Evidence for extended YFP-EGFR dimers in the absence of ligand on the surface of living cells

The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. Structural studies have revealed two distinct conformations of the ectodomain of the EGFR: a compact, tethered, conformation and an untethered extended conformation. In the context of a monomer-dimer transition model, ligand binding is thought to untether the monomeric receptor leading to exposure of a dimerization arm which then facilitates receptor dimerization, kinase activation and signaling. For receptors directed orthogonal to the local plane of the membrane surface, this would lead to a large change in the distance of the receptor N-terminus from the membrane surface. To investigate this experimentally, we produced stable BaF/3 cell lines expressing a biochemically functional yellow fluorescent protein (YFP)-EGFR chimera and determined the vertical separation of the N-terminal YFP tag from the membrane using fluorescence resonance energy transfer (FRET) techniques. Homo-FRET/rFLIM was employed to determine the presence of unliganded dimers and to measure the average distance between the N-terminal tags in those dimers. The results suggest that EGF-induced activation occurs within or between pre-formed and extended dimers with very little change in the extension of the N-terminii from the membrane surface. These results provide constraints on possible models for EGFR activation.