Investigating the activation mechanism of discoidin domain receptor 1 using advanced microscopy techniques

Abstract

The discoidin domain receptors (DDR1 and DDR2) are unusual receptor tyrosine kinases (RTKs) that function as collagen receptors and regulate cell migration, adhesion, and differentiation. Ligand binding to the extracellular region of RTKs results in kinase activity and autophosphorylation of tyrosine residues on the intracellular side. For most RTKs, the molecular mechanism of receptor activation involves ligand-induced dimerisation. However, the DDRs exist as constitutive dimers in the absence of collagen. In addition, it is thought ligand-induced conformational changes within the DDR dimer cannot be transferred to the intracellular side. The DDRs also activate very slowly, over hours rather than seconds to minutes as is typical for other RTKs. As such, the DDRs employ a unique activation mechanism that is currently poorly understood. In this thesis, I investigate the activation mechanism of DDR1 using various microscopy techniques. DDR1 was found to be distributed on the cell surface in distinct puncta that are dependent on specific transmembrane interactions. Collagen is known to bind to a binding trench on the extracellular discoidin domain of DDR1, and here the data show collagen binding requires another site, the conserved “signalling patch” residues, which may be a secondary collagen binding site or a site for DDR1-protein interactions essential for collagen binding. Within 5 minutes of collagen binding, DDR1 redistributes on the cell surface, and over 60 minutes of stimulation DDR1 increasingly aggregates. DDR1 phosphorylation correlates with the increasing aggregation. In addition, the aggregation results in DDR1 molecules moving within 10 nm of each other, and preliminary experiments suggest this occurs between DDR1 dimers. Ligand-independent aggregation of DDR1, through incubation with a pentameric IgM antibody, also results in DDR1 phosphorylation. This thesis supports an activation mechanism where collagen binding promotes DDR1 aggregation between DDR1 dimers, which may be required for phosphorylation to occur.