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 已被证明可以调节多种重要的 生物过程，例如分泌、细胞生长和 血管舒张等。 ATP 也被证明可以诱导 Cl- 气道上皮细胞中的非 CFTR 分泌物（囊性纤维化 跨膜调节蛋白）依赖性。这一观察有 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 嘌呤受体信号转导的机制 对于核苷酸的潜在治疗发展有价值 CF的治疗。用核苷酸进行 CF 处理可能会产生 长期暴露于核苷酸激动剂的条件 诱发脱敏。然而，其分子机制 P2U嘌呤受体的脱敏作用目前尚不清楚。 我们 建议认真研究长期脱敏 人表皮样 A431 细胞中的 P2U 嘌呤受体系统和 用克隆的 P2U 嘌呤受体转染的 1321N1 星形细胞瘤。这 A43l 细胞以多种方式响应细胞外 ATP 嘌呤受体。未转染的 1321N1 系统代表了理想的 阴性对照。该提案的一个具体目标是确定是否 P2U嘌呤受体的功能是通过调节 质膜上的受体数量并检查是否下降 嘌呤受体的调节发生在长期脱敏过程中。 我们将通过监测来检查嘌呤受体的下调 多种受体功能标志物：细胞内动员 钙的储存、IP3 和 DAG 的形成以及钙的刺激 通量。药理学数据也将通过结合来支持 我们将在其中检查 ATP 结合参数的实验（使用 放射性标记的配体）在脱敏之前和之后。在 此外，ATP 的光反应类似物，(3-O-(4-苯甲酰基)苯甲酰基) ATP， 和多克隆抗体（Western 分析）将用于测量 A431细胞质膜中P2嘌呤受体的数量和 转染1321N1细胞。期间受体数量 脱敏过程也可以通过调节来控制 P2U 嘌呤受体基因的转录。因此，我们也会使用 分子技术来检查表达水平 嘌呤受体。使用这些不同的方法（药理学、 生物化学和分子）我们还将检查重新敏化 长期暴露于激动剂后的细胞，以及 蛋白质合成的恢复。长的特征 P2 嘌呤受体的术语脱敏对于 核苷酸作为 CF 治疗剂的未来发展。 我们研究的未来目标将包括阐明 调节 P2 嘌呤受体信号传导的分子调控机制 和脱敏。少数民族学生（本科生、研究生） 将直接参与这项研究并将学习现代技术 用于生物技术、生物化学和分子生物学。学生 将有能力将生物技术应用到基础领域 科学生物医学问题。