Molecular Cell, Vol.
11, 507–517, February, 2003, Copyright 2003 by Cell Press
EGF Activates Its Receptor by Removing Interactions
that Autoinhibit Ectodomain Dimerization
Kathryn M. Ferguson,1,2,4,* Mitchell B. Berger,1,2
Jeannine M. Mendrola,1 Hyun-Soo Cho,3
Daniel J. Leahy,3 and Mark A. Lemmon1,2,*
1
Department of Biochemistry and Biophysics
2
Graduate Group in Biochemistry
and Molecular Biophysics
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19104
3
Department of Biophysics and Biophysical Chemistry
Howard Hughes Medical Institute
The Johns Hopkins University School of Medicine
Baltimore, Maryland 21205
Summary
Epidermal growth factor (EGF) receptor is the proto-
type of the ErbB (HER) family receptor tyrosine kinases
(RTKs), which regulate cell growth and differentiation
and are implicated in many human cancers. EGF acti-
vates its receptor by inducing dimerization of the 621
amino acid EGF receptor extracellular region. We de-
scribe the 2.8 Å resolution crystal structure of this
entire extracellular region (sEGFR) in an unactivated
state. The structure reveals an autoinhibited configu-
ration, where the dimerization interface recently iden-
tified in activated sEGFR structures is completely oc-
cluded by intramolecular interactions. To activate the
receptor, EGF binding must promote a large domain
rearrangement that exposes this dimerization inter-
face. This contrasts starkly with other RTK activation
mechanisms and suggests new approaches for de-
signing ErbB receptor antagonists.
Introduction
The mechanistic understanding of transmembrane sig-
naling by growth factor receptor tyrosine kinases (RTKs)
has become quite sophisticated in recent years (Schles-
singer, 2000). It is now well accepted that signaling is
initiated by growth factor binding to the extracellular
region of the receptor, which leads to RTK dimerization
or to the rearrangement of protomers within a preformed
dimer (Jiang and Hunter, 1999; Schlessinger, 2000). In
the active dimer, the two tyrosine kinase domains are
thought to be arranged ideally for mutual transphos-
phorylation and activation.
Numerous biophysical studies of extracellular regions
from receptors with a single transmembrane domain,
beginning with the human growth hormone (hGH) recep-
tor (de Vos et al., 1992), have led to the emergence of
a paradigm in which a bivalent ligand drives receptor
dimerization by binding simultaneously to two receptor
*Correspondence: mlemmon@mail.med.upenn.edu (M.A.L.), ferguso2@
mail.med.upenn.edu (K.M.F.)
4
Present address: Department of Physiology, University of Pennsyl-
vania School of Medicine, B400 Richards Building, 3700 Hamilton
Walk, Philadelphia, Pennsylvania 19104.
molecules. Crystallographic studies of several ligand-
bound RTK extracellular fragments have allowed visual-
ization of such dimers, including those from the Flt-1
vascular endothelial growth factor (VEGF) receptor
(Wiesmann et al., 1997), the TrkA nerve growth factor
(NGF) receptor (Wiesmann et al., 1999), and EphB2 (Hi-
manen et al., 2001). Similarly, fibroblast growth factor
(FGF) receptor dimerization involves simultaneous inter-
action of FGF•heparin complexes with two receptor
molecules (Schlessinger et al., 2000). In each of these
cases, the bound ligand contributes directly to the re-
ceptor dimerization interface and does not dramatically
alter the conformation of the RTK extracellular region.
It is now clear that the epidermal growth factor (EGF)
receptor is an exception to this rule. EGF is monomeric
and binds the extracellular region of its receptor to form
a 2:2 (ligand:receptor) dimer (Lemmon et al., 1997). Re-
cent crystal structures have shown that EGF does not
contribute at all to the interface in this dimer (Garrett et
al., 2002; Ogiso et al., 2002). Instead, each growth factor
binds to only one receptor, presumably inducing a con-
formational change that promotes dimerization through
receptor•receptor interactions.
The EGF receptor is one of four ErbB (or HER) family
RTKs (Yarden and Sliwkowski, 2001), which also include
ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4).
Under normal physiological conditions a family of 12 or
more growth factor agonists, including EGF, trans-
forming growth factor-␣ (TGF-␣), and the neuregulins,
regulate these receptors by inducing their homo- and/or
hetero-oligomerization (Yarden and Sliwkowski, 2001).
Overexpression or overactivation of ErbB receptors is
associated with several different human cancers, includ-
ing breast cancer and colon cancer. ErbB receptors
(notably ErbB2 and EGFR) are currently targets of thera-
peutic approaches that are in development or in clinical
use (Mendelsohn, 2001; Pegram et al., 2000).
The ⬇600 amino acid extracellular region of each ErbB
receptor contains four subdomains, designated do-
mains I, II, III, and IV (Lax et al., 1988a, 1988b). Domains
I and III (alternatively called L1 and L2 [Bajaj et al., 1987])
share approximately 37% amino acid sequence identity.
Domains II and IV (also termed S1 and S2 [Bajaj et al.,
1987] or CR1 and CR2 [Ward et al., 2001]) share 17%
sequence identity and are cysteine rich. Recent crystal-
lographic studies have shown how domains I, II, and III
are arranged in a dimeric complex of the EGF receptor
extracellular region with ligand. Most of domain IV was
either missing (Garrett et al., 2002) or present but disor-
dered (Ogiso et al., 2002). In both structures, the bound
growth factor interacts exclusively with domains I and
III of the same receptor molecule and is distant from the
dimer interface. Dimerization appears to be mediated
primarily by a loop or dimerization arm that projects
from domain II (Garrett et al., 2002; Ogiso et al., 2002),
and growth factor binding is thought to induce a confor-
mational change that stabilizes this arrangement.
To fully understand the nature of this conformational
change, the structure of an inactive, monomeric form
of the complete EGF receptor extracellular region
Molecular Cell
508

