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Rahul Das        Das Stamp

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

Nicholas F. Endres*, Rahul Das*, Adam W. Smith*, Anton Arkhipov, Erika Kovacs, Yongjian Huang, Jeffrey G. Pelton, Yibing Shan, David E. Shaw, David E. Wemmer, Jay T. Groves and John Kuriyan

* These authors contributed equally

Cell. 2013 Jan 31;152(3):543-56. (local copy)

Abstract / Figures from the paper / Supplemental Material / Coordinates / References


This paper is part of two coupled papers in Cell illustrating the importance of conformational coupling across the membrane in activation of EGFR. In this paper we present fluorescence microscopy data that suggests that the intracellular domain is inhibited in the unliganded receptor by interactions with the plasma membrane. NMR and other data suggest that conformational coupling between the transmembrane and juxtamembrane segments is required for relief of this inhibition upon receptor activation by ligand binding. In the companion paper, Arkhipov et al share observations from long time scale molecular dynamics of EGFR in lipid bilayers. These simulations support the conclusions of our experimental data, and also provide insights into why ligand binding to the extracellular module of EGFR is required for activation. These two papers are part of a series of papers from the lab elucidating the mechanism of activation of EGFR family receptors (Zhang et al 2006, Jura et al, 2009a, Jura et al, 2009b, Endres et al, 2011).


How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Figures from the paper.

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First figure from paper

Figure 1. Model for EGFR Activation and Domain Architecture (A) Model for monomer-dimer equilibrium of EGFR in the absence and presence of EGF (Yarden and Schlessinger, 1987).
(B) EGFR constructs used in this study.
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Second figure from paper

Figure 2. Surface Density Dependence of EGFR Activation (A) Fluorescence microscopy images of EGFR fused to mCherry expressed in Cos-7 cells (left, red) compared to phosphorylation level of EGFR at Tyr 1068 (middle panels, green, merged with expression in right panels). In bottom panels, cells were treated with EGF for 3 min at 37C prior to fixation (Experimental Procedures). (B) Relationship between EGFR surface density and phosphorylation level. In the left panel, individual data points represent the mean surface density and the mean phosphorylation level for selected cells with comparable surface density (within 100 molecules per mm2 of the mean value). Trend lines were calculated using linear and second-order polynomial fits for EGFR with and without ligand, respectively. In the right panel, bars represent the mean ratio of phosphorylation level to surface density for all cells within equal ranges of surface densities (value on x axis ±250 molecules per mm2). In these diagrams, as well as all subsequent ones, error bars represent SEM. (C) Surface density-dependent phosphorylation for a construct with extracellular domain deleted (TM-ICM) compared to EGFR with or without EGF. (D) Surface density-dependent phosphorylation levels for a construct with a flexible linker inserted between the extracellular module and the transmembrane (ECM-GlySer-TMICM) compared to EGFR. Error bars represent SEM. See also Figure S2.
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Third figure from paper

Figure 3. Activity of the Intracellular Module Localized to the Plasma Membrane with the c-Src Motif (A) Surface density dependence of phosphorylation for Myr-ICM compared to EGF-treated EGFR and Myr-GCN4-ICM. (B) Confocal fluorescence microscopy images of cells expressing EGFR (top) or the intracellular module fused to a c-Src membrane localization motif (Myr-ICM, bottom), showing expression levels (left, red) and antibody detection of phosphorylation at Tyr 1068 (middle panels, green, merged with expression in right panels). Large panels are images in the x-y plane of the basal surface of the cells (closest to the coverslip). Smaller panels are projections in the x-z plane, orthogonal to the basal surface, at the y coordinate indicated by the white arrow. Note that, whereas expression of Myr-ICM is higher than EGFR, its phosphorylation level is significantly lower. (C) Schematic model for docking of the EGFR kinase domain against the plasma membrane based on molecular dynamics simulations of unliganded EGFR in lipid bilayers (Arkhipov et al., 2013). In the kinase domain, positively charged residues that interact with negatively charged lipids during the simulations are labeled and shown as blue dots. The LRRLL motif in the JM-A segment is shown in stick form, with leucines in green and arginines in blue. (D) Surface density dependence of phosphorylation for charge-reversal mutations in the N-lobe interaction region of the intracellular domain (Myr-ICM K713E/ K715E), compared to Myr-ICM and EGF-treated EGFR. (E) Surface density dependence of phosphorylation for another set of charge-reversal mutations in the N-lobe (Myr-ICM K689E/K692E), compared to Myr-ICM and EGF-treated EGFR. The data for EGFR and Myr-ICM are the same as in Figure 3D because samples were prepared on the same day. (F) Surface density dependence of phosphorylation for ECM-TM-GlySer-ICM and EGFR in the absence and presence of EGF. Error bars represent SEM. See also Figures S1 and S3.
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Fourth figure from paper

