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  • Introduction The Discoidin Domain Receptors DDRs comprising

    2020-05-12

    Introduction The Discoidin Domain Receptors (DDRs), comprising DDR1 and DDR2, are collagen-binding receptor tyrosine kinases (RTKs) that function as microenvironmental sensors at the interface of the extracellular matrix and the intracellular signal transduction machinery [1]. In response to ligand engagement, the DDRs undergo receptor tyrosine phosphorylation and recruit SH2- and PTB-containing adaptor proteins which trigger downstream signalling [2]. The DDRs are typified by a unique delayed and sustained tyrosine phosphorylation signalling profile. In contrast to growth factor activated RTKs where receptor phosphorylation is normally initiated within seconds to minutes of ligand binding and subsequently undergoes signal degradation by negative feedback regulation, the DDRs are activated by collagen with a delayed timescale of minutes to hours and tyrosine phosphorylation signalling remains sustained for several hours to days [3,4]. The mechanistic basis of this distinctive phosphorylation signalling dynamics is unknown, although it has been suggested that receptor localisation may be important [5]. Unlike other RTK family members such the ErbB, c-Met or PDGFR receptors, the spatiotemporal localisation of the DDRs remains poorly characterised. A previous study employed Förster resonance energy transfer (FRET) imaging and fluorescence microscopy to determine the cellular location of DDR1 in HEK293 cells [6]. The authors of this study found that DDR1 is rapidly internalised within 5 min of collagen presentation. A very recent study utilized a combination of fluorescence microscopy and l lysine reviews to demonstrate that DDR1 undergoes ligand-induced clustering at the cell surface through lateral dimer association in COS-7 cells [7]. It is unknown if DDR2 displays a similar spatiotemporal profile as DDR1.
    Materials and methods
    Results
    Discussion In this study, we provide the first spatiotemporal characterisation of DDR2 localisation and tyrosine phosphorylation signalling. Our imaging data demonstrates that upon ligand presentation, a proportion of cellular DDR2 is localised to the cell surface. The localisation of DDR2 and cellular phosphorylated proteins is independent of the collagen-binding integrins and the presence of SHC1. Our experiments also find that tyrosine phosphorylated proteins are present at the interface where DDR2 is in contact with collagen and not in the early endosomes or lysosomes. Importantly we show that an intact collagen binding pocket is necessary for the spatiotemporal localisation of DDR2 at the cell surface, providing the first evidence that collagen binding is a key driver for receptor localisation. Since the collagen binding activity of DDR2 is independent of its kinase function [9], our data also led to the unexpected and novel finding that kinase activity is necessary for receptor localisation at the cell surface. In this study, we were able derive new insights into the biology of DDR2 including the demonstration that cellular tyrosine phosphorylated proteins co-localise with DDR2 and exogenously added collagen I (Fig. 1G). This finding suggests that similar to the focal adhesion complexes associated with integrin activation by collagen [21], DDR2 signalling is a spatially localised event occurring at discrete locations at the cell-surface interface where it is in close proximity with the collagen ligand. Taken together, our imaging data on DDR2 and the body of work on DDR1 trafficking supports the view that the DDRs are localised to the cell surface where these receptors bind to collagen and induce tyrosine phosphorylation [6,7,22,23]. By utilising immunofluorescence microscopy, this study reveals the spatial heterogeneity associated with DDR2 phosphorylation-mediated signalling and the functional components required for receptor localisation, ultimately uncovering new insights into DDR2 signalling in both space and time.
    Conflicts of interest
    Acknowledgments