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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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至关重要颅面发育过程。人类 PDGFRA 突变与非综合征性唇裂相关唇/腭和人类 PDGFRB 突变会导致 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，但其分子机制定义 PDGFR 信号下游生物特异性的因素仍不完全清楚。它一直结果表明，PDGFR 在颅面发育过程中可以形成功能性同二聚体和异二聚体此外，PDGFRa/b 异二聚体表现出更强大的细胞内信号传导和增强的有丝分裂与 PDGFR 同二聚体相比的反应。然而，相对时空表达不同的二聚体及其体内配体倾向仍未完全表征。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回收。目前尚不清楚这些内部化和贩运动态是否以及如何不同 PDGFR 二聚体，可能导致不同的下游反应。该提案的目的是研究 PDGFR 激活、内化和运输的时空二聚体特异性动态，如以及它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，其中分裂的金星荧光蛋白（N 端 V1 和 C 端 V2) 与各个受体融合，以允许受体对在二聚化时可视化。首先，检查PDGFR异二聚体的时空激活，金星的面积和强度将利用两种方法的组合在整个发育过程中分析小鼠中面部的表达 PDGFR-BiFC 等位基因，PdgfraV1/V1；PdgfrbV2/V2。接下来，确定 PDGFR 异二聚体的配体倾向在体内，这些等位基因将与 Pdgfbfl 和 CectTg 等位基因结合，有条件地消除 PDGF-BB 配体在咽弓外胚层中，随后将进行金星信号分析。进一步地，要检查各种 PDGFR 二聚体的内化和运输动力学，将产生稳定的细胞系 BiFC 标记的 PDGFR 的不同组合的表达，以允许每个二聚体的可视化。流动细胞计数分析将确定各种 PDGFR 的内化率，以及免疫荧光和 TIRF 显微镜将用于研究二聚体特异性运输动力学。最后，效果如下将通过流式分析内体成分对 PDGFR 二聚体内化和运输的抑制细胞计数、蛋白质印迹以及功能迁移和增殖测定。该项目将调查，首次研究了颅面发育过程中体内 PDGFR 异二聚体的时空激活，以及 PDGFR 二聚体特异性的运输动态。因此，这些研究将揭示潜在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。