Examining PDGFR dimer-specific activation and internalization dynamics

检查 PDGFR 二聚体特异性激活和内化动力学

• 批准号: 10064887
• 负责人: Madison A Rogers
• 金额: $ 3.75万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064887
• 关键词: Alleles; Area; Biochemistry; Biological; Biological Assay; Branchial arch structure; C-terminal; Cell Line; Cells; Chemicals; Chimeric Proteins; Cleft Lip; Cleft Palate; Complement; Complex; Congenital Abnormality; Craniofacial Abnormalities; Cre driver; Cultured Cells; Data; Defect; Development; Developmental Biology; Dimerization; Early Endosome; Ectoderm; Embryo; Event; Exhibits; Face; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Human; Immunofluorescence Immunologic; In Vitro; Incidence; Individual; Knock-out; Label; Lead; Ligands; Live Birth; Mesenchyme; Molecular; Morphogenesis; Morphology; Mus; Mutation; N-terminal; Outcome; PDGFRA gene; PDGFRB gene; Pathway interactions; Pattern; Platelet Activation; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor Receptor; Process; Proteins; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recycling; Regulation; Research; Research Project Grants; Role; Signal Transduction; Specificity; Structure; Syndrome; Techniques; Time; Translating; United States; Venus; Visualization; Western Blotting; base; cleft lip and palate; craniofacial; craniofacial development; dimer; experimental study; extracellular; genetic approach; in vivo; in vivo evaluation; inhibitor/antagonist; innovation; insight; migration; mouse model; novel therapeutics; platelet phenotype; platelet-derived growth factor BB; receptor; response; spatiotemporal; stable cell line; tool; trafficking

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.

项目摘要 血小板衍生生长因子受体(PDGFR)信号转导对复杂的形态结构至关重要 颅面发育过程。人类PDGFRA基因突变与非综合征性唇裂相关 唇腭裂和人类PDGFRb基因突变会导致小崎氏过度生长综合征和潘丁宁综合征。 虽然单个受体的作用已经在小鼠模型中进行了详细的研究，但其分子机制 对PDGFR信号下游生物学特异性的定义仍不完全清楚。一直以来 结果表明，PDGFRs在颅面发育过程中既能形成功能同源二聚体，又能形成异源二聚体 此外，PDGFRA/b异源二聚体表现出更强大的细胞内信号和增强的有丝分裂 与PDGFR同源二聚体相比的反应。然而，相对的时空表达， 不同的二聚体及其在体内的配基倾向仍未完全确定。此外，PDGFR 二聚体内化是受体活性调节的一个关键方面，最终导致受体 降解或循环利用。目前尚不清楚这些内部化和贩运动态是否以及如何不同于 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，AS 以及它们在体内的配基倾向。为了检测不同的二聚体，我将实现双分子荧光 互补(BIFC)，一种融合蛋白技术，通过这种技术，分裂的金星荧光蛋白(N-末端V1 和C-末端V2)与单个受体融合，以便在受体对二聚化时进行可视化。 首先，为了研究PDGFR异二聚体的时空激活，金星的面积和强度 利用两者的组合，将在整个发育过程中分析小鼠面中部的表达 PDGFR-BIFC等位基因，PdgfraV1/V1；PdgfrbV2/V2。接下来，确定PDGFR异二聚体的配基倾向 在体内，这些等位基因将与Pdgfbfl和CrectTg等位基因结合，有条件地去除PDGF-BB配体 在咽弓外胚层中，将进行后续的金星信号分析。进一步地，为了检查 不同PDGFR二聚体的内化和运输动力学，将产生稳定的细胞系 表达BIFC标记的PDGFRs的不同组合，以实现每个二聚体的可视化。流动 细胞学分析将确定各种PDGFRs的内在化速率，以及免疫荧光和 TIRF显微镜将被用来研究二聚体特定的运输动力学。最后，它的影响 内体成分对PDGFR二聚体内化和转运的抑制作用将通过Flow进行分析 细胞计数、Western blotting、功能迁移和增殖分析。这个项目将调查，为 首次研究了PDGFR异源二聚体在颅面发育过程中的时空激活。 以及PDGFR二聚体特有的贩运动态。因此，这些研究将揭示 受体酪氨酸激酶(RTK)信号在颅面发育过程中产生的生物特异性。