Structure–function relationships and supramolecular organization of the EGFR (epidermal growth factor receptor) on the cell surface

114 Biochemical Society Transactions (2014) Volume 42, part 1

Structure–function relationships andsupramolecular organization of the EGFR(epidermal growth factor receptor) on thecell surfaceSarah R. Needham*1, Laura C. Zanetti-Domingues*, Michael Hirsch*, Daniel J. Rolfe*, Christopher J. Tynan*,Selene K. Roberts*, Marisa L. Martin-Fernandez* and David T. Clarke**Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Oxford, Didcot, Oxfordshire OX11 0FA, U.K.

AbstractDimerization and higher-order oligomerization are believed to play an important role in the activation ofthe EGFR (epidermal growth factor receptor). Understanding of the process has been limited by the lackof availability of suitable methods for the measurement in cells of distances in the range 10–100 nm, tooshort for imaging methods and too long for spectroscopic methods such as FRET. In the present article,we review the current state of our knowledge of EGFR oligomerization, and describe results from a newsingle-molecule localization method that has allowed the quantitative characterization of the distributionof EGFR–EGFR distances in cells. Recent data suggest the involvement of cortical actin in regulating theformation of EGFR complexes.

IntroductionOligomerization and clustering of protein complexes in theplasma membrane are an important feature of many biologicalprocesses, including cell signalling. EGFR (epidermal growthfactor receptor) is one of the ErbB family of four receptortyrosine kinases that initiate signalling cascades promotingcell proliferation, motility and survival [1]. The extracellulardomain of this receptor has a four-subdomain structureand, in the absence of its ligand, is held in a closedor ‘tethered’ conformation [2]. Upon ligand binding, thetethered structure opens into an extended form, which iscapable of forming a dimer with another receptor [3]. Ligand-induced EGFR dimerization is believed to be the key step forthe initiation of EGFR signalling, through the formation ofan asymmetric dimer and allosteric transactivation of the twokinase domains. However, there is evidence that the situationin cells is significantly more complex than that described bythe ligand-induced dimerization model. There have been anumber of reports of the existence of dimers and higher-orderoligomers of EGFR in cells in the absence of bound ligand [4–9]. There have also been numerous suggestions that receptorconfinement within plasma membrane domains plays a rolein the regulation of receptor activation [10–12]. Conversely,oligomerization and clustering of inactive EGFR have beendismissed as an artefact of receptor overexpression in the celllines used for many of these investigations [13].

Key words: cortical actin, dimerization, epidermal growth factor receptor (EGFR), oligomerization,

single-molecule localization.

Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; F-actin,

filamentous actin; FLIP, fluorophore localization imaging with photobleaching; SNR, signal-to-

noise ratio; TIRF, total internal reflection fluorescence.1To whom correspondence should be addressed (email [email protected]).

There is therefore a requirement for methods capable ofcharacterizing receptor conformation, oligomerization andclustering on the cell surface, and environment currentlyintractable for X-ray crystallography. The distance measure-ments required to adequately characterize these phenomenaare challenging, ranging from molecular dimensions in theorder of 1–20 nm to plasma membrane organization atthe level of nanodomains and picket fences, from 20 to100 nm [14]. Methods commonly used to measure theshorter distances are FRET [15], which measures the distancebetween fluorescent probes, and EPR spectroscopy, whichmeasures distances between two magnetic probes [16], bothin the range ∼2–8 nm. Use of these methods requires theattachment of probes at suitable sites on the protein ofinterest. Unfortunately, measuring dimerization of manytransmembrane proteins, including EGFR, requires themeasurement of distances outside the FRET and EPR range,from 10 to 20 nm. Available imaging methods with, inprinciple, the required resolution have some drawbacks.Transmission EM and AFM usually require some form oflabelling to identify the proteins of interest. This is usuallyaccomplished through the use of full-length monoclonalantibodies, which can be as long as 13 nm [17], andthis limits the effective resolution to tens of nanometres,insufficient for measuring dimers and small complexes.Within the last 10 years, so-called ‘super-resolution’ opticalmicroscopy methods such as STORM (stochastic opticalreconstruction microscopy) [18], PALM (photoactivatedlocalization microscopy) [19], STED (stimulated emissiondepletion microscopy) [20] and NSOM (near-field scanningoptical microscopy) [21] have extended the capabilities ofoptical microscopy below the diffraction limit. However,

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these techniques are still limited to approximately 20 nmresolution at best, and are unsuitable for the measurementof intermolecular distances.

