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Biological processes take place through interactions between macromolecules, such as nucleic acids and proteins. It is, therefore, fundamental to understand the functions of proteins and how they form complexes in order to carry out their role. Importantly, including spatial information in the analyses of protein complexes allows us to account for cell or tissue heterogeneity, highlighting the importance of in situ studies in biologically and clinically relevant material.

To that end, methods for the analysis of protein complexes in situ have been developed, such as in situ proximity ligation assay (PLA) and proximity-dependent initiation of hybridisation chain reaction (proxHCR). Both methods depend on antibodies for target recognition and utilise oligonucleotide systems in order to generate reporter signals with fluorescence readout. While in situ PLA employs rolling circle amplification, proxHCR is an enzyme-free method that takes advantage of DNA hybridisation properties. Both methods, however, yield a polymerised reporter signal of protein complex formation.

To further the use of proxHCR, we optimised the design of the oligonucleotide system as well as the experimental procedure, so as to increase the robustness and versatility of the assay. In addition, we developed a novel method, MolBoolean, that simultaneously reports the levels of free proteins as well as protein complexes. In this way, we address limitations of earlier methods and provide the opportunity to obtain a more comprehensive picture of biological processes. Our methods provide a means to circumvent the resolution limits of light microscopy by utilising molecular tricks so that protein binding events, that occur below the resolving power of conventional instrumentation, are made visible.  

The methods presented in the present doctoral thesis provide powerful tools in the analysis of protein interactions and have applications in cell biology studies as well as in diagnostics. 

Part of this thesis was the examination of the function of 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) receptor, potentially linked to vitamin D₃. We investigated the expression and subcellular localisation of 1,25D₃-MARRS in an array of cell lines and employed siRNA-mediated depletion to examine effects on cellular processes in androgen-independent prostate cancer cell models. Our data suggest that 1,25D₃-MARRS supports cell proliferation and might have a role in cell migration. Additionally, we observe an effect on the regulation of intracellular vitamin D₃ levels. With this study, we contribute to the understanding of the role of 1,25D₃-MARRS in prostate cancer cells, that could potentially prove of value in the adaptation of therapeutic strategy for prostate carcinoma.

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.