Figure 4. FCCS Data for EGFR Constructs on the Plasma Membrane (A) Schematic of laser excitation and fluorescence detection for two-color PIE-FCCS (left). Pulse diagram (right) showing excitation pulses (top, with GFP in blue and mCherry in green) and emission (bottom, with GFP in green and mCherry in red) is shown. Note that time gating allows us to eliminate mCherry emission when GFP is excited. (B) Relative cross-correlation values for various EGFR constructs. Myr-FP is a coexpression of GFP and mCherry, each fused separately to the c-Src membrane localization motif. Data are represented as a scatterplot, with the red line representing the median value. Surface densities of EGFR constructs ranged from 100–1,000 molecules per mm2. See also Figure S4.
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Fifth figure from paper

Figure 5. NMR Structure of Transmembrane-Juxtamembrane Dimer in Bicelles and Role of N-Terminal Dimer Interface in Receptor Activation (A) Structural model of the transmembrane-juxtamembrane segment of EGFR in DMPC/DHPC bicelles as determined from NMR data. Intermolecular NOESY connectivities are shown with gray lines. Dimer interfaces are expanded in the right panels. (B) Expanded view of the transmembrane dimer interface with residues in the small-small-x-x-small-small motif highlighted. (C) Surface density dependence of phosphorylation for EGFR with four residues in the N-terminal interface mutated (4I, T624I/G625I/G628I/A629I). Both wild-type and mutant EGFR are compared with or without EGF treatment. (D) FACS data comparing EGFR expression level (x axis) to Tyr 1068 phosphorylation level for wild-type EGFR and the 4I mutant. Error bars represent SEM. See also Figures S5 and S6.
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Sixth figure from paper

Figure 6. The Effect of the I640E Mutation on Transmembrane Helix Structure and Receptor Activation (A) 1H, 15N chemical shift differences between the I640E mutant and the wild-type TM-JM segment for each residue. The solid (red) and dashed (black) horizontal lines represent the chemical shift differences expected based on the digital resolution of the spectra and calculated from the average chemical shift, respectively. The vertical red dashed lines represent the predicted membrane-spanning region, based on the sequence analysis. (B) The Ala Cb region from the 1H-13C (CT) HSQC spectra of the wild-type and I640E TM-JM segments in DMPC/DHPC bicelles, respectively. Schematic representation of the C-terminal dimer is shown at the right, with the uniformly labeled helix on the left and unlabeled one on the right. Residues examined in NMR or cell-based experiments are highlighted. (C) Representative 2D strip plot showing the Ala 637 Hb intermolecular NOE cross-peak at the 13C frequency of Ala 637 Cb, from 3D 15N-13C F1-filtered/F3-edited NOESY-HSQC spectra. (D) Surface density dependence of phosphorylation for the I640E mutation in full-length EGFR. Both wild-type and mutant EGFR are compared with or without EGF treatment. (E) FACS data comparing expression level (x axis) to Tyr 1068 phosphorylation level for the TM-ICM construct, with or without the I640E mutation. Error bars represent SEM. See also Figure S7.
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Seventh figure from paper

Figure 7. Models for Structural Coupling (A) Model for structural coupling between the transmembrane helices and the juxtamembrane segments (JM-A) at the plasma membrane, based on NMR data and molecular dynamics simulations (Arkhipov et al., 2013). The LRRLL motif in JM-A is highlighted, with leucine side chains in green and yellow and arginines in blue. (B) Model for asymmetric dimer formation at the plasma membrane. The surface of the kinase domains and the backbone of the juxtamembrane segments are shown. Residues in the LRRLL motif in the JM-A are shown as sticks, with leucine in yellow (activator) or green (receiver) and arginine in blue. Glu 666 of the receiver and Arg 949 of the activator are also shown.
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Supplemental Material

