THE INTERACTION OF THROMBIN AND ADP RECEPTORS IN PLATELETS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这个项目是基于这样的观察，即血小板中的PAR4信号依赖于P2Y12，但其潜在的基础尚不清楚。我们已经证明，在激动剂激活后，PAR4和P2Y12可以从人血小板中免疫共沉淀，并且这两种受体在HEK293T细胞中表达时直接联系在一起。我们假设PAR4对P2Y12的功能依赖至少部分是由于两个受体的物理联系，并且他们的联系促进了支架蛋白Arrestin-？2向PAR4-？P2Y12复合体的募集。我们将在以下两个具体目标中检验这一假设： 目的1评估HEK293T细胞中PAR4-βP2Y12异二聚化的特异性和结构要求。我们在HEK293T细胞中通过生物发光共振能量转移(BRET)鉴定了PAR4的第四跨膜(TM4)中与P2Y12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PAR4突变体受体的膜表达，并使用BRET和免疫共沉淀研究天然PAR4和我们的TM4-？PAR4突变体的相互作用特异性。 目的2是确定P2Y12与PAR4的关联与PAR4诱导的信号反应的相关性。通过表达突变形式的PAR4与天然形式的PAR4以及P2Y12，我们将确定arrestin-β2是否与能够与P2Y12相互作用的天然PAR4特异性地结合，而不是与P2Y12相互作用的突变形式的PAR4。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PAR4突变形式的P2Y12非依赖信号，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异二聚的调节和功能意义的理解，并将帮助我们为R01应用生成额外的初步数据，以了解PAR4-P2Y12在血小板中的功能意义。

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.