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The platelet-derived growth factor receptors (PDGFRs) play important roles in multiple cellular processes including cell survival, cell growth and cell migration. Dysregulation of PDGFRs causes aberrant PDGF signaling, which leads to various diseases. Insights from the research about PDGF/PDGFR signaling have enlightened new opportunities to understand the molecular mechanisms of cancer and other diseases. The goal of this thesis is to further investigate the mechanisms of the modulation of PDGF/PDGFR signaling to identify new ways of controlling aberrant signaling of these RTKs in various diseases, including cancer.

Ubiquitination is an important post-translational modification related to protein degradation, receptor internalization, intracellular trafficking, cell proliferation, and other cellular processes. It can be reversed by DUBs, and the overexpression of DUBs is involved in various diseases including cancers. In paper I, we identified two main DUBs working on PDGFRβ, USP17 and USP4. They affected the timing of STAT3 activation and trafficking via different mechanisms, thus fine-tuning its transcriptional activity, which further regulated the proliferative response induced by PDGF-BB.

SUMOylation is another post-translational modification that is important for the regulation of protein subcellular localization, protein stability, protein-DNA interactions, protein-protein interactions, genome organization, DNA repair and transcriptional regulation. In paper II, we have identified PDGFRα as a SUMOylation substrate and performed a characterization of the functional role of SUMOylation in PDGFRα signaling and cell proliferation.

Proteolytic cleavage of RTKs regulates their downstream signaling pathways by affecting their structure, stability, subcellular localization and interaction with other proteins. In paper III, we have identified that PDGFRβ is cleaved in the region Y579-Y857 upon ligand stimulation by a Ca2+-dependent protease, which is dependent on its internalization. The proteasomal inhibitor bortezomib blocked the internalization, as well as the cleavage of PDGFRβ, and also affected its downstream signaling.

Apart from the classic degradation in lysosomes and proteasomes, several RTKs have also been found to undergo autophagy and to be targeted in autophagosomes which further fuse with lysosomes. In liver hepatocytes (LX2) cells, it has been reported that PDGFRα, but not PDGFRβ, undergo selective autophagy. In paper IV, we identified that in certain types of cells, PDGFRβ may be involved in the autophagy pathway, which may affect the synthesis of new PDGFRβ.

To conclude, in this study, we investigated how the turnover and signaling of the RTKs PDGFR isoforms α and β are modulated by ubiquitination, SUMOylation, proteolytic cleavage and degradation.

Cell signaling is mediated by signaling proteins that relay the signal in an intricate network of interactions before the signal is translated into a biological response. Short linear motifs (SLiMs) in intrinsically disordered regions of proteins serve as docking sites for protein interaction in all aspects of cell regulation including signal transduction. SLiM-mediated interactions are transient and low affinity and can be hijacked by virus. Only a small fraction of SLiMs have been described, but many more exist. Platelet derived growth factor receptor β (PDGFRβ) belongs to the family of receptor tyrosine kinases (RTKs) and controls cell growth, proliferation and migration. Dysregulation of PDGFRβ mediated signaling pathways is seen in many cancer types. To discover and characterize protein interactions, large scale high through-put methods are needed in concert with low through-put methods, that can characterize the interaction in a cellular context. The aim of this thesis has been to study protein-protein interactions in internalization and signaling of PDGFRβ and motif-mediated host-virus interactions through the use of in situ proximity ligation assay (PLA).  

Signaling via PDGFRβ is compartmentalized and depends on receptor internalization. In paper I we investigated the effects of dynamin inhibition for activation and signaling of PDGFRβ, and found that dynamin inhibition leads to impaired dimerization of the PDGFRβ. The results indicate that membrane localization of PDGFRβ is affected by dynamin. 

In paper II we developed a new method, Molboolean, for localized simultaneous detection of both free protein and protein in complex in cells. Molboolean is based on the principles from PLA with a fluorescent read out detectable with fluorescence microscopy.       

In paper III we mapped SLiM based host-virus interactions and explored their mechanisms. Using proteomic peptide phage display, we identified 1712 potential virus-host interactions by screening a library covering intrinsically disordered regions of the proteome for 229 RNA viruses. Clathrin mediated endocytosis was found to be a common target for viral hijacking, and viral binding of clathrin impaired PDGFRβ internalization.       

Some RTKs are proteolytically cleaved following ligand activation. In paper IV we characterized the Ca2+-dependent proteolytic cleavage of PDGFRβ. The cleavage resulted in two PDGFRβ fragments and was dependent on receptor internalization. Inhibition of the proteasome with bortezomib prevented internalization and cleavage and resulted in increased activation of PLCγ and STAT3. 

This thesis provides insight in the regulation of PDGFRβ signaling and internalization, and highlights contributions of both large-scale screenings and low through-put methods for studying protein-protein interactions.