Table 1. Data Collection and Refinement Statistics

Data Collection Statisticsa

Space group C2
Unique cell dimensions a ⫽ 119.2 Å, b ⫽ 103.7 Å, c ⫽ 101.5 Å; ␤ ⫽ 119.3⬚
X-ray source CHESS F1
Resolution limit 2.8 Å
Observed/unique 90,194/26,060
Completeness 98 (95.4)
Rsymb 0.048 (0.345)
⬍I/␴⬎ 12 (2.2)

Refinement Statistics
Resolution limits 20-2.8 Å
No. of reflections/no. test set 22,435 (2563)
R factor (Rfree)c 0.24 (0.31)
Model

Protein aa 5–615 (sEGFR), aa 5–49 (EGF), 14 saccharide units

Total number of atoms 5096
Rmsd bond lengths (Å) 0.016
Rmsd bond angles (⬚) 1.89
a
Numbers in parentheses refer to last resolution shell.
b
Rsym ⫽ ⌺|Ih ⫺ ⬍Ih⬎|/⌺Ih, where ⬍Ih⬎ ⫽ average intensity over symmetry equivalent measurements.
c
R factor ⫽ ⌺|Fo ⫺ Fc|/⌺Fo, where summation is over data used in the refinement; Rfree includes only 10% of the data excluded from the
refinement.

