Blood Platelet Thromboxane Receptors

描述(由申请人提供)：本申请提出的实验涉及血小板中血栓素A2受体(TPR)信号的三个基本方面：1.血小板TPR和其他GPCR之间的交叉信号。我们的假设是，GPCR-G蛋白(GP)复合体的形成过程是基于质量作用原理的，这一原理定义并优先考虑了血小板GPCR信号和交叉信号。该模型预测了血小板GPCR信号的动态状态，该信号可能会受到包括心血管疾病在内的许多因素的干扰。我们认为，这一原理形成了一种分子机制，通过该机制，血小板可以整合它们各自独立的GPCR信号通路，并由此建立血小板GPCR信号的偏好。现在建议进行实验，以进一步研究这一动态过程的基本原理。2.映射TPR配体结合口袋。我们的假设是配体停靠在C-端ED3，并与TM5和/或N-端ED4中的残基相互作用。我们先前证明了ED3中的C183-D193形成了TPR的关键配体结合域，这些结果最近得到了核磁共振的证实。我们现在将确定ED3中参与这种配体结合的残基。基于TPR配体的有效配位半径，我们还将考察邻近残基(来自TM5和N-末端eD4)在对接/信号转导过程中的参与。利用定点突变和PG受体嵌合体，实验将测量激动剂/拮抗剂配体亲和力和功能反应作为配体对接和疗效的指标。此外，使用一种新的功能性抗体的兔模型将被用来确定TPR ED3在体内对血小板反应性的贡献。3.cAMP介导的G-α12/13磷酸化的分子和功能后果。我们的假设是，G-α12/13的构象敏感开关I区(SRI)的磷酸化在通过G-α12/13途径调节信号中起着重要作用。我们最近在G13的Thr(203)处证明了PKA介导的SW1的磷酸化。我们现在将进一步定义这种磷酸化对血小板激活和抑制的分子/功能后果。定点多肽/突变和诱导微基因将被用来确定PKA对静息/激活的血小板中G12/13-Rho信号的调节。随后的实验将确定G12/13介导的血小板功能反应，以及这些反应是如何被G-α磷酸化调节的。总的来说，拟议的实验应该提供关于TPR信号转导的新的和重要的信息。这些信息反过来应该有助于开发控制TXA2介导的血栓栓塞症的治疗方法

DESCRIPTION (provided by applicant): The present application proposes experiments which address three fundamental aspects of TXA2 receptor (TPR) signaling in platelets: 1.Cross-signaling between platelet TPRs and other GPCRs. Our hypothesis is that the process of GPCR-G protein (GP) complex formation is based on the principles of mass action, and that this principle defines and prioritizes platelet GPCR signaling and cross-signaling. This model predicts a dynamic state of platelet GPCR signaling, which can be perturbed by numerous factors including cardiovascular disease. We believe that this principle forms a molecular mechanism by which platelets can integrate their separate GPCR signaling pathways and by which platelet GPCR signaling preferences are established. Experiments are now proposed to further investigate the underlying principles of this dynamic process. 2. Map the TPR ligand-binding pocket. Our hypothesis is that ligands dock at C-terminal ED3 and interact with residues in TM5 and/or N-terminal ED4. We previously demonstrated that C183-D193in ED3 form a critical ligand binding domain of TPRs, and these results have recently been confirmed by NMR. We will now identify the residues in ED3, which participate in this ligand binding. Based on the effective coordination radius of TPR ligands, we will also examine the participation of neighboring residues (from TM5 and N-terminal ED4) in the docking/signal transduction process. Using site-directed mutagenesis and PG receptor chimeras, experiments will measure agonist/antagonist ligand affinity and functional responses as indices ligand docking and efficacy. In addition, a rabbit model using a new functional antibody will be employed to determine the contribution of TPR ED3 to platelet reactivity in vivo. 3. The molecular and functional consequences of cAMP-mediated G-alpha12/13 phosphorylation. Our hypothesis is that phosphorylation of the conformationally sensitive Switch I Region (SRI) of G-alpha12/13 plays an important role in modulating signaling through the G-alpha12/13 pathway. We recently demonstrated PKA-mediated phosphorylation within SW1 at Thr (203) of G13. We will now further define the molecular/functional consequences of such phosphorylation on platelet activation and inhibition. Site-directed peptides/mutagenesis and inducible minigenes will be used to define PKA modulation of G12/13-Rho signaling in resting/activated platelets. Subsequent experiments will identify G12/13-mediated platelet functional responses, and how these responses are modulated by G-alpha phosphorylation. Collectively, the proposed experiments should provide new and important information regarding TPR signal transduction. This information should, in turn, aid in the development of therapeutic approaches for controlling TXA2-mediated thromboembolism