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

Abstract

The platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases allows cells to communicate with one another by binding to growth factors at the plasma membrane and activating intracellular signaling pathways to elicit responses such as migration, proliferation, survival and differentiation. The PDGFR family consists of two receptors, PDGFR and PDGFR{beta}, that dimerize to form PDGFR homodimers, PDGFR/{beta} heterodimers and PDGFR{beta} homodimers. Here, we overcame prior technical limitations in visualizing and purifying PDGFR/{beta} heterodimers by generating a cell line stably expressing C-terminal fusions of PDGFR and PDGFR{beta} with bimolecular fluorescence complementation fragments corresponding to the N-terminal and C-terminal regions of the Venus fluorescent protein, respectively. We found that these receptors heterodimerize relatively quickly in response to PDGF-BB ligand treatment, with a peak of receptor autophosphorylation following 5 minutes of ligand stimulation. Moreover, we demonstrated that PDGFR/{beta} heterodimers are rapidly internalized into early endosomes, particularly signaling endosomes, where they dwell for extended lengths of time. We showed that PDGFR/{beta} heterodimer activation does not induce downstream phosphorylation of ERK1/2 and significantly inhibits cell proliferation. Further, we characterized the PDGFR dimer-specific interactome and identified MYO1D as a novel protein that preferentially binds PDGFR/{beta} heterodimers. We demonstrated that knockdown of MYO1D leads to retention of PDGFR/{beta} heterodimers at the plasma membrane, resulting in increased phosphorylation of ERK1/2 and increased cell proliferation. Collectively, our findings impart valuable insight into the molecular mechanisms by which specificity is introduced downstream of PDGFR activation to differentially propagate signaling and generate distinct cellular responses. One-sentence summaryPDGFR/{beta} heterodimer binding to MYO1D contributes to rapid internalization of the dimerized receptors, thereby negatively affecting downstream ERK1/2 signaling and cellular proliferation.