FRETting about your receptors

Abstract

Signaling through the epidermal growth factor (EGF) pathway plays an important role in many cellular and developmental processes. Activation of the ErbB1 receptor by EGF is traditionally thought to occur by ligand binding, which promotes receptor dimerization, allowing cross-phosphorylation of the cytoplasmic domains of the receptor. The activated receptors then recruit signal effectors such as SHC and Grb2, triggering the signalling cascade. Verveer et al.1xQuantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane. Verveer, P et al. Science. 2000; 290: 1567–1570CrossRef | PubMedSee all References1 now show that this might be an oversimplification.Using a series of microscopy techniques, the authors show that localized stimulation of a cell leads to receptor activation that rapidly spreads across the whole cell. Using fluorescence lifetime imaging microscopy (FLIM), they measured fluorescence resonance energy transfer (FRET) between green-fluorescent protein (GFP)-tagged ErbB1 receptor and an injected Cy3-labeled phosphospecific antibody that only binds to the receptor in its active state. This technique shows which receptors are active in real time in live cells 2xFluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Bastiaens, P et al. Trends Cell Biol. 1999; 9: 48–51Abstract | Full Text | Full Text PDF | PubMed | Scopus (351)See all References2.Using this system, the authors show that, when cells are stimulated locally using EGF-coated beads, ErbB1 receptors become activated and that this activation spreads rapidly across the plasma membrane. By photobleaching the region around the beads, they showed that the receptors bound to the ligand (and beads) are unable to move, suggesting strong binding of the ligand to its receptor. Furthermore, using a mixture of unlabelled receptor and labelled kinase-dead receptor, which can be activated but cannot activate other receptors, they show that a small portion of functional receptors could activate a much larger fraction of the kinase-dead receptors than would be expected if simple dimerization occurred. They also demonstrate that this lateral activation does not depend on cytoplasmic proteins and occurs more rapidly in the presence of a protein phosphatase inhibitor. This implies that a certain threshold of receptor activation is needed to overcome the basal phosphatase activity and that high localized stimulation – e.g. by contact with a neighbouring cell – can produce activation across the whole cell.This model also suggests that EGF is only required for the initial local activation and is not required for propagation of the signal across the rest of the cell. These results could lead to potential new drug targets to modulate ErbB signalling in cancer. The methods used here could also easily be applied to other signalling pathways.