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.
Craniofacial defects, such as cleft lip and palate, are among the most prevalent birth defects in humans, with an estimated incidence in the United States of 1/940 live births resulting in cleft lip and 1/1,574 live births resulting in cleft palate. Mutations in human PDGFRA are associated with non-syndromic cleft lip/palate, and mutations in human PDGFRB cause Kosaki overgrowth syndrome and Pentinnen syndrome. The studies proposed in this research project will impart valuable insight into the mechanisms underlying craniofacial development by examining the spatiotemporal dynamics of PDGFR dimer-specific activation, internalization, and trafficking and, ultimately, provide new therapeutic directions for the treatment of human craniofacial birth defects.