Ligand-activated signal transduction is a process critical to cell survival and function as it serves as a means of communication between the cells and their environment. Endocytosis is generally thought to down-regulate incoming signals by reducing the surface availability of receptors. However, increasing evidence in many systems suggests a notion which is referred to as the „signalling endosome" hypothesis - that endocytosis can also actively contribute to signalling apart from clearance of activated receptors and thereby attenuation of signalling. The functional aspect of signalling endosomes has been well-characterized in several pathways including RTK and TGF-β signalling. There are, however, various other signalling pathways where the active mechanism of endocytotic regulation is yet to be understood. In this study, we probe this aspect in the cytokine signalling system, where the receptors are known to internalize but the significance of such internalization and precise mechanism is unclear. My thesis aims to elucidate the function and molecular details of internalization of cytokine receptor using interleukin-4 receptor (IL-4R) signalling as a model. IL-4 and IL-13 ligands can induce assembly of three distinct complexes: IL4 induced IL-4Rα – IL-2Rγ (type I), IL-4 induced IL-4Rα – IL-13Rα1 (type II) or the IL-13 induced IL-13Rα1-IL-4Rα (type II). The formation of any of these complexes triggers signalling through the JAK/STAT pathway. However, models of how the oligomerization of the transmembrane receptors and activation takes place are very diverse and lack a clear molecular and biophysical understanding of the underlying receptor dynamics. Previous results of the lab had shown that the affinities between subunits are low, precluding complex formation at the plasma membrane at physiological concentrations. In addition, IL-4R subunits localize in to endosomal structures adjacent to the plasma membrane. It had already been shown that the shared IL-4R subunit IL-2Rγ is internalized by a specific, actin dependent, Rac1/Pak1 regulated endocytosis route in the IL-2 context. We could show that pharmacological suppression of this endocytosis pathway also prevented IL-4 induced JAK/STAT signalling, placing endocytosis upstream of signalling. Here I show using immuno-EM techniques that these endosomal structures are multivesicular bodies. Importantly, I could show that receptor subunits are highly enriched in the limiting membrane of these endosomes relative to the adjacent plasma membrane. Using quantitative loading assays I could furthermore demonstrate that this enrichment is achieved by constitutive internalization of receptors from the cell surface into cortical endosomes. The trafficking kinetics of the receptor subunits is independent of ligand occupancy. Pharmacological inhibition shows that receptors and ligand traffic via the previously identified Rac1/Pak1 pathway. Finally, Vav2 was identified as a candidate Guanine Exchange Factor (GEF) that may regulate Rac1 activity and thereby control the actin polymerization cascade driving IL-4R endocytosis. Immunoprecipitations showed that Vav2 interacts both with the cytoplasmic tail region of the receptors and the receptor associated 2 kinase JAK3. Vav2 may thus couple the receptor/JAK complexes to the Rac1/Pak1 mediated endocytosis route. Taken together, our results suggests that stable „signalling endosomes‟ adjacent to the plasma membrane act as enrichment centres, where ligand and receptor concentrations are locally increased by constitutive trafficking. The confined environment of the endosome then compensates for the weak affinities between the ligand and receptor and facilitates ligand-mediated receptor dimerization. Importantly, overexpression of both type II IL-4R subunits renders signal transduction resistant to endocytosis inhibition, strongly suggesting that the critical factor effecting signalling is sufficient concentration, which the endosomes facilitate achieving. The endosomes are thus dispensable as signalling scaffolds when the receptors are in sufficient concentration, where activated receptors could interact with downstream pathway components. Endocytosis thus provides a crucial means for the signalling process to overcome the thermodynamic hurdles for receptor oligomerization. In conclusion, our data propose a novel, purely thermodynamic role of endosomes in regulating cytokine receptor signalling not seen in any other signalling pathway.

Neuropilin-2 (NRP2) is a single transmembrane receptor and was first found in the nervous system to play a role in axon guidance. Interestingly, NRP2 was also found on many tumor cells and various studies showed that NRP2 is associated with a poor prognosis in different cancers and is involved in migration and therapy resistance. We investigated the prognostic potential of NRP2 in the pancreatic ductal adenocarcinoma (PDAC) and found out that in contrast to other kinds of cancer a high expression of NRP2 is associated with a longer cancer specific survival. We hypothesized that this effect could be either triggered through an expression of different interaction partners of NRP2. Both semaphorine 3F and VEGFs can bind to NRP2 but have different effects on cancer cells. Semaphorine 3F was found to have a great potential as a cancer inhibitor in pancreatic cancer whereas VEGFs are often associated with a worse prognosis. Both compete for the binding to NRP2. Furthermore, we found high expression of NRP2 in tumor-associated macrophages (TAMs) in PDACs. Until now, NRP2 expression and function is poorly analyzed in the immune system. Therefore, we next focused on the investigation of NRP2 in the immune system during cancer progression. We used LysM:cre-NRP2LoxP/LoxP (conditional knock-out of NRP2 in macrophages) and Vav:cre-NRP2LoxP/LoxP (conditional knock-out in all immune cells) for our experiments. We showed that NRP2 is upregulated during the differentiation/maturation of macrophages. Next, we injected LLC cells subcutaneously to analyze the effect of NRP2 knock-out in macrophages (LysM:cre) or in the all immune cells (Vav:cre). No difference was detected in tumor size, but the vascularization was impaired in both mouse models. Different tumor models with extended tumor growth times and metastasis should be performed next to proof the importance of NRP2 in immune cells during tumor progression. Due to the broad expression of NRP2 in the immune system we used the Vav:cre-NRP2LoxP/LoxP mouse to investigate the role of NRP2 during an immune response. We used a mild allergic inflammation model of the lung and analyzed the different immune cell populations. Interestingly, T cells and eosinophils were reduced during the inflammation indicating, that the conditional knock-out of NRP2 is inhibiting the immune response. We further analyzed the role of NRP2 in T cells and found out, that the expression of NRP2 is very different in the various T cell populations. CD8+ T cells express ca. 10 times as much mRNA for NRP2 compared to CD4+ T cells. Also, the CD4 subpopulation showed a diverse expression of NRP2. Th2 and Th17 express a lot of NRP2 and Treg and Th1 very low levels. These results suggest an important role of NRP2 in certain cells. The knock-out of NRP2 in Th2 cells leads to an upregulation of IL-13, IL-5 and IL10. We first showed the importance of NRP2 during an immune response and found interesting regulations in immune cell populations and important cytokines. More work needs to be done to understand the functions of NRP2 during an immune response.

Eph receptors are known to be overexpressed in various types of cancer and are therefore promising targets for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of Eph-overexpressing tumors, monitoring responses to therapy directed toward Eph, and thus improvement in patient outcomes. This work report the synthesis and evaluation of several fluorine-18-labeled peptides containing the SNEW and SWLAY amino acid motif, with high affinity for the EphA2 and B2 receptor, for their potential as radiotracers in the non-invasive imaging of cancer using PET. For the purposes of radiofluorination, EphA2- and EphB2-antagonistic peptides were varied at the C-terminus by the introduction of L-cysteine, and further by alkyne- or azide-modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [18F]AFP and [18F]BFP were applied, and their capacity to introduce fluorine-18 was compared with that of the established building block [18F]FBAM. Copper-assisted Huisgen 1,3-dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide- or alkyne-modified peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.