Characterization of PDGFR alpha/beta heterodimers

PDGFR α/β 异二聚体的表征

• 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
• 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

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。 这个过程中的缺陷导致常见的颅面畸形，如唇腭裂。信令 通过血小板衍生生长因子受体（PDGFRs）在人类和小鼠中起着关键作用， 颅面发育PDGFRa已被证明在NCC迁移中起主导作用，有助于 促进妊娠中期面部间充质细胞增殖，促进成骨细胞分化。可选择地， PDGFRb主要调节妊娠中期后面部间充质的增殖。此外，PDGFRa和 PDGFRb已被证明在颅面间充质中发生遗传和物理相互作用， 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的方法，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、运输和下游信号传导动力学。该提案的目标是充分描述 这些动力学PDGFR α/B异源二聚体在体外和体内，并确定PDGFR二聚体特异性 介导各种PDGFR二聚体的差异运输的相互作用蛋白。第一，PDGFR a/B 异源二聚体将使用GFP-陷阱纳米抗体免疫沉淀，以响应PDGF的时程。 BB配体刺激以检查PDGFR a/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细胞的共定位。 PDGFR a/B异源二聚体与各种内体区室的标志物响应于时间进程 PDGF-BB配体刺激以检查PDGFR a/B异二聚体的运输动力学。这些发现 将与我们以前的PDGFR同源二聚体的结果进行比较。第二，BiFC标记的PDGFRa 使用GFP-捕获物纯化PDGFR α/B同源二聚体、PDGFR α/B异源二聚体 通过使用纳米抗体，并通过质谱分析以鉴定PDGFR二聚体特异性相互作用蛋白。小说 在受体运输中具有已证实作用的蛋白质将在细胞中过度表达和被抑制。 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 将如上评估二聚体。最后，Venus表达将在E8.5的颅面结构中进行分析- E16.5胚胎，其对于PdgfraV 1和PdgfrbV 2 BiFC敲入等位基因均为双纯合， 通过荧光显微镜在冠状冰冻切片中观察 颅面发育过程中PDGFR α/B异源二聚体的形成这里提出的研究将确定 如何将生物特异性引入个体PDGFR二聚体的下游，并提供有价值的见解 哺乳动物颅面发育的机制