Examining PDGFR dimer-specific activation and internalization dynamics

项目摘要 血小板源性生长因子受体（PDGFR）信号传导对于复杂的形态学过程至关重要。 颅面发育的过程。人PDGFRA突变与非综合征性唇腭裂相关 人PDGFRB的突变导致Kosaki过度生长综合征和Pentinnen综合征。 虽然已经在小鼠模型中详细研究了单个受体的作用，但分子机制 定义PDGFR信号传导下游的生物特异性仍然不完全清楚。已经 显示PDGFRs在颅面发育过程中可以形成功能性同源二聚体和异源二聚体 并且，进一步地，PDGFR a/B异二聚体表现出更强的细胞内信号传导和增强的促有丝分裂性 与PDGFR同源二聚体相比的反应。然而，相对时空表达的 不同的二聚体和它们在体内的配体倾向仍然没有完全表征。此外，PDGFR 二聚体内化是调节受体活性的一个关键方面，最终导致受体活化。 降解或再循环。目前尚不清楚这些内部化和贩运动态在以下国家之间是否存在差异，以及如何存在差异： PDGFR二聚体，可能导致不同的下游反应。这项建议的目的是 研究PDGFR激活、内化和运输的时空二聚体特异性动力学， 以及它们在体内的配体倾向。为了检测不同的二聚体，我将使用双分子荧光 互补（BiFC），一种融合蛋白技术，由此使分裂的Venus荧光蛋白（N-末端V1） 和C-末端V2）与单个受体融合，以允许受体对在其二聚化时的可视化。 首先，为了检查PDGFR异二聚体的时空激活，金星的面积和强度 在整个发育时间内，将利用两种方法的组合分析鼠中面的表达 PDGFR-BiFC等位基因，PdgfraV 1/V1; PdgfrbV 2/V2。接下来，为了确定PDGFR异二聚体的配体倾向， 在体内，这些等位基因将与Pdgfbfl和CrectTg等位基因组合以条件性地消融PDGF-BB配体 在咽弓外胚层，随后金星信号分析将进行。此外，为了检查 通过调节各种PDGFR二聚体的内化和运输动力学，将产生稳定的细胞系， 在BiFC标记的PDGFR的不同组合的表达中，以允许每个二聚体的可视化。流 细胞计数分析将确定各种PDGFR的内化率，以及免疫荧光和 TIRF显微镜将被用来调查二聚体特定的运输动力学。最后，影响 将通过流式细胞仪分析内体组分对PDGFR二聚体内化和运输的抑制作用 流式细胞术、蛋白质印迹和功能性迁移和增殖测定。该项目将调查，为 第一次，PDGFR异源二聚体在颅面发育过程中的体内时空激活， 以及PDGFR二聚体特异性运输动力学。这些研究将揭示潜在的机制 在颅面发育过程中由受体酪氨酸激酶（RTK）信号传导产生的生物特异性。

Project Summary Platelet-derived growth factor receptor (PDGFR) signaling is critical to the complex morphological process of craniofacial development. Mutations in human PDGFRA are associated with non-syndromic cleft lip/palate, and mutations in human PDGFRB cause Kosaki overgrowth syndrome and Pentinnen syndrome. While the roles of individual receptors have been studied in detail in mouse models, the molecular mechanisms that define biological specificity downstream of PDGFR signaling remain incompletely understood. It has been shown that the PDGFRs can form both functional homodimers and heterodimers during craniofacial development and, further, that PDGFRa/b heterodimers exhibit more robust intracellular signaling and enhanced mitogenic responses in comparison to PDGFR homodimers. However, the relative spatiotemporal expression of the different dimers and their ligand propensities in vivo remain incompletely characterized. Furthermore, PDGFR dimer internalization is a critical aspect in the regulation of receptor activity, ultimately leading to receptor degradation or recycling. It is unknown if and how these internalization and trafficking dynamics differ between PDGFR dimers, potentially leading to differential downstream responses. The aim of this proposal is to investigate the spatiotemporal dimer-specific dynamics of PDGFR activation, internalization, and trafficking, as well as their ligand propensities in vivo. To detect distinct dimers, I will implement bimolecular fluorescence complementation (BiFC), a fusion protein technique whereby a split Venus fluorescent protein (N-terminal V1 and C-terminal V2) is fused to individual receptors to allow visualization of receptor pairs upon their dimerization. First, to examine the spatiotemporal activation of PDGFR heterodimers, the area and intensity of Venus expression will be analyzed in the murine midface throughout developmental time utilizing combinations of two PDGFR-BiFC alleles, PdgfraV1/V1;PdgfrbV2/V2. Next, to determine the ligand propensity for PDGFR heterodimers in vivo, these alleles will be combined with Pdgfbfl and CrectTg alleles to conditionally ablate the PDGF-BB ligand in the pharyngeal arch ectoderm, and subsequent Venus signal analyses will be performed. Further, to examine internalization and trafficking dynamics of the various PDGFR dimers, stable cells lines will be generated to result in the expression of different combinations of BiFC-tagged PDGFRs to allow for visualization of each dimer. Flow cytometry analyses will determine internalization rates of the various PDGFRs, and immunofluorescence and TIRF microscopy will be employed to investigate dimer-specific trafficking dynamics. Finally, the effects of inhibition of endosomal components on PDGFR dimer internalization and trafficking will be analyzed via flow cytometry, Western blotting, and functional migration and proliferation assays. This project will investigate, for the first time, the spatiotemporal activation of PDGFR heterodimers in vivo during craniofacial development, as well as PDGFR dimer-specific trafficking dynamics. These studies will thus uncover mechanisms underlying biological specificity generated by receptor tyrosine kinase (RTK) signaling during craniofacial development.