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: 10214555
• 负责人: Madison A Rogers
• 金额: $ 3.8万
• 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/10214555
• 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 血小板来源的生长因子受体（PDGFR）信号对于复杂形态至关重要 颅面发育的过程。人PDGFRA中的突变与非综合裂解有关 唇/pa和人PDGFRB中的突变会导致科萨基过度生长综合征和五本宁综合征。 虽然在小鼠模型中详细研究了单个受体的作用，但分子机制 该定义PDGFR信号下游的生物学特异性仍然不完全理解。它一直 表明PDGFR可以在颅面发育过程中形成功能均匀二聚体和异二聚体 此外，PDGFRA/B异二聚体表现出更强的细胞内信号传导和促有丝分裂的增强 与PDGFR同二聚体相比，响应。但是，相对时空表达 体内不同的二聚体及其配体倾向仍未完全表征。此外，PDGFR 二聚体内在化是受体活性调节的关键方面，最终导致受体 退化或回收利用。这些内在化和运输动态是否以及在之间有何不同 PDGFR二聚体，可能导致差异下游响应。该提议的目的是 研究PDGFR激活，内在化和运输的时空二聚体特异性动力学 以及他们在体内的配体倾向。为了检测不同的二聚体，我将实施双分子荧光 互补（BIFC），一种融合蛋白技术，裂开金星荧光蛋白（N末端V1） 和C末端V2）与单个受体融合，以使受体对二聚体的可视化。 首先，要检查PDGFR异二聚体的时空激活，金星的区域和强度 在整个发育时间中，将在鼠中的中间分析表达，并利用两个组合 PDGFR-BIFC等位基因，PDGFRAV1/V1; PDGFRBV2/V2。接下来，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等位基因结合起来，以有条件地消除PDGF-BB配体 将在咽弓外胚层中进行，随后的金星信号分析将进行。此外，要检查 各种PDGFR二聚体的内在化和运输动力学，将产生稳定的细胞线以结果 在表达BIFC标记的PDGFR的不同组合中，允许每个二聚体的可视化。流动 细胞仪分析将确定各种PDGFR的内在化速率以及免疫荧光和 TIRF显微镜将用于研究二聚体特异性运输动力学。最后，影响的影响 将通过流量分析抑制PDGFR二聚体内在化和运输的内体成分 细胞仪，蛋白质印迹以及功能迁移和增殖测定法。该项目将调查，因为 第一次是颅面发育过程中PDGFR异二聚体在体内的时空激活， 以及PDGFR二聚体特异性运输动力。因此，这些研究将发现基础的机制 颅面发育过程中受体酪氨酸激酶（RTK）信号传导产生的生物特异性。