to a gene marker was found, defining an approximately 9 cM region harboring the disease locus. The specific aims of this proposal are: 1) to minimize the region of linkage by developing and applying new genetic markers to a large outbred linkage pedigree in order to identify a subset of genes in the homologous region of the human genome as positional candidates; 2) to clone and analyze positional candidates for mutations; and 3) to initiate functional analyses of the disease-causing gene product. Genes will be chosen for new marker development in an interative process from the locality of markers showing close linkage to the disease locus until a gene marker is found which exhibits no recombination with the disease locus. The I-GS gene will be identified by mutation analysis, and antibody will be generated to study intracellular localization and determine binding partners. Results of these efforts are expected to open a window onto a new aspect of nutrient absorption and membrane receptor biology.

描述（由申请方提供）：钴胺素（维生素B 12）是维持正常代谢的必需微量营养素。人类和其他动物已经进化出一种高度特异性的机制，将膳食钴胺素集中在远端小肠上皮细胞的吸收表面。钴胺素的内吞作用是由cubilin介导的，cubilin是一种顶端膜的大型多配体受体。Imerslund-Grasbeck综合征（I-GS）是一种常染色体隐性遗传性状的选择性钴胺素吸收不良和蛋白尿，由于不能表达功能性cubilin在回肠肠上皮细胞和肾近端小管细胞的顶膜。Cubilin突变在一个人群中引起人类I-GS，但其他原因仍有待确定。在犬I-GS模型中已经描述了cubilin向顶膜运输的遗传缺陷，但是cubilin被排除为疾病基因，这表明未知的辅助活性对于肠和肾上皮细胞中的极化cubilin表达是必不可少的。我们的目标是利用这种独特的，自然发生的动物模型，以更好地了解受体表达的极化上皮细胞生物学。由于缺乏功能性候选基因，我们启动了一种比较位置候选基因的方法来确定I-GS疾病基因。显着的连锁基因标记被发现，定义一个约9厘米的区域窝藏的疾病位点。该建议的具体目标是：1）通过开发新的遗传标记并将其应用于大型远交连锁谱系，以将人类基因组同源区域中的基因子集鉴定为位置候选者，从而使连锁区域最小化; 2）克隆并分析突变的位置候选者;以及3）启动致病基因产物的功能分析。在一个交互过程中，从显示与疾病基因座紧密连锁的标记的位置，直到发现不与疾病基因座重组的基因标记，选择基因用于新标记的开发。将通过突变分析鉴定I-GS基因，并将产生抗体以研究细胞内定位并确定结合伴侣。这些努力的结果有望为营养吸收和膜受体生物学的新方面打开一扇窗户。