Characterization of PDGFR alpha/beta heterodimers

PDGFR α/β 异二聚体的表征

基本信息

批准号：
10676610
负责人：
Maria B Campana
金额：
$ 7.46万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10676610
关键词：
Ablation Adult Alleles Binding Biological Biological Assay C-terminal Cartilage Cell Line Cleft Lip Cleft Palate Complement Complex Congenital Abnormality Craniofacial Abnormalities Defect Development Dimerization Disease Embryo Endosomes Face Fluorescence Fluorescence Microscopy Frozen Sections Genes Goals Human Immunoprecipitation In Vitro Incidence Individual Knock-in Laboratories Life Ligands Ligation Live Birth Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Mesenchyme Molecular Mus Mutation N-terminal Neural Crest Cell Outcome Study Phosphorylation Plasmids Platelet-Derived Growth Factor Platelet-Derived Growth Factor Receptor Platelet-Derived Growth Factor alpha Receptor Play Pregnancy Process Proliferating Proteins Proteomics Recycling Repression Role Signal Pathway Signal Transduction Signaling Molecule Skeleton Small Interfering RNA Specificity Syndrome Terminator Codon Testing Tissues Transfection United States Vascular Diseases Venus Western Blotting bone cell motility cleft lip and palate craniofacial craniofacial development craniofacial structure dimer experimental study in vivo innovation insight knock-down nanobodies novel novel therapeutics osteoblast differentiation overexpression palatal shelves platelet-derived growth factor BB receptor response spatiotemporal stable cell line trafficking

项目摘要

Project Summary Craniofacial development is a complex process that requires various signaling pathways to mediate cross-talk between tissues that eventually differentiate into the cartilage and bone of the frontonasal skeleton. Defects in this process result in common craniofacial malformations, such as cleft lip and palate. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in both human and mouse craniofacial development. PDGFRa has been shown to play a predominant role in NCC migration, contribute to proliferation of the facial mesenchyme at mid-gestation and promote osteoblast differentiation. Alternatively, PDGFRb primarily regulates proliferation of the facial mesenchyme past mid-gestation. Further, PDGFRa and PDGFRb have been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers, though the mechanism and function of signaling through these heterodimers remains unknown. We have used an innovative approach, bimolecular fluorescence complementation (BiFC), to explore individual, activated PDGFR dimers, which has revealed preliminary differences in dimer-specific activation, trafficking and downstream signaling dynamics. The goal of this proposal is to fully characterize these dynamics for PDGFRa/b heterodimers in vitro and in vivo, and to identify PDGFR dimer-specific interacting proteins that mediate differential trafficking of the various PDGFR dimers. First, PDGFRa/b heterodimers will be immunoprecipitated using a GFP-Trap nanobody in response to a timecourse of PDGF- BB ligand stimulation to examine the dimerization and autophosphorylation dynamics of PDGFRa/b heterodimers. Then, fluorescence microscopy experiments will be performed to examine co-localization of PDGFRa/b heterodimers with markers of various endosomal compartments in response to a timecourse of PDGF-BB ligand stimulation to examine the trafficking dynamics of PDGFRa/b heterodimers. These findings will be compared to our previous results for the PDGFR homodimers. Second, BiFC-tagged PDGFRa homodimers, PDGFRb homodimers and PDGFRa/b heterodimers will be purified using the GFP-Trap nanobody and analyzed by mass spectrometry to identify PDGFR dimer-specific interacting proteins. Novel proteins with demonstrated roles in receptor trafficking will be both overexpressed and repressed in the relevant PDGFR-BiFC stable cell line in the presence of PDGF ligand, and trafficking of the various PDGFR dimers will be assessed as above. Finally, Venus expression will be analyzed in craniofacial structures of E8.5- E16.5 embryos that are double-homozygous for PdgfraV1 and PdgfrbV2 BiFC knock-in alleles both in whole mount and in coronal frozen sections by fluorescence microscopy to examine the timing and localization of PDGFRa/b heterodimer formation during craniofacial development. The studies proposed here will determine how biological specificity is introduced downstream of individual PDGFR dimers and provide valuable insight into the mechanisms underlying mammalian craniofacial development.

项目概要颅面发育是一个复杂的过程，需要多种信号通路来介导最终分化为额鼻骨骼的软骨和骨的组织之间的串扰。这一过程的缺陷会导致常见的颅面畸形，例如唇裂和腭裂。信令通过血小板衍生生长因子受体（PDGFR）在人类和小鼠中发挥着关键作用颅面发育。 PDGFRa 已被证明在 NCC 迁移中发挥主导作用，有助于促进妊娠中期面部间质的增殖并促进成骨细胞分化。或者， PDGFRb 主要调节妊娠中期后面部间质的增殖。此外，PDGFRa和 PDGFRb 已被证明在颅面间充质中通过遗传和物理相互作用形成功能性异二聚体，尽管通过这些异二聚体的信号传导机制和功能仍然存在未知。我们使用了一种创新方法，即双分子荧光互补 (BiFC) 探索个体、激活的 PDGFR 二聚体，这揭示了二聚体特异性的初步差异激活、运输和下游信号动态。该提案的目标是充分描述这些 PDGFRa/b 异二聚体在体外和体内的动态，并鉴定 PDGFR 二聚体特异性介导各种 PDGFR 二聚体差异运输的相互作用蛋白。首先，PDGFRa/b 将使用 GFP-Trap 纳米抗体对异二聚体进行免疫沉淀，以响应 PDGF- 的时间进程。 BB 配体刺激以检查 PDGFRa/b 的二聚化和自磷酸化动力学异二聚体。然后，将进行荧光显微镜实验来检查共定位 PDGFRa/b 异二聚体，具有响应时间过程的各种内体区室的标记 PDGF-BB 配体刺激以检查 PDGFRa/b 异二聚体的运输动态。这些发现将与我们之前的 PDGFR 同二聚体结果进行比较。二、BiFC标记的PDGFRa 同二聚体、PDGFRb 同二聚体和 PDGFRa/b 异二聚体将使用 GFP-Trap 进行纯化纳米抗体并通过质谱分析来鉴定 PDGFR 二聚体特异性相互作用蛋白。小说已证明在受体运输中发挥作用的蛋白质将在 PDGF配体存在下的相关PDGFR-BiFC稳定细胞系，以及各种PDGFR的运输二聚体将如上所述进行评估。最后，将分析E8.5-颅面结构中的Venus表达 PdgfraV1 和 PdgfrbV2 BiFC 敲入等位基因均为双纯合的 E16.5 胚胎通过荧光显微镜安装并在冠状冰冻切片中检查时间和定位颅面发育过程中 PDGFRa/b 异二聚体的形成。这里提出的研究将决定如何将生物特异性引入个体 PDGFR 二聚体下游并提供有价值的见解深入研究哺乳动物颅面发育的机制。