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.