Blood Platelet Thromboxane Receptors

血小板血栓素受体

• 批准号: 6836442
• 负责人: GUY C LEBRETON
• 金额: $ 34.32万
• 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 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6836442
• 关键词: G protein; biological signal transduction; cell surface receptors; chimeric proteins; cyclic AMP; laboratory mouse; laboratory rabbit; phosphorylation; platelet activation; prostaglandin receptor; protein kinase A; receptor binding; site directed mutagenesis; thromboxanes

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

描述（由申请人提供）：本申请提出了解决血小板中TXA 2受体（TPR）信号传导的三个基本方面的实验：1.血小板TPR和其它GPCR之间的交叉信号传导。我们的假设是，GPCR-G蛋白（GP）复合物形成的过程是基于质量作用原理，并且该原理定义并优先考虑血小板GPCR信号传导和交叉信号传导。该模型预测了血小板GPCR信号传导的动态状态，其可以被包括心血管疾病在内的许多因素干扰。我们认为，这一原则形成了一种分子机制，通过这种机制，血小板可以整合其单独的GPCR信号通路，并通过这种机制建立血小板GPCR信号偏好。现在提出实验来进一步研究这个动态过程的基本原理。2.绘制TPR配体结合口袋。我们的假设是，配体停靠在C-末端ED 3，并与TM 5和/或N-末端ED 4中的残基相互作用。我们以前证明了ED 3中的C183-D193形成TPR的关键配体结合结构域，这些结果最近被NMR证实。我们现在将鉴定ED 3中参与这种配体结合的残基。基于TPR配体的有效配位半径，我们还将研究邻近残基（来自TM 5和N-末端ED 4）在对接/信号转导过程中的参与。使用定点诱变和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功效的指标。此外，使用新的功能性抗体的兔模型将用于确定TPR ED 3对体内血小板反应性的贡献。3. cAMP介导的G-α 12/13磷酸化的分子和功能后果。我们的假设是G-α 12/13的构象敏感开关I区（SRI）的磷酸化在通过G-α 12/13途径调节信号传导中起重要作用。我们最近证明了PKA介导的磷酸化在SW 1的Thr（203）的G13。我们现在将进一步确定这种磷酸化对血小板活化和抑制的分子/功能后果。定点肽/诱变和诱导型小基因将用于定义静息/活化血小板中G12/13-Rho信号传导的PKA调节。随后的实验将确定G12/13介导的血小板功能反应，以及这些反应如何通过G-α磷酸化调节。总的来说，拟议的实验应该提供新的和重要的信息TPR信号转导。反过来，这些信息应该有助于开发控制TXA 2介导的血栓栓塞的治疗方法