CELLULAR REGULATION OF KIDNEY EPITHELIAL CELL POLARITY

肾上皮细胞极性的细胞调节

基本信息

批准号：
2142654
负责人：
Dennis Brown
金额：
$ 24.27万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-08-01 至 1996-07-31
项目状态：
已结题

项目摘要

Physiological regulation of kidney epithelial cell function is achieved by the regulated recycling of vesicles that carry proteins between the cytoplasm and distinct plasma membrane domains. The cellular control of this cycling process is poorly understood. This proposal aims to unravel the mechanisms that are involved in the polarized insertion of transport proteins into epithelial cell membranes. We will concentrate on intracellular pathways that are of particular importance to kidney function, i.e., post-Golgi vesicles that deliver proteins to either apical or basolateral plasma membranes, and endocytotic vesicles that are involved in the recycling of membrane proteins. Our specific aims are: 1) to examine selected cellular pathways and mechanisms involved in the sorting and the selective insertion of apical and basolateral plasma membrane proteins. We postulate that apical and basolateral plasma membrane proteins are segregated after leaving the Golgi apparatus into discrete vesicles that interact differently with the microtubular apparatus of epithelial cells, and with different plasma membrane domains. We will examine physiologically important molecules such as proton pumps, anion exchangers, and glucose transporters under conditions that alter intracellular trafficking, including microtubule disruption, acid-base manipulations and manoeuvres that block protein export from the Golgi to the cell surface. We will ask whether membrane and secreted proteins ride in the same vesicles, and whether lipid-anchored membrane proteins are handled differently by epithelial cells. Finally, we will examine the subcellular distribution and assembly of the proton pump using specific antibodies. 2) to examine the role of GTP-binding proteins in transport vesicle function. We postulate that the apically and basolaterally-directed vesicles whose pathways will be mapped, contain or can interact with specific G-proteins and/or other proteins that are involved in vesicle function and targeting. Using available antibody and cDNA probes for specific G-proteins, we will examine the role of G-proteins in vesicle trafficking using high-resolution morphological techniques combined with biochemical assays and functional studies in intact kidney and on isolated vesicles. Much of the published work on epithelial polarity has been obtained using virally-infected cultured cells by following expression and membrane insertion of exogenous viral proteins. An important and unique aspect of the proposed studies is that they will mostly be carried out in situ using markers of endogenous proteins that are of functional importance in kidney physiology.

肾上皮细胞功能的生理调节是通过调节的囊泡的回收细胞质和不同的质膜结构域。细胞控制这种循环过程知之甚少。该建议旨在解散传输插入涉及的机制蛋白质进入上皮细胞膜。我们将集中精力对肾脏特别重要的细胞内途径功能，即后囊泡，将蛋白质输送到顶端或基底外侧质膜和涉及的内吞囊泡在膜蛋白的回收中。我们的具体目的是：1）检查选定的细胞途径和分类所涉及的机制以及顶端和基底外侧质膜的选择性插入蛋白质。我们假设顶端和基底外侧质膜蛋白质在离开高尔基体后分离蛋白质与微管设备不同相互作用的囊泡上皮细胞，具有不同的质膜结构域。我们将检查生理上重要的分子，例如质子泵，阴离子在改变的条件下交换器和葡萄糖转运蛋白细胞内运输，包括微管破坏，酸碱操纵和操纵者阻止蛋白质从高尔基出口到细胞表面。我们会询问膜和分泌蛋白是否乘坐在相同的囊泡中，以及脂质锚定的膜蛋白是否是通过上皮细胞的处理方式不同。最后，我们将研究使用特定的质子泵的亚细胞分布和组装抗体。 2）检查GTP结合蛋白在运输中的作用囊泡功能。我们假设这是顶端的基底方向的囊泡，其途径将被映射，包含或可以与特定的G蛋白和/或其他蛋白质相互作用参与囊泡功能和靶向。使用可用的抗体和特定G蛋白的cDNA探针，我们将检查G蛋白的作用在囊泡运输中，使用高分辨率形态学技术结合完整肾脏的生化测定和功能研究并在孤立的囊泡上。上皮的大部分已发表的工作极性是使用病毒感染的培养细胞获得的遵循外源性病毒蛋白的表达和膜插入。拟议的研究的一个重要而独特的方面是，他们将通常使用内源性蛋白的标记在原位进行肾脏生理学的功能重要性。