Investigation of Platelet G-Protein Coupled Receptors

 描述(申请人提供)：虽然5-羟色胺5-羟色胺2 A受体(5HT2AR)和ADP P2Y1受体的信号在血栓性疾病发病机制中的作用已有很好的文献记载，但目前还没有针对这两种途径的拮抗剂可用于临床。这在一定程度上是因为缺乏关于受体信号和结构的知识。本申请提出的实验涉及血小板5HT2a和P2Y1受体的结构生物学和信号转导的基本方面：1.5HT2AR配体结合区(即第二细胞外环；EL2)拮抗在预防血栓性疾病中的生物学意义。我们的假设是5HT2AR的EL2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5HT2AR的EL2在血小板激活中发挥重要作用。增强激动剂诱导的血小板活化。随后的研究中，我们将调查EL2Ab和EL2多肽疫苗对出血时间和血栓形成的影响。2.P2Y1受体的G蛋白偶联结构域。我们的假设是，P2Y1的胞内结构域包含单独的区域，这些区域限制与特定G蛋白的偶联。在这方面，我们的一个有趣的结果是，一个模拟P2Y1第二细胞内环(IL2)(简称MyR-N-IL2pep)N端的肽阻断了ADP诱导的聚集。这一发现表明，P2Y1的N-IL2结构域参与了GQ的受体偶联。同样，其他IL区域在G蛋白偶联中的作用将通过检测其对应的多肽对ADP触发的血小板激活的影响来确定。此外，还将研究任何具有生物活性的白介素肽对出血时间和血栓形成的影响。总而言之，这些研究的结果将提供关于5HT2AR和P2Y1受体的生物学和结构的基本信息，并可能定义新的治疗靶点和/或辅助用于治疗血栓性疾病状态的有机衍生物/药物的分子模拟研究预测。

 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.