Understanding the requirements of DDR1 clustering in DDR1 activation
File(s)
Author(s)
Feng, Yuxuan
Type
Thesis
Abstract
Discoidin domain receptors (DDRs), DDR1 and DDR2, are unique receptor tyrosine kinases that perform essential roles in both physiological and pathological conditions. They are constitutive dimers on the cell surface and respond to collagen binding, leading to the activation of the kinase and several downstream signalling pathways. Previous biochemical and cellular studies have shown that collagen-induced DDR1 clustering is essential for DDR1 activation, and that the transmembrane domain (TMD) plays a crucial role in mediating DDR1 dimerization and autophosphorylation. However, the detailed molecular mechanisms remain unclear. Here, a variety of studies were conducted to bring further insights into the DDR1 dimerization and activation mechanisms. The activation of DDR1 was enforced by a ligand-independent optical approach using Cryptochrome Circadian Regulator 2 (Cry2), and ligand-induced DDR1 activation was modulated by co-expression with transmembrane peptides in a second approach. Furthermore, the TMD interaction interfaces were analyzed by disulphide cross-linking studies via cysteine scanning mutagenesis, and the dimerization motifs within the DDR1 TMD were validated using a series of mutagenesis studies and a Venus fluorescent protein-based bimolecular fluorescence complementation (BiFC) assay. This project demonstrates that collagen-induced DDR1 activation can be boosted by co-expression with one of the transmembrane peptides (DDR1-TMD) generated in this project, demonstrating the ability of the TMD to regulate DDR1 activation. Although no ligand-specific TMD dimerization motifs were revealed by the presented experiments, a strong cross-linking within the N-terminal part of DDR1 TMD was shown to constitutively exist and contribute to the DDR1 intrinsic interactions. Overall, this project provides further understanding and approaches for future investigations in studying DDR1 dimerization and collagen-induced receptor activation.
Version
Open Access
Date Issued
2024-05-10
Date Awarded
01/11/2024
License URL
Advisor
Leitinger, Birgit
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)