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First supplimental figure
Fig. S1. Estimation of the Cytoplasmic Fraction of Myr-ICM, Related to Figure 3 (A) Diagram demonstrating how the contribution of cytoplasmic protein to total intensity depends on the thickness of the cell. Light green shading represents diffuse cytoplasmic fluorescence, solid green lines represent stronger membrane fluorescence, and the blue ellipses represent the shape of the confocal beam. At the edge of the cell, where the thickness is less than the height of the beam, two membranes will contribute to the total fluorescence intensity and the contribution of the cytoplasmic protein will be significantly reduced compared its contribution in the center of the cell. (B) Confocal images of EGFR (left), Myr-ICM (center) and the intracellular module without the c-Src localization sequence, ICM (right). For each group of images, the large center image is a top view in the x-y plane (the basal surface). The smaller images on the right and left are projections in the y-z plane and x-z planes, respectively. Note that cytoplasmic fluorescence is stronger for Myr-ICM-expressing cells compared to EGFR-expressing cells. (C) Fluorescence intensity as a function of z-position for cells expressing Myr-ICM (green curve). Note the presence of two peaks for Myr-ICM, indicating localization to basal and apical membranes. The cytoplasmic fluorescence intensity (Ic) is assumed to be the minimal intensity, which occurs when the beam is entirely contained within the cell. Membrane intensity (Im) is assumed to be the difference between the peak value and the cytoplasmic value (Ic). Ic and Im are used to estimate the contribution of cytoplasmic Myr-ICM to the overall fluorescence intensities measured in the single-cell assays (Extended Experimental Procedures). For comparison, fluorescence intensity for cells expressing the intracellular domain without the c-Src localization sequence is also shown, (ICM, blue curve). Note one central peak, indicating only cytoplasmic localization. (D) Fluorescence intensity as a function of z-position for cells expressing GFP (blue curve, measured in the center of the cell, and green curve, measured at the periphery), compared to a lipid bilayer containing Bodipy-labeled lipids (dotted green curve). The difference in full width at half maximum between the bilayer and GFP at the cell periphery (Hp $200 nm) represents thickness of the cell in peripheral regions, which is used to estimate the contribution of cytoplasmic Myr-ICM to the overall fluorescence intensities measured in the single cell assays (Extended Experimental Procedures). (E) Surface-density dependent activation for Myr-ICM compared to EGF-stimulated EGFR, with or without correcting for an estimated cytoplasmic population of 15% (Extended Experimental Procedures). This correction for cytoplasmic Myr-ICM does not affect the conclusion that Myr-ICM is strongly inhibited relative to EGF-stimulated EGFR, nor does it significantly affect the apparent surface-density dependence of Myr-ICM. Errors bars represent the standard error of the mean.
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Second supplimental figure
Figure S2. Controls for the Surface-Density Dependence of EGFR Phosphorylation, Related to Figure 2. (A) Scatter plot for the phosphorylation level of Tyr 1068 as a function of the surface density of the receptor. Trend lines were calculated using linear and second-order polynomial fits for EGFR with and without ligand, respectively. This plot shows the raw data that were used to generate the averaged values shown in Figure 2B. (B) Surface-density dependent phosphorylation of Tyr 1173 for EGFR, with or without EGF-treatment. Note the pattern of ligand-independent and EGF-stimulated phosphorylation is similar to that observed for Tyr 1068 (Figure 2B). The two panels are as explained in Figure 2B. (C) Surface density-dependent phosphorylation for the V924R mutant compared to wild-type EGFR, with or without EGF treatment. Note that the V924R mutation, which inhibits formation of the asymmetric dimer (Zhang et al., 2006), strongly suppresses both ligand-independent and ligand-dependent activation, regardless of surface density. (D) FACS data comparing EGFR expression level (x axis) to Tyr 1068 phosphorylation. (E) Cos-7 cells viewed by fluorescence microscopy, expressing EGFR (top panels), the extracellular module deletion construct (TM-ICM, bottom panels), and the construct with a flexible linker inserted between the extracellular module and the transmembrane helix (ECM-GlySer-TMICM, middle panels). Expression level (left column, red) and phosphorylation level at Tyr 1068 (middle column, green) are shown. Expression and phosphorylation channels are merged in the right column. For the TM-ICM construct, note that at the cell periphery despite weak localization of TM-ICM, the phosphorylation of Tyr 1068 is still strong. Errors bars represent the standard error of the mean.
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Third supplimental figure