A technique that has showed promise for inter- and intra-molecular distance measurements on the required lengthscales is single-fluorophore localization. This technique relieson the fact that the position of a single emitter can bedetermined to high precision through fitting a profile tothe diffraction-limited spot [22]. Given a sufficiently highSNR (signal-to-noise ratio), localization can be achievedwith a precision of better than 2 nm. A number of practicalmethods have been developed to exploit single fluorophorelocalization, including SHRImP (single-molecule high-resolution imaging with photobleaching) [23] and NALMS(nanometre-localized multiple single-molecule microscopy)[24]. These methods have demonstrated measurement offluorophore separations of approximately 10 nm in so-called ‘DNA rulers’, i.e. immobilized dsDNA labelledwith a fluorophore at each end. The methods workby measuring the shift in position of the centroid ofthe diffraction-limited spot following photobleaching ofone of the two fluorophores. These methods have notbeen applied to measurements in cells, probably becauseof limitations such as the presence of inhomogeneouspopulations with an indeterminate number of fluorophore–fluorophore separations, and poor SNR due to backgroundautofluorescence. The latter is minimized through the useof TIRF (total internal reflection fluorescence) microscopy,which only illuminates fluorophores within approximately200 nm of the coverslip [25]. However, the use of TIRFintroduces an additional problem in the form of a widerange of SNR values as a result of various excitation fieldstrengths due to various distances of the fluorophores fromthe coverslip. Recently, we have described a variant of existingsingle-fluorophore localization techniques that uses Bayesianalgorithms for single-molecule spot detection and intensitytracking, correction for sample drift and Monte Carlobootstrapping to determine errors in molecule localization[26]. This method, FLIP (fluorophore localization imagingwith photobleaching), produces more accurate errors, andmeasures distance distributions with confidence intervals,without the need for prior knowledge of the number ofdiscrete fluorophore–fluorophore separations present in thesample. This technique has been used to provide newinformation on the spatial distribution of inactive and activeEGFR molecules in cells.

In the present article, we survey the current state ofour knowledge of EGFR oligomerization and clustering incells. We describe FRET data, placing it in the context ofhigh-resolution structural information obtained from X-raycrystallography of fragments of the receptor, and discuss thelatest results obtained using the new FLIP technique.

Early studies of EGFR complexesThe first high-resolution structures of EGFR fragments werepublished approximately 10 years ago [2,3,27]. However,

before this, a significant quantity of data on the initialsteps of EGFR activation had already been obtained. Earlystudies used cross-linking techniques to show that both ligandbinding and receptor dimerization were required for EGFRactivation [28–31]. SAXS was used to demonstrate that EGF(epidermal growth factor) induces the formation of EGFRdimers, and that each dimer contains two EGF molecules[32]. The formation of not only dimers, but also trimers andlarger multimers, in response to EGF was shown by a numberof techniques including SDS/PAGE, SEC (size-exclusionchromatography) and electron microscopy [33]. The modelof EGFR activation through EGF-induced dimerization (oroligomerization) was therefore well established before theavailability of high-resolution structural data.

As well as demonstrating the existence of EGF-induceddimers, early studies also provided evidence for the presenceof EGFR dimers in the absence of ligand. These studiesgenerally employed A431 cells, a human epidermoid car-cinoma cell line that overexpresses EGFR with up to 2×106

receptors per cell [34]. The existence of pre-formed dimerswas demonstrated by density gradient centrifugation andcross-linking [30,35,36]. Close associations between EGFRmolecules in the absence of ligand were also demonstratedthrough the use of FRET, which showed the presenceof distances between EGFR molecules between 5 and8 nm, a distance apparently consistent with dimerizationand/or oligomerization [8,9,37]. However, the use of A431cells raises the question of whether the dimers observedcould also be explained by receptor overexpression and/orstimulation by autocrine EGF produced by the cells [38].

EGFR complexes in the light of structuralstudiesA number of crystallographic studies have shed light on theconformation of the extracellular domain of EGFR. How-ever, they have also raised questions about the interpretationof the FRET data described above. The preferred dimerstructure from crystallography is the so-called ‘back-to-back’ dimer [3]. This structure shows an inter-EGF distancegreater than 11 nm (Figure 1), a distance that would beundetectable by FRET and which is inconsistent with the5–8 nm distance measured in earlier FRET experiments.However, shorter distances have regularly been measuredusing fluorescent EGF ligands as probes in a number of celllines using both ensemble and single-molecule FRET andother techniques such as fluorescence correlation microscopy[7,39]. For example, Figure 2 shows the distribution of single-pair FRET efficiencies measured in three different cell lines.One explanation for this apparent anomaly is the existence ofhigher-order oligomers, such as tetramers. Given the presenceof short distances from the EGF-binding pocket to theplasma membrane [40], one plausible model to explain thedata appears to be a tetramer consisting of two back-to-back dimers joined by an asymmetric head-to-head interface,giving a ligand–ligand distance of approximately 4 nm. Thisinterface has been observed in the crystal structures, although

C©The Authors Journal compilation C©2014 Biochemical Society


Figure 1 Model of the extracellular domains of the ‘back-to-back’

crystallographic dimer of EGFR

Ligand EGF molecules are shown in yellow, with fluorescent labels shown

as green spheres. The ligand–ligand separation is approximately 11 nm.

it was thought to be of questionable biological relevance [3],and involves the EGFR ectodomains adopting a positionlying flat on the membrane rather than standing aboveit [39,41]. MD simulations support this model, indicatinga significant increase in the stability of the head-to-headinteraction when the tetramer is relaxed on the membrane.The orientation of the receptor with respect to the membranewas investigated more extensively using a combinationof FRET microscopy, Monte Carlo simulations and MDsimulations, showing that high-affinity EGFR adopts the‘flat-on-the-membrane’ conformation [40]. The asymmetryof this structure and similarities with the Drosophila EGFRprovides some basis for negative co-operativity of EGFbinding in the human receptor, which was not explained bythe pseudo-two-fold symmetry of the crystal structure ofthe extracellular EGFR dimer. This has begun to answer aquestion that has been open for several decades.

