Novel insights into discoidin domain receptor 1 activation and regulation
File(s)
Author(s)
Juskaite, Victoria
Type
Thesis
Abstract
The discoidin domain receptors, DDR1 and DDR2, are intriguing receptor tyrosine kinases (RTKs) that signal in response to collagen with unusually delayed autophosphorylation kinetics. Both DDRs play essential roles in development and regulate cell adhesion, migration, invasion, proliferation, and survival. The DDRs are attractive drug targets for atherosclerosis, arthritis, organ fibrosis, and various forms of cancer, but how collagen binding translates to DDR activation is not well understood. Previous studies suggested that DDR1 activation occurs in intracellular compartments following collagen-induced endocytosis. To define this mechanism further, I used biochemical assays and immunofluorescence. Contrary to the proposed activation mechanism, collagen did not induce DDR1 internalisation, and ligand-induced DDR1 phosphorylation was limited to the plasma membrane, supporting a mechanism whereby DDR1 is activated on the cell surface. Activation of the majority of RTKs occurs by ligand-induced dimerisation. However, the DDRs are constitutive, non-covalent dimers, which led to the hypothesis that activation of DDRs arises by collagen-induced lateral association (clustering) of DDR dimers on the cell membrane. To investigate this mechanism, different types of signalling-incompetent DDR1 mutants (‘receiver’) were co-expressed with functional DDR1 (‘donor’), and phosphorylation of receiver DDR1 by donor DDR1 was monitored by SDS-PAGE and Western blotting, or by immunofluorescence. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor activation, I demonstrate that the donor phosphorylates receiver DDR1 dimers in trans in a process that requires kinase activity of the donor but not the receiver. Intriguingly, DDR1 phosphorylation in trans is not dependent on ectodomain contacts, specific intracellular regions, the fibrillar nature of collagen, or a particular mode of activation, but requires intact transmembrane domain interactions. Moreover, collagen induces redistribution of DDR1 into a more compact structure, and mutant DDR1 that cannot bind collagen is recruited to collagen when co-expressed with a functional receptor. In summary, this thesis uncovered a mechanism of DDR1 activation whereby collagen induces clustering of DDR1 dimers on the cell surface, which leads to phosphorylation between dimers.
Version
Open Access
Date Issued
2017-12
Date Awarded
2018-03
Advisor
Leitinger, Birgit
Sponsor
Medical Research Council (Great Britain)
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)