(sEGFR) is also required. In this paper we describe such
a structure of sEGFR, which includes domains I, II, III,
and IV, as well as an EGF molecule bound with very low
affinity (at low pH) in a mode that cannot induce receptor
dimerization. The structure reveals an autoinhibited con-
figuration, in which the domain II dimerization arm is
completely occluded by intramolecular interactions with
domain IV. We present a model for how high-affinity
EGF binding to the EGF receptor may remove the dimer-
ization arm from its intramolecular binding site, thus
promoting receptor dimerization and activation.
Results and Discussion
Structure Determination
We produced the entire extracellular region of human
EGFR (sEGFR: amino acids 1–618 of the mature protein
with a C-terminal hexahistidine tag) as described (Fergu-
son et al., 2000) by secretion from Sf9 cells infected
with recombinant baculovirus. Crystals that diffracted
to 2.8 Å resolution were grown from a 1.2:1 mixture of
EGF and sEGFR in 12% PEG 3400 at pH 5.0 or lower
and could only be obtained in the presence of excess
EGF. We determined the structure by molecular replace-
ment using search models based on coordinates of the
ErbB3 (HER3) extracellular region (sErbB3) (Cho and
Leahy, 2002) and refined the model to 2.8 Å resolution.
One EGF•sEGFR complex is present in the asymmetric
unit. Our final model contains amino acids 5–615 of
sEGFR, amino acids 5–49 of human EGF, and 14 carbo-
hydrate residues. Crystallographic and refinement sta-
tistics are given in Table 1.
Overall Structure
The arrangement of the four structural domains in
sEGFR is shown in Figure 1A. Domains I and III (blue
and yellow in Figure 1A) have the ␤ helix or solenoid
topology expected from the previously determined
structure of the sequence-related extracellular region of
the insulin-like growth factor-1 (IGF-1) receptor (Garrett
et al., 1998). Domains I and III stretch from amino acids
1–165 and 310–481 respectively, and consist of six turns
of ␤ helix that form a barrel-like structure capped off at
each end by an ␣ helix. As their sequence similarity
suggests, these two domains are also closely related to
one another in structure and are both clearly implicated
in EGF binding by crystallographic (Garrett et al., 2002;
Ogiso et al., 2002) and biochemical experiments (Kohda
et al., 1993; Lax et al., 1988a, 1989; Summerfield et al.,
1996; Woltjer et al., 1992; Wu et al., 1990). In our struc-
ture, EGF is bound exclusively to domain I of sEGFR
(Figure 2) in a very low-affinity mode (at low pH) that
cannot induce sEGFR dimerization (see below).
As expected from the IGF-1 receptor structure and
from chemical analysis of sEGFR disulfide bond struc-
ture (Abe et al., 1998), the cysteine-rich domains II
(amino acids 166–309) and IV (amino acids 482–618)
both contain a succession of small disulfide-bonded
modules that comprise an extended rod-like structure.
Two types of disulfide-bonded module are seen in each
domain (Ward et al., 2001). In one type (C1), a single
disulfide bond constrains an intervening bow-like loop.
In the other type (C2), two disulfide bonds link four suc-
cessive cysteines in the pattern Cys1-Cys3 and Cys2-
Cys4 to give a knot-like structure. Domain II of sEGFR
contains three consecutive C2 modules followed by five
C1 modules, listed C2IIa-C2IIb-C2IIc-C1IIa-C1IIb-C1IIc-C1IId-
C1IIe where each module is subscripted for its domain
and position in that domain. Domain IV contains seven
disulfide-bonded modules, arranged in the order C2IVa-
C1IVa-C1IVb-C2IVb-C1IVc-C1IVd-C2IVc (Figure 1A).
Intramolecular Domain II/IV Interactions Constrain
the sEGFR Structure
A striking feature of the sEGFR structure is the extensive
intramolecular contact between domains II and IV (Fig-
Autoinhibition of EGF Receptor Dimerization
509
ure 1), which buries a total solvent accessible surface
area of 922 Å2 (461 Å2 from each domain) in an interface
with a shape complementarity parameter (Sc) value
(Lawrence and Colman, 1993) of 0.66 (comparable to
that seen in antibody/antigen interfaces). At the center
of this interface a prominent 20 residue ␤-hairpin/loop
(residues 240–260) projects from the domain II C1IIb mod-
ule to insert between the equivalent (but shorter) loops
of the two most C-terminal C1 modules in domain IV
(C1IVc and C1IVd, which correspond to residues 561–569
and 572–585, respectively). Several side chain-to-back-
bone and backbone-to-backbone hydrogen bonds are
Figure 1. Structure of the Autoinhibited
sEGFR Monomer
(A) Ribbon representation of sEGFR, with do-
main I (L1) colored blue, domain II (S1) green,
domain III (L2) yellow, and domain IV (S2) red.
N and C termini are labeled. Disulfide bonds
in domains II and IV that constitute C1 disul-
fide-bonded modules are black, while those
in C2 modules are gray. Individual C1 and C2
modules are labeled in domain IV as de-
scribed in the text. Disulfide bonds in do-
mains I and III are colored yellow. The domain
I-bound EGF molecule (see Figure 2) has
been omitted for clarity.
(B) Detail of the region boxed in (A), showing
autoinhibitory intramolecular interactions be-
tween the second C1 module of domain II
(C1IIb) and the C1IVc and C1IVd modules of do-
main IV. Parts of domains II and IV shown
have green and red surfaces, respectively,
with backbones represented by green and
red worms. Interdomain hydrogen bonds are
drawn as white dashed lines.
formed between these regions of domains II and IV, as
shown in Figure 1B, and there are additional contribu-
tions from carbohydrate. Moreover, the side chain of a
tyrosine (Y246) close to the tip of the domain II ␤-hairpin/
loop makes hydrogen bonds with the side chains of
D563 and K585 from the C1IVc and C1IVd modules, respec-
tively, of domain IV (Figure 1B). An almost identical inter-
domain tether occurs within the extracellular region of
unliganded HER3/ErbB3 (sErbB3), burying 810 Å2 (Cho
and Leahy, 2002) but is absent from the structure of the
HER2/ErbB2 (sErbB2) extracellular region (Cho et al.,
2003).
Molecular Cell
510

Figure 2. Space-Filling Representation of

sEGFR with Domain I-Bound EGF
sEGFR is shown in CPK representation, with
subdomains colored as in Figure 1. Bound
EGF is magenta. EGF is intimately associated
with domain I (blue) but does not come closer
than 4 Å away from domain III. The side chain
of Y14 (projecting from the left-hand side of
EGF) is too distant from domain III to partici-
pate in hydrogen bonding.