Figure S3. Controls for the Intracellular Module with c-Src Localization Sequence, Related to Figure 3 (A) Relationship between surface density and phosphorylation level for an intracellular module construct with a flexible linker (GGGSGGGT) inserted in between the c-Src membrane localization sequence and the juxtamembrane segment (Myr-GlySer-ICM), compared to myr-GCN4-ICM and EGFR. (B) Confocal images of cells expressing Myr-ICM (top panels), myr-GCN4-ICM (middle panels) and Myr-ICM K713E/K715E (bottom panels) showing phosphorylation levels at Tyr 1068 (Experimental Procedures). Expression is shown in left panels, phosphorylation in middle panels and the two channels are merged in the right panel. Large boxes are views in the x-y plane of the basal surface of cells closest to coverslip. Small boxes are projections in the x-z plane, orthogonal to the basal surface of the cell. Note strong plasma membrane localization for all three constructs, indicating that differences in localization are not responsible for differences in phosphorylation levels. Errors bars represent the standard error of the mean.
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Fourth supplimental figure
Figure S4. Controls for Fluorescence Cross-Correlation Studies, Related to Figure 4 (A) Sample two-color pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) spectrum for the Myr-GCN4-ICM construct. Fluorescence correlation spectroscopy (FCS) spectra, shown in red (Gr(t)) and green (Gg(t)), are calculated from the autocorrelation of the fluorescence-intensity fluctuations for mCherry and GFP, respectively. By fitting the data to a 2D diffusion model (plotted as solid lines, Experimental Procedures), we can determine the number of mCherry and GFP fluorescent molecules in the laser spot (Nr, Ng, respectively, as shown in equation). The fluorescence cross-correlation spectroscopy (FCCS) spectrum, shown in blue, is calculated from the cross correlation of the mCherry and GFP fluorescence intensity fluctuations. From the auto and cross-correlation functions, we calculate a relative cross-correlation value, (F), which represents the extent to which the diffusion of GFP and mCherry-labeled molecules is correlated (as shown in equation). (B) Scatter plot comparing cross-correlation values derived from fluorescence fluctuations with time-gating as described (labeled PIE, Extended Experimental Procedures), compared to the same data analyzed without time-gating (labeled no PIE, Extended Experimental Procedures). The median value (red line) of the EGFR distribution without the PIE time gate is now much closer to the dimerization control, Myr-GCN4-ICM. This demonstrates that spectral crosstalk between the mCherry and GFP channels can have a significant effect on the cross-correlation value. (C) Scatter plot of PIE-FCCS data, comparing relative cross-correlation values for a fusion of GFP and mCherry in one protein (GFP-mCh-Kras), to Myr-GCN4-ICM, and a co-expression of mCherry and GFP both fused to the c-Src localization sequence (Myr-FP). Note that the median cross-correlation value (red line) for Myr-GCN4-ICM is roughly half of the value for the fusion, consistent with it being a dimer. The fusion construct (GFP-mCh-Kras) was localized to the plasma membrane with a localization sequence from Kras instead of the c-Src sequence (Myr). This construct is localized to the plasma membrane to a comparable extent to constructs fused to the c-Src sequence. (D) Scatter plot of cross-correlation values as a function of surface density for intracellular module constructs Myr-ICM and Myr-GCN4-ICM. Note that for Myr-ICM the majority of cross-correlation values are less than 0.03, and do not increase with surface density in the range of densities examined. (E) 1-D scatter plot of PIE-FCCS data, comparing relative cross-correlation values for EGFR constructs with our without EGF treatment. The increase in median cross-correlation values (red lines) we observe when we treat EGFR with EGF, is not significantly altered when EGFR is mutated to be catalytically inactive (D813N), or the entire intracellular domain is deleted (ECM-TM). This indicates that EGF-induced increases in cross correlation are not heavily influenced by large scale clustering in response to receptor phosphorylation and recruitment of downstream receptors by the activated kinase domain.
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Fifth supplimental figure
