Single-molecule analysis of epidermal growth factor binding on the surface of living cells

Abstract

Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediately with a less than 1% occupancy among tens of thousands of EGFR molecules on single cell surface. Activation of EGFR requires the formation of a signaling dimer of EGFR bound with a single ligand to each molecule. How sufficient numbers of signaling dimers are formed at such low occupancy rate is still not known. Here, we have analyzed the kinetics of EGF binding and the formation of the signaling dimer using single-molecule imaging and mathematical modeling. A small number of EGFR on the cell surface formed dimeric binding sites, which bound EGF two orders of magnitude faster than the monomeric binding sites. There was a positive cooperative binding of EGF to the dimeric binding sites through a newly discovered kinetic intermediate. These two mechanisms facilitate the formation of signaling dimers of EGF/EGFR complexes.

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

“…1). In this sense, the HRG receptors behaved similarly to ErbB1 (19). The association rate constant for the interaction between the ligand and the receptor monomers (k 1 ) was 0.004 (EGF) or 0.002ðHRGÞ nM −1 s −1 .…”

“…Using an oblique illumination fluorescence microscope (19) (Fig. 1A and Movie S1), we observed the associations between TMR-HRG and its receptors on the apical surfaces of MCF-7 cells in culture as single molecules.…”

“…Single-molecule imaging (17,18) is a suitable technique for addressing this question because the observation of individual ligand molecules on the cell surface allows the simultaneous detection of the ligandreceptor interactions and the dimerization of the liganded receptor molecules. Using this technique, we studied the association between EGF and ErbB1 in HeLa cells (19). The results suggest that a small fraction of ErbB1 molecules form predimers, which rapidly associate with EGF in contrast to the slower rate of association observed for the monomers.…”

“…The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al (2006) EMBO J 25:4215-4222], suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow.…”

Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis

“…Measurements of the distributions and temporal fluctuations in the duration of individual molecular recognition processes using the single-molecule technique have revealed the static and dynamic disorders (20)(21)(22)(23) and memory landscapes (24,25) of enzymatic reactions, as well as dynamic polymorphisms of protein structures (26)(27)(28). In living cells, single-molecule measurements have even been used to clarify spatially inhomogeneous kinetics in receptor-ligand (29) and protein-protein (30) dissociations, and to identify previously unknown reaction intermediates (31).…”