FIG 4.

The vIVb deletion increases EGFR phosphorylation only when present in both the activator and receiver of the active asymmetric dimer. (A) Flow cytometry analysis of activation loop phosphorylation using cotransfected EGFR mutants. Pairs of EGFR constructs consisting of activator-impaired, mCherry-tagged EGFR, and receiver-impaired, Cerulean-tagged EGFR were cotransfected into HEK-293T cells. After EGF stimulation, cells were analyzed for mCherry, Cerulean, and FITC fluorescence, reflecting the expression levels of each construct and antiphosphotyrosine staining for Tyr 845. Data were binned according to mCherry and Cerulean intensity and represented as a two-dimensional histogram, with the color of each bin corresponding to the intensity of phosphotyrosine staining for cells within that bin. Phosphorylation at Tyr 845 is compared between EGFR pairs with none (wild type), one, or both dimer partners containing the vIVb deletion [Δ(vIVb), X symbol], as indicated. (B) Flow cytometry analysis of phospho-Erk1/2 (pErk) in cells expressing mutant EGFR asymmetric dimer pairs. Cells containing the pairs of constructs shown in panel A were treated with EGF or without EGF and stained for pErk. Each Δ(vIVb) combination (deletion in the activator-impaired construct, receiver-impaired construct, or both) is plotted separately and overlaid on the data for the intact-tail pair (wild type).