Platelet-derived growth factor receptors (α and β) are included in the family of receptor tyrosine kinases (RTKs). The dimeric PDGF ligands bind to the PDGF receptors and this results in receptor dimerization, autophosphorylation, and thus activation. The autophosphorylated receptor dimer activates numerous signaling pathways. The responses of the cell to the activation of these pathways include proliferation, migration and survival. After ligand stimulated activation, PDGF receptors are internalized mainly via clathrin-coated pits and subsequently degraded in the lysosomes. However, the proteasomes have also been reported to play a role in PDGFRβ degradation. The main aim of this project was to study the mechanism of ligand-stimulated cleavage of PDGFRβ and to explore its possible consequences. By using antibodies that recognize the extracellular and intracellular parts of the receptor, respectively, we showed that ligand stimulation of PDGFRβ leads to the formation of an extracellular (~130 kDa) and an intracellular (~70 kDa) fragment. These fragments were shown to be specific fragments of the receptor located at the plasma membrane and not newly synthesized receptor. The presence of calcium ions intracellularly was found to be essential for cleavage. Interestingly, we observed that with the use of the proteasomal inhibitor called bortezomib, receptor cleavage was inhibited. On the other hand, lysosomal inhibition did not inhibit PDGFRβ fragmentation. Subsequently, we showed that cleavage occurs after the receptor has been endocytosed. Then, we examined whether blocking PDGFRβ cleavage had any effect on the downstream signaling of the receptor. We revealed an increase in receptor, PLCγ and STAT3 phosphorylation and a decrease in Erk phosphorylation upon bortezomib treatment. Moreover, we observed that PDGFRβ undergoes another type of cleavage upon ligand stimulation in fibroblasts, known as ectodomain shedding. Finally, we showed that receptor internalization is decreased upon bortezomib treatment, suggesting that bortezomib blocks receptor cleavage by inhibiting its internalization. The cleavage site, responsible enzyme, as well as potential functional consequences of ligand-stimulated PDGFRβ cleavage, remain to be determined.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-502607 |
| Date | January 2023 |
| Creators | Tsiatsiou, Agni Karolina |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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