PDGFR dimer-specific activation, trafficking and downstream signaling dynamics

Abstract

Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFR and PDGFR, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein. We find that PDGFR receptors homodimerize more quickly than PDGFR receptors in response to PDGF ligand, with increased levels of autophosphorylation. Further, we demonstrate that PDGFR homodimers are trafficked and degraded more quickly, while PDGFR homodimers are more likely to be recycled back to the cell membrane. We show that PDGFR homodimer activation results in a greater amplitude of phospho-ERK1/2 and phospho-AKT signaling, as well as increased proliferation and migration. Finally, we demonstrate that inhibition of clathrin-mediated endocytosis leads to changes in cellular trafficking and downstream signaling, particularly for PDGFRa homodimers. Collectively, our findings provide significant insight into how biological specificity is introduced to generate unique responses downstream of PDGFR engagement.

PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

“…2C and D). As we previously observed, the anti-PDGFR blots contained two bands representing the non-glycosylated and glycosylated forms of the receptors (7,24), while the anti-phospho-PDGFR blots possessed a single band representing the glycosylated, phosphorylated versions of the receptors upon ligand treatment (24).…”

“…PDGFRb homodimers via Dyngo-4a treatment resulted in increased phospho-ERK1/2 levels and decreased phospho-AKT levels at early timepoints of ligand stimulation (24), indicating that engagement of these signaling molecules occurs at the same subcellular platforms for PDGFRa/b heterodimers and PDGFRb homodimers. Alternatively, we previously observed that Dyngo-4a treatment of the PDGFRa homodimer cell line had a negative effect on ERK1/2 and AKT phosphorylation, resulting in delayed and reduced peaks of activation (24). These results demonstrated that rapid internalization of PDGFRa homodimers plays an important role in the propagation of downstream signaling and suggested that this dimer primarily signals from early endosomes.…”

“…Further, the PDGFRa/b heterodimers had considerably reduced levels of autophosphorylation compared to either homodimer, particularly at early ligand stimulation timepoints (24). Next, PDGFRa/b heterodimers were internalized into early endosomes more quickly and dwelled at that location for longer periods of time than either homodimer (24).…”

“…To investigate PDGFRa/b-specific dynamics, and compare these to what we previously observed for PDGFRa homodimers and PDGFRb homodimers (24), we stably integrated PDGFRa-V1 and PDGFRb-V2 sequences (Fig. S1A) into the human-derived HCC15 cell line via lentiviral transduction.…”

“…Following dimerization and activation at the cell membrane in response to ligand binding, PDGFRs are internalized and trafficked through the early endosome before being trafficked to the lysosome via late endosomes for degradation or recycled to the membrane via recycling endosomes for continued signaling (24,(29)(30)(31)(32)(33). To investigate the trafficking dynamics of PDGFRa/b heterodimers in response to ligand treatment, we examined colocalization of the Venus signal in our PDGFRaV1/bV2 cell line with markers of various subcellular compartments.…”