Molecular Mechanism of Polarized Cubilin Expression

描述(申请人提供)：钴胺(维生素B12)是维持正常新陈代谢所必需的微量营养素。人类和其他动物已经进化出一种高度特异的机制，将饮食中的钴胺浓缩在远端小肠肠道细胞的吸收表面。钴胺的内吞作用是由顶膜上的一种大的多配体受体Cubilin介导的。Imerslund-Grasbeck综合征(I-GS)是一种选择性钴胺吸收不良和蛋白尿的常染色体隐性遗传特征，原因是回肠和肾近端小管上皮细胞顶膜上不表达功能性Cubilin。Cubilin突变导致一个人群中的人类I-GS，但其他原因仍有待确定。在犬I-GS模型中，Cubilin转运到顶膜的遗传缺陷已被描述，但Cubilin被排除为疾病基因，表明未知的辅助活动是肠和肾上皮细胞Cubilin极化表达的关键。我们的目标是利用这一独特的、自然发生的动物模型来更好地了解极化上皮中受体表达的细胞生物学。由于缺乏功能基因候选，我们启动了一种比较位置候选基因的方法来确定I-GS病基因。发现了与一个基因标记的显著连锁，定义了一个大约9 cM的含有该疾病位点的区域。这项建议的具体目标是：1)通过开发和应用新的遗传标记来将连锁区域最小化，以便将人类基因组同源区域中的基因子集识别为位置候选；2)克隆和分析突变的位置候选；以及3)启动致病基因产物的功能分析。在从与病位紧密连锁的标记的位置到找到与病位没有重组的基因标记的迭代过程中，将选择基因来开发新的标记。将通过突变分析鉴定I-GS基因，并产生抗体以研究细胞内定位和确定结合伙伴。这些努力的结果有望为了解营养吸收和膜受体生物学的新方面打开一扇窗。

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.