SIGNAL TRANSDUCTION BY PURINORECEPTORS--REGULATION BY DESENSITIZATION

嘌呤受体的信号转导——脱敏调节

• 批准号: 5211643
• 负责人: FERNANDO A GONZALEZ
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211643
• 关键词: affinity labeling; biological signal transduction; cell membrane; clone cells; human tissue; neoplastic cell culture for noncancer research; purinergic receptor; receptor expression; receptor sensitivity; transfection; western blottings

Extracellular ATP has been shown to modulate a variety of important biological processes, such as, secretion, cellular growth, and vasodilation, among others. ATP has also been shown to induce Cl- secretion in airway epithelial cells that is non-CFTR (cystic ficrosis transmembrane regulatory protein)-dependent. This observation has raised the possibility of the use of nucleotides for the treatment of cystic fibrosis (CF). Characterization of the fundamental molecular mechanisms that control P2 purinoceptors signal transduction will be valuable for the potential therapeutic development of nucleotides in the treatment of CF. CF treatment with nucleotides may produce conditions of prolonged exposure to the nucleotide agonists that could induce desensitization. However, the molecular mechanisms of desensitization of the P2U purinoceptor are currently unclear. We propose to rigorously investigate the long-term desensitization of the P2U purinoceptor system in the human epidermoid A431 cells and in 1321N1 astrocytomas transfected with a cloned P2U purinoceptor. The A43l cells respond to extracellular ATP in a variety of ways by means of purinoceptors. Untransfected 1321N1 system represent an ideal negative control. A specific aim of this proposal is to determine if the function of P2U purinoceptors is controlled by modulation of the number of receptors on the plasma membrane and to examine if down- regulation of purinoceptors occurs during long-term desensitization. We will examine the down regulation of the purinoceptor by monitoring a variety of receptor function markers: mobilization of intracellular stores of calcium, formation of IP3 and DAG, and stimulation of calcium fluxes. The pharmacological data will be supported also by binding experiments in which we will examine the ATP binding parameters (using radioactively labeled ligands) before and after desensitization. In addition, a photoreactive analog of ATP, (3-O-(4-benzoyl)benzoyl) ATP, and polyclonal antibodies (Western analysis) will be used to measure the amount of P2 purinoceptors in the plasma membrane of A431 cells and transfected 1321N1 cells. The amount of receptors during the desensitization process may also be controlled by regulation of transcription of the P2U purinoceptor gene. Therefore, we will also use molecular techniques to examine the level of expression of the purinoceptor. Using these various approaches (pharmacological, biochemical and molecular) we will also examine the resensitization of the cells after chronic exposure to agonist, and the involvement of protein synthesis in the recovery. The characterization of the long- term desensitization of the P2 purinoceptor will be important for the future development of nucleotides as therapeutic agents against CF. Future goals of our research will include the elucidation of the molecular regulatory mechanisms that modulate P2 purinoceptor signaling and desensitization. Minority students (undergraduate and graduate) will directly participate in this study and will learn modem techniques used in biotechnology, biochemistry and molecular biology. Students will become competent in the application of biotechnology to a basic science-biomedical problem.

细胞外ATP已显示可调节各种重要 生物过程，例如分泌，细胞生长和 血管舒张等。 ATP也已显示诱导Cl- 非CFTR的气道上皮细胞的分泌 跨膜调节蛋白）依赖性。这个观察结果 提出了使用核苷酸治疗的可能性 囊性纤维化（CF）。基本分子的表征 控制P2 Purinoceptors信号转导的机制将是 对于核苷酸在潜在的治疗发展中有价值的 CF的处理。 CF用核苷酸处理可能会产生 长时间暴露于核苷酸激动剂的条件 诱导脱敏。但是， P2U Purinoceptor的脱敏目前尚不清楚。 我们 建议严格研究 人类表皮A431细胞中的P2U Purinoceptor系统和 用克隆的P2U Purinoceptor转染的1321N1星形胶质细胞瘤。这 A43L细胞以多种方式对细胞外ATP响应 Purinoceptors。未转染的1321N1系统代表理想 阴性对照。该提案的具体目的是确定是否是否 P2U Purinoceptor的功能由调制控制 质膜上的受体数量，检查是否下降 Purinoceptor的调节发生在长期脱敏期间。 我们将通过监视purinoceptor的下降调节 多种受体功能标记：动员细胞内 钙的存储，IP3和DAG的形成以及钙的刺激 通量。药理数据也将通过结合来支持 我们将检查ATP结合参数的实验（使用 脱敏之前和之后的放射性标记配体）。在 另外，ATP的光反应类似物（3-O-（4-苯甲酰苯甲酰）ATP， 和多克隆抗体（西方分析）将用于测量 A431细胞质膜中的P2 Purinoceptor的量和 转染1321N1细胞。在 脱敏过程也可以通过调节 P2U Purinoceptor基因的转录。因此，我们也将使用 分子技术检查的表达水平 purinoceptor。使用这些各种方法（药理学， 生化和分子）我们还将检查 长期暴露于激动剂后的细胞，并参与 恢复中的蛋白质合成。长期表征 P2 Purinoceptor的术语脱敏对 核苷酸作为对CF的治疗剂的未来开发。 我们研究的未来目标将包括阐明 调节P2 Purinoceptor信号传导的分子调节机制 和脱敏。少数族裔学生（本科生和毕业生） 将直接参与这项研究，并学习调制解调器技术 用于生物技术，生物化学和分子生物学。学生 将在将生物技术应用于基本的应用方面有能力 科学生物医学问题。