Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single‐Molecule Diffusivity in the Membrane of Living Cells

Abstract

We present as ingle-molecule diffusional-mobilityshift assay(smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells.Wefound that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and badrenergic receptors,asdetermined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from af ew tens of kilodaltons to several hundred kilodaltons.This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants.s mDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.Onthe surface of ap lasma membrane,t he interactions of membrane proteins with ligands are crucial to the communication between the intra-and extracellular environments. [1] Va rious types of ligand-receptor interactions occur in the membrane of living cells,yet many of these interactions have not been elucidated.[2] Because the crowded and heterogeneous cellular membrane generates unique biochemical conditions for biomolecular reactions, [3] membrane-protein interactions should be investigated in the intact membrane of living cells if their genuine biochemical properties are to be understood.

Influence of the extracellular domain size on the dynamic behavior of membrane proteins

“…There have been several corrections to this model [29][30][31][32][33][34][35] ; however, they are mainly focused on the impact of lipid environment, TMD and ICD of the proteins on lateral diffusion, with exception of GPI-anchored proteins where some hints of influence of protein size on diffusion were observed, 36,37 . Most recently, the computational simulations 38 and experimental methods 39 have predicted that the diffusion of MPs might also be influenced by the ECDs.…”