Mapping EGFR complexes in the basal stateusing single-molecule localizationRecently, a development of single-molecule localizationtechniques (FLIP) has allowed the measurement of inter-

Figure 2 Single-pair FRET efficiencies in different cell types

Distribution of single-pair FRET efficiencies between EGFR in (A) A431

cells (2×106 receptor copies per cell) [39], (B) HeLa cells (50 000

receptor copies per cell) and (C) T47D cells (7000 receptor copies per

cell).

EGFR distances in cells, in the distance range required tomap long-range intermolecular interactions, and clusteringat the level of membrane nanodomains [26]. This methodbuilds on previous work using single-step photobleaching,but has overcome a number of problems inherent in makingthis type of measurement in cells. In particular, it addressesthe problem of poor and varying SNRs, and the question ofhow to determine the distribution of distances that best fitsthe complex picture of the cellular environment. In short, themethod achieves this by assigning confidence intervalsto the individual separation density functions of the



fluorophores. This allows the objective determination of thedistribution of intermolecular separations, and takes intoaccount the SNRs of every individual position measurement.

FLIP has been used to characterize the separations betweenEGFR molecules in T47D cells. This cell line expresses EGFRat physiological levels (∼7–15×103 copies per cell) [42,43],avoiding the possibility that oligomers are the result ofreceptor overexpression. For the investigation of EGFR in itsbasal state, cells were labelled with a fluorescent anti-EGFRAffibody antagonist shown not to cause receptor activation.The distribution of separations between inactive EGFR isshown in Figure 3(A). This shows a range of separations withpeaks at 8, 22, 37, 46 and 57 nm. This could be explainedthrough the formation of a linear polymer with a repeatperiodicity consistent with EGFR–EGFR separation in adimer, with the shortest peak corresponding to the dimerobserved crystallographically. A model of this structure isshown in Figure 3(B). These data appear to unequivocallydemonstrate the existence of dimers and higher-order EGFRoligomers in the basal state, and the low EGFR expressionlevel in the cell line used rules out the possibility that thiscould be an overexpression artefact.

These authors speculate that the presence of the higher-order oligomers might be due to the interaction of EGFRwith actin filaments, for which there is prior evidencein the literature [44,45]. The separations measured wouldbe consistent with receptor binding to cortical F-actin(filamentous actin), which is a left-handed helix with arepeat of 35.9 nm [46]. We have recently obtained datathat provide supporting evidence for this hypothesis; actinpolymerization is disrupted by the depletion of plasmamembrane cholesterol, probably through interactions withPtdIns(4,5)P2 [47]. Figure 3(C) shows the distribution ofEGFR separations in T47D cells following treatment withthe cholesterol-depleting agent methyl-β-cyclodextrin. The36 nm repeat suggested to be associated with cortical F-actinbinding is no longer present, compared with the data foruntreated cells shown in Figure 3(A). A similar effect has beenobserved for GPI (glycosylphosphatidylinositol)-anchoredproteins, for which a mechanism of complex formationregulated by cortical actin activity has been suggested [48].

ConclusionsConsiderable advances have been made in the last decadein the understanding of EGFR dimerization and oligomer-ization in cells. These advances have been informed byhigh-resolution structures, but enabled by the developmentof new techniques for measuring in cells intermoleculardistances ranging from 1 to 100 nm. In particular, single-pairFRET techniques have been valuable for characterizing shortdistances and single-molecule localization techniques vitalfor measuring the longer distances important in oligomerformation. There now appears to be unequivocal datashowing that EGFR dimers and higher-order complexes existin cells in the basal state, and that these complexes are notan artefact of receptor overexpression. Application of these

Figure 3 Characterization of ECFR–antibody complexes using FLIP

(A) Separations of surface EGFR–Affibody complexes in T47D cells,

measured using FLIP. (B) Model of EGFR homopolymer consistent with

the distances observed in (A). (C) Separations of surface EGFR–Affibody

complexes in T47D cells, measured using FLIP, following treatment with

the cholesterol-depleting agent methyl-β-cyclodextrin, showing the loss

of the ∼36 nm repeat.

new techniques should now begin to shed more light on thedetails of the polymerization process, and therefore on EGFRactivation mechanisms.

Funding

This work was supported by the Biotechnology and Biological

Sciences Research Council [grant number BB/G006911/1].

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Received 7 October 2013doi:10.1042/BST20130236


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Structure–function relationships and supramolecular organization of the EGFR (epidermal growth factor receptor) on the cell surface