Comparative Real-Time Kinetics of Ligand-Receptor Interactions Using Immobilization-Based Sensing Readouts

Interactions between receptor tyrosine kinases and their specific growth factor ligands are crucial for cell signaling. Although much research has focused on these processes, quantitative attention to their earliest kinetics has been limited. Here, we used two immobilization-based sensing methods, biolayer interferometry (BLI) and surface plasmon resonance (SPR), to analyze the real-time binding kinetics of three high-affinity ligands with the full extracellular domain of various epidermal growth factor receptor (EGFR) isoforms. We observed that BLI measurements show both fast and slow dissociation phases, regardless of the EGFR isoform. SPR experiments, despite using a different detection readout, confirm the existence of two binding substates, indicating that this is a common feature of ligand-EGFR interactions. Furthermore, our data systematically demonstrate that the type of immobilization-based sensing technique influences not only the magnitude of kinetic and affinity parameters but also the relative interactions among different ligands and EGFR isoforms. Using these methods, we also found that glycan side chains at position N151 within the canonical ligand-binding site do not affect overall interactions with the three common high-affinity growth factors. Finally, we show that an extensively deglycosylated EGFR isoform binds all tested ligands with significantly lower affinity. Our approach could be applied to other ligand-receptor systems to directly evaluate how specific posttranslational modifications impact their interactions.