P2Y Receptor Trafficking in Epithelial Cells

上皮细胞中的 P2Y 受体运输

• 批准号: 6531275
• 负责人: ROBERT A NICHOLAS
• 金额: $ 32.11万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6531275
• 关键词: G protein; MDCK cell; cellular polarity; chimeric proteins; intracellular transport; protein kinase; protein localization; protein protein interaction; protein structure function; protein transport; proteomics; purinergic receptor; radioimmunoassay; receptor binding; receptor coupling; receptor expression; respiratory epithelium; site directed mutagenesis; yeast two hybrid system

DESCRIPTION (provided by applicant): The proper targeting of proteins to either the apical or basolateral surface of polarized airway epithelial cells is critical for normal lung function. An important example is cystic fibrosis, a disease caused in large part by a single amino acid mutation in the CFTR Cl- channel that prevents its transport to the apical surface of airway cells. Other important proteins, such as G protein-coupled P2Y receptors for extracellular nucleotides, are expressed in airway and other polarized epithelial cells and are targeted to different membrane domains of these cells. Thus, the P2Y1 receptor is expressed exclusively at the basolateral surface of polarized epithelial cells, whereas the P2Y2 receptor is expressed at the apical surface. Very little is known about the sorting signals and mechanisms by which G protein-coupled receptors in general, and P2Y receptors in particular, are targeted to their final destination in polarized cells. In this application, we propose to identify the mechanisms by which P2Y1 and P2Y2 receptors are targeted to opposite surfaces of airway and other polarized epithelial cells. We will first establish the routes by which P2Y1 and P2Y2 receptors are delivered to the basolateral and apical domains of polarized cells. In the second aim, we will identify the targeting domains that direct polarized expression of the two receptors by utilizing both confocal microscopy and quantification of cell-surface expression in MDCK cells. We will delimit the regions that are responsible for polarized expression of the receptors, define the amino acids that are most critical, and determine whether the sorting signals direct targeting of non-sorted receptors. Finally, we will identify proteins that interact with the targeting domains of these receptors by yeast 2-hybrid and proteomics approaches. Once identified, we will characterize these proteins to determine if they direct polarized targeting. These experiments will provide important information into the mechanism by which P2Y receptors achieve their steady-state localization in polarized epithelial cells and generate insights into how G protein-coupled receptors in general are targeted to different membrane domains.

描述（由申请方提供）：蛋白质正确靶向极化气道上皮细胞的顶端或基底侧表面对于正常肺功能至关重要。 一个重要的例子是囊性纤维化，这是一种在很大程度上由CFTR Cl-通道中的单个氨基酸突变引起的疾病，该突变阻止其转运到气道细胞的顶端表面。 其他重要的蛋白质，如细胞外核苷酸的G蛋白偶联P2 Y受体，在气道和其他极化上皮细胞中表达，并靶向这些细胞的不同膜结构域。 因此，P2 Y1受体仅在极化上皮细胞的基底外侧表面表达，而P2 Y2受体在顶端表面表达。 对于G蛋白偶联受体，特别是P2 Y受体在极化细胞中靶向其最终目的地的分选信号和机制知之甚少。 在本申请中，我们提出确定P2 Y1和P2 Y2受体靶向气道和其他极化上皮细胞的相对表面的机制。 我们将首先建立P2 Y1和P2 Y2受体递送到极化细胞的基底外侧和顶端区域的途径。 在第二个目标中，我们将通过利用共聚焦显微镜和MDCK细胞中细胞表面表达的定量来确定指导两种受体的极化表达的靶向结构域。 我们将界定负责受体极化表达的区域，定义最关键的氨基酸，并确定分选信号是否直接靶向未分选的受体。 最后，我们将通过酵母双杂交和蛋白质组学方法鉴定与这些受体的靶向结构域相互作用的蛋白质。 一旦确定，我们将表征这些蛋白质，以确定它们是否直接极化靶向。 这些实验将提供重要的信息到P2 Y受体实现其在极化上皮细胞中的稳态定位的机制，并产生洞察G蛋白偶联受体一般是如何靶向不同的膜结构域。