Single Quantum Dot Tracking Coupled to a Three-State HMM Provides New Mechanistic Insight Into erbB1 Homodimerization

Abstract

The erbB family of trans-membrane receptor tyrosine kinases serves as the prototypical model for dimerization-induced signal transduction. The current paradigm for signal initiation by erbB1 homo-dimers is based on ligand-induced conformational changes, which expose the dimerization arms of activated receptors. To track erbB1 dynamics at the molecular level, we used two-color single quantum dot (QD) tracking to determine diffusion and dimerization characteristics of resting, ligand-bound and kinase-inhibited receptors. Long-lived erbB1 homo-dimers were directly visualized between EGF-QD-bound receptors, characterized by a 50 nm separation. However, we also observed dimer interactions whose close approach was punctuated by periods of excursion up to hundreds of nanometers apart, suggesting a third domain-confined receptor state. For each condition, transition rates between free, domain-confined, and dimer states were extracted using a modified three-state Hidden Markov Model (HMM). This analytical model uses separation of pairwise QD trajectories to determine probabilities of state transitions, whose rates are fit over many candidate interactions. Diffusional behavior was determined by state and showed slowing upon dimer formation; EGF-bound monomeric receptors confined to the same domain showed two-fold slower diffusion than free receptors and showed a further three and a half-fold slowing upon dimerization. Resting or kinase inhibited receptors did not show this dramatic mobility change. While the small molecule tyrosine kinase inhibitor, PD153035 that blocks receptor phosphorylation altered receptor diffusion, it did not change the dimer off rate. Resting erbB1 dimers tracked via monovalent heavy-chain only antibody fragments (VhH) did not demonstrate correlated motion and had a >6-fold higher off-rate. Our studies provide new mechanistic insight into erbB1 dimerization, demonstrate a role for membrane domains in promoting protein-protein interactions, and suggest a reduction in receptor mobility as a feature of signaling competent erbB1 dimers.