Role of Na, K-ATPase beta isoforms in pump sorting and epithelial integrity

描述(由申请人提供)：该项目的长期目标是确定固有的分选信号和必要的相互作用蛋白，使必要的转运酶Na，K-ATPase能够不同地运输到正常上皮细胞和受疾病影响的上皮细胞的顶端和/或基侧膜。在胃癌、肾癌和多囊肾肾小管上皮细胞中，-31亚基水平降低和(32-亚基亚基)亚基丰度高与泵从基侧向顶端定位的转变和正常细胞-细胞黏附的丧失有关。我们推测，与Na，K-ATPase的(31-亚基)亚基连接的N-糖链对于稳定侧膜上的泵，从而维持上皮细胞间的黏附是重要的。另一方面，Na，K-ATPase(32-异构体)所特有的N-糖链对于泵的顶端输送是重要的。其具体目的是：1.阐明Na，K-ATPase(31-和32-亚基)的单个N-糖链在培养的极化肾细胞顶膜和基底侧膜中泵的极化分选、运输和稳定中的作用。2.研究Na，K-ATPase(31-亚基)及其糖基化在正常和可逆性缺血损伤肾细胞间黏附中的作用。我们将研究这两种(3-亚基)糖基化缺陷突变体在MDCK细胞中的表达对泵的囊泡运输、泵在顶膜和基侧膜之间的分布、泵与细胞骨架的结合以及细胞-细胞黏附的影响。我们还将确定(3-亚基)中单个N-糖链的碳水化合物组成的差异，并确定能特异性识别N-糖链并促进Na，K-ATPase(3-亚基)异构体的运输、分选和膜保留的凝集素蛋白。我们将确定哪种类型或哪种碳水化合物的N-糖链更有利于细胞接触的形成。这些观察将扩展到缺血条件下培养的细胞，以将这些观察与肾脏的病理联系起来。阐明Na，K-ATPase分选和鉴定促进适当转运的蛋白质的细节将使我们更好地理解肾纤维化、癌症、多囊肾病和肾缺血中细胞-细胞接触中断的潜在机制，并可能影响新疗法的发展。

DESCRIPTION (provided by applicant): The long-term goals of this project are to determine the intrinsic sorting signals and necessary interacting proteins that enable differential trafficking of an essential transport enzyme, the Na,K-ATPase, to the apical and/or basolateral membranes of normal epithelial cells and those affected by disease. Reduced level of the -31-subunit and high abundance of the (32-subunit isoform in gastric and renal carcinoma and renal tubular cells of polycystic kidneys correlates with a switch from basolateral to apical localization of the pump and loss of normal cell-cell adhesion. We hypothesize that the N-glycans linked to the (31-subunit isoform of the Na,K-ATPase are important for stabilization of the pump on the lateral membranes and therefore for the maintenance of intercellular adhesion in epithelia. On the other hand, the N-glycans unique to the (32-isoform of the Na,K-ATPase are important for apical delivery of the pump. The specific aims are: 1. Elucidate the role of individual N-glycans of the Na,K-ATPase (31- and (32-subunits in polarized sorting, trafficking, and stabilization of the pump in the apical and basolateral membranes of polarized renal cells in culture. 2. Investigate the role of the (31-subunit of the Na,K-ATPase and its glycosylation in intercellular adhesion of renal cells in normal conditions and upon reversible ischemic injury. We will study the effect of expression of glycosylation-deficient mutants of the two (3-subunit isoforms in MDCK cells on vesicular trafficking of the pump, its distribution between apical and basolateral membranes, association of the pump with the cytoskeleton, and in cell-cell adhesion. We will also determine the differences in the carbohydrate composition of the individual N-glycans in the (3-subunits and identify the lectin proteins that specifically recognize N-glycans and facilitate trafficking, sorting and membrane retention of the Na,K-ATPase (3-subunit isoforms. We will determine which type or which carbohydrate composition of N-glycans is preferable for cell contact formation. These observations will be extended to cells in culture under ischemic conditions to relate these observations to pathology of the kidney. Elucidation of the details of the Na,K-ATPase sorting and identification of the proteins that facilitate proper trafficking will give us a better understanding of the mechanism underlying disruption of cell-cell contacts in renal fibrosis, cancer, polycystic kidney disease and renal ischemia and could impact the development of novel therapeutics.