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.

项目摘要颅面开发是一个复杂的过程，需要各种信号通路来调节最终分化为额骨骨骼的软骨和骨骼的组织之间的串扰。在此过程中的缺陷导致常见的颅面畸形，例如唇裂和口感。信号通过血小板来源的生长因子受体（PDGFR）在人和小鼠中都起着关键作用颅面发展。 PDGFRA已显示在NCC迁移中起主要作用，贡献在妊娠中期，面部间充质的扩散并促进成骨细胞分化。或者， PDGFRB主要调节妊娠中期面部间充质的增殖。此外，PDGFRA和 PDGFRB已显示在颅面间充质中遗传和物理相互作用以形成功能异二聚体，尽管通过这些异二聚体信号的机理和功能仍然存在未知。我们使用了一种创新的方法，双分子荧光互补（BIFC）探索个体，激活的PDGFR二聚体，该二聚体揭示了二聚体特异性的初步差异激活，运输和下游信号传导动力学。该提议的目的是完全表征这些在体外和体内的PDGFRA/B异二聚体的动力学，并识别PDGFR二聚体特异性相互作用的蛋白质介导了各种PDGFR二聚体的差异运输。首先，PDGFRA/b 使用GFP-trap纳米病毒对杂种二聚体进行免疫沉淀，以响应PDGF-的时间 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，以检查检查的共定位 PDGFRA/B异二聚体具有各种内体隔室标记的响应 PDGF-BB配体刺激以检查PDGFRA/B异二聚体的运输动力学。这些发现将与我们先前的PDGFR同型二聚体结果进行比较。其次，BIFC标记的PDGFRA 同型二聚体，PDGFRB同型二聚体和PDGFRA/B杂二聚体将使用GFP-trap纯化通过质谱法分析纳米机，以鉴定PDGFR二聚体特异性相互作用蛋白。小说在受体运输中表现出的作用的蛋白质将过表达和压抑相关的PDGFR-BIFC稳定细胞系在存在PDGF配体的情况下以及各种PDGFR的运输二聚体将如上评估。最后，将在E8.5-的颅面结构中分析金星表达。 e16.5 e16.5 e16.5 pdgfrav1和pdgfrbv2 bifc bifc敲入等位基因的胚胎均与通过荧光显微镜进行安装和冠状冷冻切片，以检查颅面发育过程中PDGFRA/B异二聚体形成。这里提出的研究将确定如何在单个PDGFR二聚体下游引入生物学特异性并提供宝贵的见解进入哺乳动物颅面发育的基础机制。