Biogenesis of the multivesicular body (MVB) organelle is an important process for regulation of signalling in the cell. Signal receptors embedded within the outer MVB membrane can be sorted into intralumenal vesicles which bud away from the cytosol to within the MVB preventing further signalling. Sorting of receptors, invagination of the membrane and release of vesicles into the MVB lumen are mediated by the endosomal sorting complexes required for transport (ESCRT) along with a range of accessory proteins including histidine domain protein tyrosine phosphatase (HD-PTP). HD-PTP is a multidomain protein which makes several interactions with ESCRT partners, including ESCRT-0, ESCRT-I and ESCRT-III. This thesis focusses specifically on the interaction between HD-PTP CC domain and Ubap1 (ESCRT-I), and the two interactions of HD-PTP Bro and PRR domains with STAM2 (ESCRT-0) SH3 and Core domains. To address the structure of HD-PTP, multiple techniques were used: X-ray crystallography, which gives high resolution structural information; small angle X-ray scattering (SAXS), which gives low resolution data for large non-crystallisable units in their solution state; and double electron-electron resonance (DEER) spectroscopy, which gives high resolution nanometre-range distance constraints between cysteines labelled with methanethiosulfonate spin label (MTSL). It was shown by X-ray crystallography that HD-PTP has an elongated CC domain, in stark contrast to its homologues ALIX and Bro1 which both have V-shaped CC domains. The CC domain showed limited flexibility both by SAXS and DEER. Further investigation showed that there was no significant conformational change upon binding its ESCRT-I partner Ubap1. The multidomain structure of HD-PTP Bro1-CC-PRR was described by SAXS, showing that these domains form an extended arrangement in solution. In addition, SAXS was also used to analyse the structure of these domains in complex with STAM2 (ESCRT-0), which showed that STAM2 is simultaneously tethered by the Bro1 domain and PRR. The Bro-CC-PRR portion of HD-PTP, has 9 cysteines, so with the aim of measuring local structural information in the CC domain alone, alternative spin labelling methods were investigated. Use of a bromoacrylaldehyde spin label (BASL), instead of MTSL, allowed more selective labelling of surface exposed cysteines, and avoided labelling most of the cysteines in the Bro1 domain. This novel method allowed the shape of the CC domain to be monitored during STAM2 binding and showed that there is no induced conformational change.

Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth. We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a decreased level of PDGFR ubiquitination (paper II). Consequently, internalized PDGFR could not be sorted properly and escaped degradation. This resulted in enhanced activation of phospholipase C γ (PLCγ) and changed kinetics of signal transducer and activator of transcription (STAT) 3 signalling, which further increased colony formation of HD-PTP silenced cells in soft agar, indicating a tumor suppressor role of HD-PTP. Activation of PDGFR leads to stimulation of downstream pathways. We identified Fer kinase as a critical signal transducer downstream of PDGFR in a proteomic screen. We showed that Fer kinase is essential for PDGF-induced STAT3 activation; as a result (paper III), Fer depletion severely blunted the ability of PDGFR signalling to promote anchorage-independent growth in soft agar and delayed tumor initiation in a mouse model. The crosstalk between host and tumor plays a critical role in tumor progression. At present most anti-cancer drugs are targeting tumor cells; we were interested in how targeting tumor host cells affects the efficacy of anti-tumor therapy. We found that selective PDGFRβ inhibition in host cells exerted tumor inhibitory effects on growth and vascularization of tumors with autocrine PDGF signaling, whereas tumors lacking such stimulation show only minor response on tumor growth (paper IV). Meanwhile, we demonstrated that PDGF/PDGFRβ signalling promotes expression of NG2, a marker for pericytes.

Platelet-derived growth factor (PDGF) isoforms, which bind to closely related a- and b-tyrosine kinase receptors, induce migration, proliferation, survival and differentiation of mesenchymal cells. They signal by the active receptor attracting Src homology 2 (SH2) domain containing proteins, which subsequently initiate a set of signaling pathways. The aim of this thesis was to elucidate regulatory mechanisms involved in PDGFRb signaling. In the first two projects we investigated the roles in downregulation of PDGFRb of two related adaptor proteins, i.e. ALG-2 interacting protein X (Alix) and His-domain containing protein tyrosine phosphatase (HD-PTP) functions of. We found that Alix and HD-PTP influence ubiquitination of PDGFRb following PDGF stimulation, by affecting the E3 ligase c-Cbl. Alix enhances complex formation between c-Cbl and PDGFRb, increases c-Cbl phosphorylation and decreases its stability. Interestingly, while both HD-PTP and Alix participate in degradation of PDGFRb, only Alix affects receptor internalization. Moreover, we demonstrated that absence of HD-PTP promotes cell proliferation. In conclusion, we suggest that both Alix and HD-PTP are important adaptor proteins in regulation of PDGFRb downregulation, although the observed differences between their actions suggest that Alix and HD-PTP exert their functions via different mechanisms. The third study explored the importance of tyrosine residue 857 in the activation loop of PDGFRb. We report that, in vitro the tyrosine residue 857 to phenylalanine (Y857F) mutant receptor kinase activity is diminished while in vivo it does not affect the phosphorylation of PDGFRb. The phosphorylation pattern of PDGFRb revealed that most sites in the Y857F mutant receptor were phosphorylated similarly as in the wild-type receptor. However, tyrosine residue 771 was found to be hyperphosphorylated in the Y857F mutant receptor. This may be due to defective phosphorylation and activation of SHP-2, since it has been shown to dephosphorylate the receptor at Y771. In addition, activation of the Erk1/2 and Akt pathways was defective downstream of the Y857F mutant receptor. Interestingly, the Y857F mutant receptor was able to mediate cell migration, but not proliferation. The last study investigated a role of the tyrosine kinase Fer in PDGF signaling. We showed that Fer interacted with and was activated by PDGFRb in a ligand-dependent manner. In cells depleted of Fer, receptor phosphorylation was decreased and phosphorylation of Stat3 was abolished, whereas Stat5, Erk1/2 and Akt were activated normally. Colony formation in soft agar was abolished in cells depleted of Fer, but no effect was seen on cell proliferation and migration. Since Stat3 has been shown to be involved in transformation, we speculate that phosphorylation of Stat3 in Fer-depleted cells, affects the ability of cells to form colonies.