Analysis of DDR1 function at epithelial cell contacts

Abstract

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase (RTK) family, and binds to collagen in the extracellular matrix (ECM). It therefore plays an important role in relaying information from outside the cell to intracellular components. Accordingly, DDR1 contributes to many cellular processes including migration and differentiation amongst others. In malignant states, cell-matrix interactions are often deregulated, resulting in the pro-invasive phenotype characteristic of tumours. Increased DDR1 expression is a negative prognostic marker for many cancers, however the molecular mechanisms are not fully understood. Interestingly, novel ligand-independent roles of DDR1 have recently emerged that potentially implicate the receptor at epithelial cell contacts. In this thesis, I show that during new keratinocyte contact formation, DDR1 is recruited after E-cadherin. In contrast to previous literature, DDR1 does not form a complex with E-cadherin, and distinct separate clusters of DDR1 and E-cadherin are observed at mature cell contacts. DDR1 depletion decreases the junctional E-cadherin and actin levels during cell contact formation. This phenotype is independent of actin recruitment to clustered E-cadherin receptors. Actin thin bundles are also visibly disrupted during contact formation with DDR1 depletion, which is further linked to a reduction in Rho-ROCK signalling and actomyosin contractility. Not only are the levels of phosphorylated myosin light chain and myosin phosphatase reduced, but ROCK1 levels are also reduced by DDR1 knockdown, suggesting that DDR1 has a regulatory role upstream of ROCK1. Preliminary experiments demonstrate potential binding between DDR1 and some members of the catenin protein family, however the significance of these interactions requires further investigation. Data collected from keratinocytes and a series of lung cancer cell lines, suggest that E-cadherin-mediated cell contacts inhibit collagen-mediated DDR1 activation, possibly by preventing DDR1 ligand accessibility. Overall, my results suggest that DDR1 stabilizes epithelial cell contacts through regulation of actomyosin contractility.