Live cell measurements of interaction forces and binding kinetics between Discoidin Domain Receptor 1 (DDR1) and collagen I with atomic force microscopy

Abstract

BackgroundDiscoidin Domain Receptors (DDRs) are membrane-tethered proteins of the receptor tyrosine kinase family, which signal in response to collagen. DDR expression is associated with human diseases, including fibrosis and cancer. The role of DDRs in human pathogenesis is mediated by dysregulated receptor function in response to the collagenous milieu. Thus, understanding DDR-collagen interactions is important for developing novel therapeutic strategies against DDRs. MethodsWe developed a biophysical method to isolate and measure specific interactions between DDR1 and collagen in live cells at the single molecule level using atomic force microscopy. This new method is capable of providing density and kinetics of membrane receptors in live cells. ResultsWe isolated DDR1-collagen interactions and quantified the association and dissociation rates of the DDR1-collagen I complex. We estimated separate binding probabilities of collagen I to DDR and integrin, and by combining kinetic and binding probability data, we were able to estimate the density of receptors in two cancer cell types. We also tested the viability of a DDR1 blocking antibody and determined its efficacy in suppressing DDR1-collagen binding. ConclusionsThe new method shows promise in quantifying receptor-ligand kinetics and receptor density on live cells. General significanceThe new approach is applicable to other receptor-ligand systems and allows the determination of critical parameters at the single cell/single molecule level – in particular, the direct determination of kinetic and density differences of receptors in different cell types. This capability should prove to be useful in cancer research and drug design.