Characterization of PDGFR alpha/beta heterodimers

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.

Grant Number: 5F32DE032554-03
NIH Institute/Center: NIH
Principal Investigator: Maria Campana