DESENSITIZATION OF P2Y2 NUCLEOTIDE RECEPTOR & CF THERAPY

P2Y2 核苷酸受体的脱敏

• 批准号: 6325854
• 负责人: FERNANDO A GONZALEZ
• 金额: $ 11.03万
• 依托单位: UNIVERSITY OF PUERTO RICO RIO PIEDRAS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325854
• 关键词: affinity labeling; biological signal transduction; clone cells; cystic fibrosis; enzyme activity; human tissue; immunoprecipitation; neoplastic cell culture for noncancer research; nucleotides; phosphorylation; protein kinase; purinergic receptor; receptor coupling; receptor expression; receptor sensitivity; respiratory disease /disorder therapy; respiratory epithelium; transfection; western blottings

Description (Adapted from Application): Extracellular nucleotides activate P2Y2 receptors that modulate important biological processes, such as ion secretion, cellular growth, and vasodilatation, among others. UTP (a P2Y2 receptor agonist) has been shown to induce calcium-dependent anion secretion in airway epithelial cells expressing a defective cystic fibrosis transmembrane conductance regulator, a cAMP-dependent chloride channel. This observation suggests that nucleotides may be efficacious in the treatment of cystic fibrosis. One potential limitation of this therapy is that prolonged exposure to nucleotides causes desensitization of the P2Y2 receptor leading to receptor sequestration and downregulation. Considering the therapeutic potential for P2Y2 receptor activation in vivo, the investigators will attempt to understand the molecular mechanisms underlying receptor desensitization. Preliminary results suggest that desensitization is caused by phosphorylation of amino acid residues in the intracellular C-terminal domain of the receptor. In the present application, the PI proposes to apply molecular, biochemical and pharmacological approaches to study the mechanisms of desensitization of a recombinant P2Y2 receptor expressed in human 1321N1J astrocytoma cells that lack endogenous nucleotide receptors. This transfection system will be used to express a variety of P2Y2 nucleotide receptor constructs (e.g., deletion and point mutants) to identify the specific phosphorylation sites and protein kinases that regulate agonist-induced desensitization and sequestration (i.e., uncoupling and internalization). To accomplish this goal they will express a series of epitope-tagged P2Y2 receptor mutants in 1321N1J cells followed by metabolic labeling of the cells and purification of receptor protein to directly determine the sites of phosphorylation. After identifying the P2Y2 receptor phosphorylation sites and kinases that regulate agonist-induced and heterologous desensitization, they will use the accumulated information to address the hypothesis that P2Y2 receptor uncoupling and internalization are distinct biochemical events. Therefore, they will determine how specific P2Y2 receptor domains and phosphorylation sites relate to receptor activation, signaling, desensitization (uncoupling), and sequestration (internalization). The information generated by the proposed experiments will provide an understanding of the molecular mechanisms for desensitization of P2Y2 receptors after acute and chronic exposure to nucleotides These results will be important for the future development of nucleotide therapies for cystic fibrosis and other diseases by elucidating means to optimize the beneficial effects of drug administration (i.e., signaling and anion secretion) while minimizing or eliminating the deleterious uncoupling and internalization of the receptor.

描述（改编自应用）：细胞外核苷酸激活P2 Y2 调节重要生物过程，如离子分泌， 细胞生长和血管舒张等。UTP（P2 Y2受体 激动剂）已显示诱导气道中钙依赖性阴离子分泌 表达缺陷性囊性纤维化跨膜的上皮细胞 电导调节剂，cAMP依赖性氯离子通道。该观察结果 表明核苷酸可能有效地治疗囊性 纤维化这种疗法的一个潜在局限性是， 核苷酸导致P2 Y2受体脱敏，导致受体 隔离和下调。考虑到治疗潜力 P2 Y2受体在体内的激活，研究人员将试图了解 受体脱敏的分子机制。初步 结果表明，脱敏是由氨基酸磷酸化引起的 受体的胞内C-末端结构域中的残基。本 应用，PI建议应用分子，生物化学和 研究药物脱敏机制的药理学方法 在人1321 N1 J星形细胞瘤细胞中表达的重组P2 Y2受体， 缺乏内源性核苷酸受体。该转染系统将用于 表达多种P2 Y2核苷酸受体构建体（例如，缺失和 点突变体）以鉴定特异性磷酸化位点和蛋白质 调节激动剂诱导的脱敏和隔离的激酶（即， 解偶联和内化）。为了实现这一目标，他们将表达一个 在1321 N1 J细胞中的一系列表位标记的P2 Y2受体突变体， 细胞的代谢标记和受体蛋白的纯化， 直接决定磷酸化位点。在识别出P2 Y2之后， 受体磷酸化位点和调节激动剂诱导的激酶， 异源脱敏，他们将使用积累的信息， 解决P2 Y2受体解偶联和内化是 不同的生化事件因此，他们将确定P2 Y2 受体结构域和磷酸化位点与受体活化有关， 信号传导、脱敏（解偶联）和螯合（内化）。 拟议的实验产生的信息将提供一个 了解P2 Y2受体脱敏的分子机制 在急性和慢性暴露于核苷酸后这些结果将是重要的 用于囊性纤维化的核苷酸疗法的未来发展， 通过阐明手段优化药物的有益效果， 给药（即，信号传导和阴离子分泌），同时最小化或 消除有害的受体解偶联和内化。