Investigation of Platelet G-Protein Coupled Receptors

 描述（由申请人提供）：虽然有充分的文献证明了5-羟色胺5 HT 2A受体（5 HT 2AR）和ADP P2 Y1受体的信号传导参与血栓性疾病的发病机制，但目前还没有可用于临床的拮抗剂，并且其靶向这些途径中的任何一种。这部分源于缺乏关于受体信号传导和结构的知识。本申请提出了解决血小板5 HT 2A和P2 Y1受体的结构生物学和信号传导的基本方面的实验：5 HT 2AR配体结合区拮抗作用的生物学意义（即，第二细胞外环; EL 2）在预防血栓性疾病中的作用。我们的假设是，5 HT 2AR的EL 2在血小板活化中起重要作用。为了解决这一假设，我们的实验将评估一个假设的能力，即5 HT 2AR的EL 2在血小板活化中起重要作用。增强的激动剂诱导的血小板活化。随后的研究我们将研究EL 2Ab和基于EL 2肽的“疫苗”对出血时间和血栓形成的影响。2. P2 Y1受体的G蛋白偶联结构域。我们的假设是，P2 Y1的细胞内结构域包含单独的区域，限制耦合到一个特定的G蛋白。在这方面，我们感兴趣的结果之一是，模拟P2 Y1的第二胞内环（IL 2）的N-末端的肽（缩写为Myr-N-IL 2 pep）阻断ADP诱导的聚集。这一发现表明P2 Y1的N-IL 2结构域参与受体与Gq的偶联。类似地，其他IL区在G蛋白偶联中的作用将通过检查其相应肽对ADP触发的血小板活化的作用来确定。此外，将研究任何生物活性IL肽对出血时间和血栓形成发展的影响。总的来说，从这些研究中获得的结果将提供有关5 HT 2AR和P2 Y1受体生物学和结构的基本信息，并可能定义新的治疗靶点和/或有助于治疗血栓性疾病状态的有机衍生物/药物的分子建模研究预测。

 DESCRIPTION (provided by applicant): While the involvement of signaling of the serotonin 5HT2A receptor (5HT2AR) and the ADP P2Y1 receptor in the pathogenesis of thrombotic diseases is well documented, there are no antagonists that are currently available for clinical use, and which target either of these pathways. This derives, in part, from lack of knowledge regarding receptor signaling and structure. The present application proposes experiments that address fundamental aspects of the structural biology and signaling of the platelet 5HT2A and P2Y1 receptors: 1. the biological significance of antagonism of 5HT2AR ligand-binding region (i.e., the second extracellular loop; EL2) in the prevention of thrombotic diseases. Our hypothesis is that EL2 of 5HT2AR plays an important role in platelet activation. To address this hypothesis, our experiments will evaluate the ability of a hypothesis is that EL2 of 5HT2AR plays an important role in platelet activation. enhanced, agonist-induced platelet activation. Subsequent studies we will investigate the effects of EL2Ab and an EL2 peptide-based "vaccine" on bleeding time, and thrombosis development. 2. The G-protein coupling domains of P2Y1 receptor. Our hypothesis is that the intracellular domains of P2Y1 contain separate regions that confine coupling to a specific G-protein. In this regard, one of our interesting resuls is that a peptide mimicking the N-terminus of the second intracellular loop (IL2) of P2Y1 (abbreviated Myr-N-IL2pep) blocked ADP-induced aggregation. This finding suggests that the N-IL2 domain of P2Y1 participates in receptor coupling to Gq. Similarly, the role of other IL regions in G-protein coupling will be determined by examining the effects of their corresponding peptides on ADP-triggered platelet activation. Also, the effects of any biologically-active IL peptides on bleeding time and thrombosis development will be investigated. Collectively, results obtained from these studies will provide fundamental information concerning 5HT2AR and P2Y1 receptor biology and structure, and may define new therapeutic targets and/or aid molecular modeling study predictions for organic derivatives/agents for treating thrombotic disease states.