BLOOD PLATELET THROMBOXANE RECEPTORS

血小板血栓烷受体

• 批准号: 6183587
• 负责人: GUY C LEBRETON
• 金额: $ 27.28万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6183587
• 关键词: G protein; antibody; biological signal transduction; calcium flux; cyclic AMP; extracellular matrix proteins; goats; hemostasis; laboratory mouse; laboratory rabbit; membrane structure; phosphorylation; platelet activation; platelets; protein structure; receptor binding; site directed mutagenesis; thrombin; thromboembolism; thromboxanes

The rationale for the widespread use of aspirin in preventing coronary or cerebrovascular thrombosis is based on its ability to block the synthesis of TXA2. Thus, TXA2 signal transduction plays a critical role in the sequelae of events leading to certain occlusive vascular disorders. However, in spite of the clear biological importance of TXA2, significant questions remain concerning its signaling mechanisms. In the present application, we propose experiments which will address three fundamental aspects of TXA2-mediated signal transduction. Specific Aim I. will Investigate Signaling Between Platelet Seven-Transmembrane Receptors. This aim derives from our hypothesis that G-proteins serve as communication vectors within the platelet receptor pool. We believe this is a significant finding because it provides a molecular mechanism for the integration of the separate receptor signaling pathways in platelets (or other cells). This signaling process will now be further defined relative to its existence between different platelet/endothelial receptors, its effects on ligand binding affinities, and its molecular mechanism of action. Specific Aim 2. will Investigate the Platelet TXA2 Receptor Ligand Binding Domain(s). This aim is directed at testing our hypothesis that the ligand binding domain(s) of the TXA2 receptor protein resides in the extracellular region of the protein. Recently, we have succeeded in developing a new class of biotinylated photoaffinity receptor probes, and are currently applying these probes to investigate the receptor binding pocket. Two complementary approaches to this Specific Aim will also be taken: a. site- directed mutagenesis of the receptor; and b. site-specific antibodies against the receptor. It is believed that the combined information obtained from each approach will provide more definitive information than could be obtained by the utilization of either photolabeling, mutagenesis or antibody targeting alone. Finally, Specific Aim 3. will Investigate cAMP-Mediated Phosphorylation of TXA2 receptor-associated G-alpha13. This aim will test our hypothesis that TXA2 receptor signaling through G- alpha13 is coupled to IP3-independent Ca2+ fluxes in platelets; and that phosphorylation of G-alpha13 is associated with the peculiar sensitivity of TXA2 signaling to cAMP levels. Recently, we have demonstrated that the G-alpha13 subunit is associated with endogenous platelet TXA2 receptors, and that this alpha-subunit is phosphorylated by cAMP. Experiments are now proposed to study the function of G-alpha13 (i.e. Ca2+ fluxes), its localization in platelets (surface or internal membrane), and the biochemical consequences of its phosphorylation on the stability of the TXA2 receptor-G-alpha13 complex and the stability of the G-protein heterotrimer complex. In summary, the results obtained from the proposed experiments should provide new and important information regarding signal transduction through the TXA2 receptor pathway. This information will, in turn, be of significant benefit to the development of more specific and effective pharmacological approaches to the control of TXA2-mediated thromboembolism.

阿司匹林广泛用于预防冠状动脉或脑血管血栓形成的基本原理是基于其阻断TXA 2合成的能力。因此，TXA 2信号转导在导致某些闭塞性血管疾病的事件的后遗症中起关键作用。然而，尽管TXA 2具有明确的生物学重要性，但关于其信号传导机制仍存在重大问题。在本申请中，我们提出的实验将解决TXA 2介导的信号转导的三个基本方面。具体目标一。将研究血小板7跨膜受体之间的信号传导。这一目标源于我们的假设，即G蛋白作为血小板受体池内的通信载体。我们认为这是一个重要的发现，因为它为血小板（或其他细胞）中单独的受体信号通路的整合提供了分子机制。这个信号传导过程现在将进一步定义相对于它的存在之间的不同血小板/内皮受体，其对配体结合亲和力的影响，其分子作用机制。具体目标2。将研究血小板TXA 2受体配体结合结构域。这一目的旨在检验我们的假设，即TXA 2受体蛋白的配体结合结构域位于蛋白的胞外区。最近，我们成功地开发了一类新的生物素化的光亲和受体探针，目前正在应用这些探针来研究受体结合口袋。为实现这一具体目标，还将采取两种互补办法：a.所述受体的定点诱变;和B.针对受体的位点特异性抗体。据信，从每种方法获得的组合信息将提供比单独利用光标记、诱变或抗体靶向获得的信息更明确的信息。第三，具体目标。将研究cAMP介导的TXA 2受体相关G-α 13磷酸化。这一目的将检验我们的假设，即通过G-α 13的TXA 2受体信号传导与血小板中IP 3非依赖性Ca 2+通量偶联; G-α 13的磷酸化与TXA 2信号传导对cAMP水平的特殊敏感性相关。最近，我们已经证明，G-α 13亚基与内源性血小板TXA 2受体，并且该α-亚基被cAMP磷酸化。现在提出实验来研究G-α 13的功能（即Ca 2+通量）、其在血小板（表面或内膜）中的定位以及其磷酸化对TXA 2受体-G-α 13复合物的稳定性和G-蛋白异源三聚体复合物的稳定性的生物化学后果。总之，从拟议的实验中获得的结果应该提供新的和重要的信息，通过TXA 2受体途径的信号转导。这些信息将反过来对开发更特异和有效的药理学方法来控制TXA 2介导的血栓栓塞具有显著的益处。