Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

ABSTRACT: Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transient residence in the plasma membrane, Rhodamine-conjugated EGF (EGF-Rh) and Grb2-YFP were rapidly internalized and accumulated in endosomes. Quantitative image analysis revealed that on average two Grb2-YFP molecules were colocalized with one EGF-Rh in cells stimulated with 2 ng/ml EGF-Rh, and the e

SUBMITTER: Fortian A 

PROVIDER: S-EPMC3889400 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature