Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor

Abstract

To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.

Figure 1

Unliganded EGFR adopts a monomeric tethered conformation. (a) Superose 6 gel filtration chromatograms of EGFR with the indicated pretreatments. D, dimer; M, monomer. In preparations with EGF, the amount of monomer present varied in independent preparations with the same mutation or drug, and thus variations in monomer content in the profiles in a are not meaningful. (b) Representative EM class averages of unliganded EGF receptors. (c) The ectodomain after masking. (d) Bottom, the best cross-correlating projections from the unliganded ectodomain crystal structure. Top, enlarged ribbon diagrams in same orientation. (e) The best correlating projections with a monomer from the dimeric, liganded ectodomain crystal structure. Cross-correlation coefficients are shown below the projections. Scale bars, 10 nm.

Figure 2

The liganded EGF receptor ectodomain can link to multiple kinase-domain dimerization states. (a,b) Representative EM class averages of EGF-bound receptors with asymmetric (a) or symmetric (b) kinase-domain dimers, with masked regions and cross-correlations for each average shown below in c–n. (c,d) Class averages after masking of all but the ectodomain. (e,f) Best-correlating ectodomain projections. Enlarged ribbon diagrams are shown in upper parts of m,n. (g,h) Class averages after masking of all but the kinase domain. (i–l) Best-correlating asymmetric kinase domain (i,j) and symmetric kinase domain (k,l) crystal structure projections. Cross-correlation scores are shown below projections. Scale bars,10 nm. (m,n) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures, determined by cross-correlation with masked class averages in c,d,g,h. Structures are shown as ribbon diagrams enlarged relative to the class averages while maintaining spatial relationships. The better-correlating asymmetric kinase dimer is shown in m, and the better-correlating symmetric kinase dimer in n. (o) Schematic diagram of the asymmetric kinase dimer in which one kinase (green) activates the other (orange). The position of Val924 is marked with a star. Modified from reference .

Figure 3

Mutational disruption of the asymmetric kinase dimer. (a,b) Representative class averages of EGFR V924R mutant in complex with EGF with unassociated kinase monomers (a) or symmetric kinase dimers (b). (c,d) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures as in Figure 2m,n, determined using masked class averages in a,b. (e,f) Representative class averages of EGFR T669D S671D mutant in complex with EGF with unassociated kinase monomers (e) or symmetric kinase dimers (f). (g,h) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures as in Figure 2m,n, determined using masked class averages from e,f as described in Figure 2. Scale bars, 10 nm.

Figure 4

Kinase inhibitors gefitinib and PD168393 cooperate with EGF in promoting the asymmetric kinase dimer. (a,b) Representative class averages of EGFR in complex with EGF and gefitinib or PD168393. Representative asymmetric dimeric (1), symmetric dimeric (2) and unassociated kinase domains (3) are shown. (c,d) The kinase domain after masking. (e–j) Best-correlating projections with asymmetric (e,f) and symmetric dimers (g,h) from kinase crystal structure. Cross-correlation scores are shown below each projection. Scale bars, 10 nm. (i,j) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures determined using masked class averages as in Figure 2. (k) Fraction of asymmetric-like kinase dimers, symmetric-like kinase dimers and kinase monomers in the presence of EGF and indicated inhibitors or mutations, calculated using all class averages with well-resolved kinase domains and the number of particles in each class average. WT, wild-type.

Figure 5

Effect of the inhibitors lapatinib and HKI-272 on kinase-domain dimerization. (a,b) Representative class averages of EGFR in complex with EGF and lapatinib as symmetric kinase dimers (a) or unassociated kinase monomers (b). (c) Top, representative class averages of EGFR in complex with EGF and HKI-272. Scale bars, 10 nm. Bottom, positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures, as determined using masked class averages as in Figure 2.