Quantifying the Interaction between EGFR Dimers and Grb2 in Live Cells Nuala Del Piccolo, Kalina Hristova  Biophysical Journal  Volume 113, Issue 6, Pages 1353-1364 (September 2017) DOI: 10.1016/j.bpj.2017.06.029 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 An overview of the methodology used to quantify the interactions between membrane proteins and cytoplasmic proteins. (A) Representative images (insets) of a CHO cell under reversible osmotic stress and fluorescence emission spectra collected using the FSI technique. FSI yields approximation-free measurement of donor (EGFR-mTurquoise) concentration, acceptor (Grb2-YFP) concentration, and FRET efficiency in selected regions of a cell under reversible osmotic stress (see (37) for details). (B) Cartoon representation of the thermodynamic equilibrium reactions in the presence of saturating concentrations of the EGF ligand (right) and in the absence of ligand (left). The receptors are shown in blue, the ligand in green, the phosphorylation sites in orange, and the adaptor proteins in red. The adaptor protein Grb2 exists in a monomer-dimer equilibrium in the cytoplasm, where the dimerization constant ζ is known (23,51). EGFR kinase domains are believed to adopt either an active asymmetric dimer configuration that is phosphorylated and capable of interacting with Grb2, or an inactive symmetric configuration (64–66). In the presence of saturating amounts of ligand, we assume that all receptors exist as active dimers that are either free or bound to Grb2, and we determine the association constant, γ. In the absence of ligand, we assume that the receptors are found as monomers, inactive dimers, active dimers, and RTK dimer-adaptor monomer complexes. We determine the conformational parameter, β, describing the transition from the inactive dimer to the active dimer configuration. (C) Possible oligomer sizes and geometries for the assembly of an RTK dimer with monomeric adaptor proteins. The FRET efficiency, Ecomplex, in each complex of fluorophores is calculated as a function of the pairwise FRET efficiency, Ep, as described previously (74). To see this figure in color, go online. Biophysical Journal 2017 113, 1353-1364DOI: (10.1016/j.bpj.2017.06.029) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 FRET data for the interaction between EGFR and Grb2, under saturating ligand conditions. (A) A representative image of a CHO cell under reversible osmotic stress, in the presence of EGF. Fluorescence in the donor excitation image indicates the location of EGFR; fluorescence in the acceptor excitation image denotes Grb2. (B) In the left-hand plot, FRET efficiency in the plasma membrane is shown as a function of Grb2 (acceptor) concentration in the cytoplasm. The right-hand plot shows EGFR (donor) concentration in the membrane versus Grb2 (acceptor) concentration in the cytoplasm. (C) The MSE for fits of Eq. 20 to the experimental data when the number of monomeric adaptor proteins, n, is varied between 0 and 4. The model with one Grb2 monomer per EGFR dimer gives the best fit. (D) The theoretical and the experimental concentrations of the RTK dimer-adaptor monomer complex, [Rtot∗Amon,n], are calculated using Eqs. 18 and 19, respectively. The best-fit model (yellow surface) and experimental data (green diamonds) are plotted as a function of both total receptor concentration, [Rtot], and adaptor protein concentration, [Atot], on linear (left) and semi-log (right) scales. (E) The fraction of receptors found in active dimers with no adaptors bound (red line) and in RTK dimer-adaptor monomer complexes (yellow line) for the best-fit model as a function of total receptor concentration (left) and adaptor protein concentration (right). To see this figure in color, go online. Biophysical Journal 2017 113, 1353-1364DOI: (10.1016/j.bpj.2017.06.029) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 FRET data for the interaction between EGFR and Grb2 in the absence of ligand. (A) A representative image of a CHO cell under reversible osmotic stress, in the absence of EGF. Fluorescence in the donor excitation image indicates the location of EGFR; fluorescence in the acceptor excitation image shows Grb2. (B) In the left-hand plot, FRET efficiency in a plasma membrane region is shown as a function of Grb2 (acceptor) concentration in the cytoplasm. The right-hand plot shows EGFR (donor) concentration in the membrane versus Grb2 (acceptor) concentration in the cytoplasm. (C) We fit Eq. 23 to the experimental data to determine the parameter, β, describing the conformational switch from inactive symmetric RTK dimers to active asymmetric RTK dimers (see Fig. 1 B). The theoretical and experimental RTK dimer-adaptor monomer complex concentrations are calculated using Eqs. 23 and 19, respectively. The best fit model (yellow surface) is plotted alongside experimental data (green diamonds) as a function of both total receptor concentration, [Rtot], and adaptor protein concentration, [Atot], on both a linear (left) and semi-log (right) scale. (D) The fractions of receptors found as monomers, in dimers in an inactive configuration, dimers in an active configuration, and active RTK dimer-adaptor monomer complexes are graphed as functions of total receptor concentration (left) and adaptor protein concentration (right). To see this figure in color, go online. Biophysical Journal 2017 113, 1353-1364DOI: (10.1016/j.bpj.2017.06.029) Copyright © 2017 Biophysical Society Terms and Conditions