Figure S5. Structural Analysis of the Transmembrane-Juxtamembrane Construct by NMR, Related to Figure 5 (A) Summary of NOESY connectivity for the transmembrane-juxtamembrane construct (TM-JM) in lipid bicelles plotted against the residue number. Water accessibility of the TM-JM protein was assessed in two ways. First, residues exhibiting H/D exchange are shown with boxes, with filled boxes for residues that exchange slowly with deuterium, and open boxes for residues whose H/D exchange rate is fast. Second, residues that show NOEs or chemical exchange cross-peaks at the water frequency in the 15N edited NOESY-HSQC spectra are indicated with circles. The red dashed lines represent the boundaries of the transmembrane helix based on sequence analysis. The NMR-derived secondary structure for the TM-JM protein is shown at the top of this panel, with the LRRLL motif in the JM-A labeled. (B) Ca secondary chemical shift differences are plotted against the residue number. Positive and negative DCa values indicate preference for a helix and b strand, respectively. DCa values close to zero indicates random coil structure. (C) Secondary structure as predicted by TALOS+ (Shen et al., 2009) as a function of residue number. Positive values on the y axis indicate a propensity to form an a-helix, while values near zero indicate that the residue is part of a random coil. Note that the critical LRRLL motif in the juxtamembrane segment (JM-A) is estimated to have a $30% a-helical propensity (discussed in Arkhipov et al., 2013). (D) Order parameters (S2), derived from the random coil index (RCI), are plotted against the residue number, showing increased dynamics in the juxtamembrane segment relative to the transmembrane helix. (E) 2D 15N-1H TROSY spectra of the TM-JM protein in DMPC/DHPC bicelles acquired at a 900 MHz 1H frequency and 312 K. (F) Representative ensemble of ten low-energy structures calculated from the NMR data using simulated annealing (Experimental Procedures). (G) 2D strip plot showing the Ala 629 Hb NOE cross-peak observed at the 13C frequency of Ala 629 Cb from 3D 15N-13C F1-filtered/F3-edited NOESY-HSQC spectra (Experimental Procedures). A schematic representation of the N-terminal transmembrane dimer is shown at the top. The transmembrane helices are represented as cylinders with the labeled helix shown on the left and the unlabeled helix on the right. On the labeled helix, Ala 629, which generates the intermolecular NOE, is shown (green circle) along with alanine residues that do not produce any intermolecular NOEs (red circles). On the unlabeled helix, residues participating in intermolecular NOEs with Ala 629 are shown (blue circles).
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Sixth supplimental figure
Figure S6. NOE Connectivities for Valine 636 in the Wild-Type TM-JM Construct, Related to Figure 6 (A) Representative 2D strip plot showing Val 636Hg interchain NOEs that are observed only in methyl 13C-edited NOESY-HSQC experiments. The sample for this experiment was prepared by mixing unlabeled and selectively labeled (Val-1Hg-methyl and Leu-1Hd-methyl) protein (Experimental Procedures). (B) Histogram showing the intensity of NOESY cross-peaks measured from methyl 13C-edited NOESY-HSQC. (C) Val 636 is shown on the NMR-derived structure of the wild-type TM-JM segment. The black dotted lines connect the atoms in Val 636 that show NOE connectivity and are omitted from the structure calculation.
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Seventh supplimental figure
Figure S7. Structural Analysis of I640E Mutant in the Transmembrane-Juxtamembrane Construct, Related to Figure 6 (A) The sequential NOE connectivity (dNN(i,i+1)) of the transmembrane-juxtamembrane protein (TM-JM) of EGFR in bicelles plotted against residue number. Connectivities for the I640E mutant (top row) are compared to those for the wild-type TM-JM (bottom row). The red dashed lines represent the boundaries of the transmembrane helix based on sequence analysis. The NMR-derived secondary structure for the wild-type TM-JM protein is indicated at the top of this panel, with the LRRLL motif in the JM-A labeled. The absence of NOE connectivity in the LRRLL motif of the mutant TM-JM indicates a lack of secondary structure. (B) The 13Ca secondary chemical shift differences (DCa) for the I640E mutant (top panel) and the wild-type TM-JM construct (bottom panel) are plotted against the residue number. The decrease in DCa values for the residues at the juxtamembrane segment in the I640E mutant compared to the wild-type indicates the mutation leads to a loss of secondary structure in this segment. (C) A cross section of the 2D NMR spectra, showing the backbone amide region of glycines from the 15N-1H HSQC spectra of the wild-type (top panel) and I640E mutant (bottom panel). The positions of the glycine residues are highlighted as spheres on a schematic representation of our NMR-derived transmembrane-juxtamembrane structure (left). These residues are uniquely positioned at the N-terminal dimer interface (G625 and G628) and C-terminal dimer interface (G639 and G640), and thus are useful as probes for transmembrane conformational change. The position of the I640E mutation in each helix is indicated with arrows. Selective line broadening of the Gly 639 and Gly 641 amide resonances in the mutant spectrum suggests that the C-terminal dimer interface is less stable, than the N-terminal dimer. This line broadening could be caused by conformational exchange between the two inactive states shown in Figure 7A.
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Coordinates in the Protein Data Bank:

EGFR transmembrane - juxtamembrane (TM-JM) segment in bicelles: MD guided NMR refined structure.


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