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：
3244180
负责人：
Dennis Brown
金额：
$ 25.08万
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在细胞之间携带蛋白质的囊泡的受监管回收细胞质和不同的质膜结构域。细胞控制人们对这个循环过程知之甚少。该提案旨在解开参与运输极化插入的机制蛋白质进入上皮细胞膜。我们将专注于对肾脏特别重要的细胞内途径功能，即高尔基体后囊泡将蛋白质输送到任一顶端或基底外侧质膜，以及参与的内吞囊泡膜蛋白的回收利用。我们的具体目标是：1）检查参与分选的选定细胞途径和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被分离成离散的囊泡与微管装置的相互作用不同上皮细胞，并具有不同的质膜结构域。我们将检查生理上重要的分子，如质子泵、阴离子交换器和葡萄糖转运蛋白在改变的条件下细胞内运输，包括微管破坏、酸碱阻止蛋白质从高尔基体输出到细胞表面。我们将询问膜蛋白和分泌蛋白是否骑行在相同的囊泡中，以及脂质锚定膜蛋白是否上皮细胞的处理方式不同。最后，我们将检查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。我们假设顶部和基底外侧定向的囊泡，其路径将被绘制、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。使用可用的抗体和针对特定 G 蛋白的 cDNA 探针，我们将检查 G 蛋白的作用使用高分辨率形态学技术进行囊泡运输结合完整肾脏的生化测定和功能研究和分离的囊泡。许多已发表的关于上皮细胞的工作使用病毒感染的培养细胞获得了极性外源病毒蛋白的表达和膜插入后。拟议研究的一个重要且独特的方面是，它们将大多数是使用内源蛋白标记物在原位进行的在肾脏生理学中具有重要的功能。