THE INTERACTION OF THROMBIN AND ADP RECEPTORS IN PLATELETS

血小板中凝血酶和 ADP 受体的相互作用

• 批准号: 8364951
• 负责人: DONNA S WOULFE
• 金额: $ 1.86万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364951
• 关键词: ADP Receptors; Address; Agonist; Amino Acids; Arrestins; Bioluminescence; Blood Platelets; Cells; Centers of Research Excellence; Co-Immunoprecipitations; Complex; Data; Dependence; Energy Transfer; Ensure; Funding; G Protein-Coupled Receptor Genes; Grant; Heterodimerization; Human; Immunofluorescence Microscopy; Membrane; Membrane Proteins; National Center for Research Resources; PAWR gene; Phosphorylation; Principal Investigator; Production; Regulation; Research; Research Infrastructure; Resources; Scaffolding Protein; Signal Transduction; Source; Specificity; Testing; Thrombin Receptor; United States National Institutes of Health; arrestin 2; base; cost; mutant; receptor; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project is based upon the observation that PAR4 signaling in platelets is dependent on P2Y12, but the underlying basis for this is not understood. We have shown that PAR4 and P2Y12 can be co-¿immunoprecipitated from human platelets following agonist activation and that the two receptors directly associate when expressed in HEK293T cells. We hypothesize that the functional dependence of PAR4 on P2Y12 is at least in part due to the physical association of the two receptors and that their association promotes the recruitment of the scaffolding protein, arrestin-¿2, to the PAR4-¿P2Y12 complex. We will test this hypothesis in the following 2 specific aims: Aim 1 is to assess the specificity and structural requirements for PAR4-¿P2Y12 heterodimerization in HEK293T cells. We have identified a 3-¿amino acid determinant in the fourth transmembrane (TM4) of PAR4 required for interaction with P2Y12 assessed by bioluminescence resonance energy transfer (BRET) in HEK293T cells. We will confirm this result using co-immunoprecipitation studies, characterize membrane expression of the PAR4 mutant receptor using immunofluorescence microscopy and address the interaction specificity of both native PAR4 and our TM4-¿PAR4 mutant using BRET and co-immunoprecipitation studies. Aim 2 is to define the relevance of P2Y12 association with PAR4 to PAR4-¿induced signaling responses. By expressing mutant versus native forms of PAR4 together with P2Y12, we will determine whether arrestin-¿2 specifically associates with native PAR4 that is capable of interacting with P2Y12, but not a mutant form of PAR4 that fails to interact with P2Y12. We will then evaluate arrestin-¿PI3K association and Akt phosphorylation under the same conditions. Finally, we will assess P2Y12-independent signaling of the mutant form of PAR4 to ensure the structural integrity of the mutant receptor. The project has the potential to impact our understanding of the regulation and functional significance of GPCR heterodimerization, and will help us to generate additional preliminary data for an R01 application to understand the functional significance of PAR4-¿P2Y12 association in platelets.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 该项目基于血小板中PAR 4信号传导依赖于P2 Y12的观察，但其潜在基础尚不清楚。我们已经证明，PAR 4和P2 Y12可以从激动剂激活后的人血小板中共免疫沉淀，并且当在HEK 293 T细胞中表达时，这两种受体直接缔合。我们假设PAR 4对P2 Y12的功能依赖性至少部分是由于两种受体的物理结合，并且它们的结合促进了支架蛋白arrestin-<$2向PAR 4-<$P2Y12复合物的募集。我们将在以下两个具体目标中检验这一假设： 目的1是评估HEK 293 T细胞中PAR 4-<$P2Y12异源二聚化的特异性和结构要求。我们在HEK 293 T细胞中通过生物发光共振能量转移（BRET）评估了PAR 4的第四跨膜（TM 4）中与P2 Y12相互作用所需的3-<$氨基酸决定簇。我们将使用免疫共沉淀研究证实这一结果，使用免疫荧光显微镜表征PAR 4突变体受体的膜表达，并使用BRET和免疫共沉淀研究解决天然PAR 4和我们的TM 4-<$PAR 4突变体的相互作用特异性。 目的2是确定P2 Y12与PAR 4的关联与PAR 4-γ诱导的信号应答的相关性。通过表达突变体与天然形式的PAR 4以及P2 Y12，我们将确定arrestin-β 2是否特异性地与能够与P2 Y12相互作用的天然PAR 4相关联，而不是与P2 Y12不能相互作用的PAR 4的突变形式。然后，我们将在相同条件下评估arrestin-<$PI3K结合和Akt磷酸化。最后，我们将评估PAR 4突变形式的P2 Y12非依赖性信号传导，以确保突变受体的结构完整性。 该项目有可能影响我们对GPCR异源二聚化的调控和功能意义的理解，并将帮助我们为R 01应用生成额外的初步数据，以了解PAR 4-<$P2Y12在血小板中的功能意义。