Intramolecular Domain II/IV Interactions Autoinhibit
sEGFR Dimerization
Remarkably, the same ␤-hairpin/loop from domain II that
makes intramolecular contacts in Figure 1 also plays a
dominant role in intermolecular receptor•receptor inter-
actions in the recently described crystal structures of
ligand-induced sEGFR dimers (Garrett et al., 2002; Ogiso
et al., 2002). Ogiso and colleagues have termed this loop
the “dimerization arm.” The side chain of Y246 (from the
dimerization arm) is completely buried in the interface
between sEGFR molecules in the ligand-induced dimer
and makes two interreceptor hydrogen bonds. In our
structure, the same side chain instead plays a key role
in driving intramolecular interactions (Figure 1B). These
observations can only be reconciled if the structure
shown in Figures 1 and 2 represents a monomeric form
of sEGFR, in which receptor self-association is autoin-
hibited by intramolecular domain II/IV interactions that
sequester the dimerization arm. A dramatic ligand-stabi-
lized domain rearrangement in sEGFR could free the
dimerization arm from these intramolecular contacts
and allow formation of the activated dimer described
by Garrett et al. (2002) and Ogiso et al. (2002).
tions of up to 50 ␮M also failed to show EGF-induced
sEGFR dimerization (data not shown), whereas dimer-
ization is complete at pH 8.0 (Ferguson et al., 2000). The
pH sensitivity of ligand binding can be explained from
the structure of the EGF•sEGFR complex reported by
Ogiso et al. (2002). In this complex (crystallized at pH
8.4) the interactions of EGF with domain I are essentially
identical to those seen in our structure. The EGF/domain
I interface primarily involves backbone hydrogen bonds
and van der Waal’s contacts, buries 1388 Å2 in our struc-
ture (1440 Å2 in the dimer structure), and includes no
interactions that would be affected significantly by re-
ducing pH from 7.5 to 5. By contrast, the interface
formed between EGF and domain III of sEGFR at neutral
pH (Ogiso et al., 2002) involves three histidine side

pH Sensitivity of EGF Binding to sEGFR

As mentioned above, our crystals contain a weakly
bound EGF molecule that is intimately associated with
domain I of sEGFR but makes no interactions with do-
main III (Figure 2). We are confident that this mode of
EGF binding does not induce receptor dimerization. In-
deed, under the relatively low pH conditions (pH 5.0)
that yielded crystals, EGF binds very weakly to its recep-
tor and does not induce significant activation (Nunez et
al., 1993). As shown in Figure 3, high-affinity EGF binding
by sEGFR is substantially compromised at pH values Figure 3. Binding of sEGFR to EGF Is Reduced at Low pH
below 7.5, and cannot be detected (at this concentra- Binding of sEGFR (at 150 nM) to immobilized EGF was measured at
tion) in BIAcore studies below pH 6.0. Analytical ultra- different pH values using BIAcore as described in the Experimental
centrifugation studies at pH 5 with protein concentra- Procedures. The midpoint of this titration is at approximately pH 7.3.
Autoinhibition of EGF Receptor Dimerization
511

Figure 4. Model for EGF-Promoted Domain Rearrangement in sEGFR, Leading to Dimerization

The domain rearrangement promoted by bivalent binding of EGF to both domains I and III was approximated as described in the text and is
depicted with ribbon models and cartoon forms. Modeled structures (B–D) are shown on a gray background.
(A) sEGFR, in the same orientation and with the same coloring as in Figure 1A. Domain I-bound EGF is colored magenta. Key residues in EGF
that must be docked onto domain III in the dimer (R41 and L47) are surrounded by an ellipse labeled “iii”. A hydrophobic pocket on domain
III, into which L47 projects in the EGF•sEGFR dimer (Garrett et al., 2002; Ogiso et al., 2002), is circled and marked “i”. The side chain of D355
on domain III, which hydrogen bonds with R41 of EGF in the dimer, is also circled and marked “ii”. Domain I-bound EGF can be correctly
docked on to domain III by 130⬚ anticlockwise rotation of the domain I/II rigid body (plus EGF) about the axis marked by a filled black circle,
plus a translation along this axis (into the page) of 20 Å.
(B) This domain rearrangement allows EGF binding to both domains I and III of an extended sEGFR molecule in which the domain II dimerization
loop is exposed (and faces out of the page in this view). We have not attempted to model the polypeptide in the region of the rotation axis.
(C) The entire model for extended sEGFR has been rotated 90⬚ about the vertical axis marked (from its orientation in [B]), to provide a view
in which exposure of the domain II dimerization arm (C1IIb loop) and domain IV C1IVd loop (marked with an asterisk) can be appreciated.
(D) The extended sEGFR molecule, with EGF bound to domains I and III, dimerizes through domain II-mediated interactions, with possible
additional contributions from domain IV (Berezov et al., 2002; Schlessinger, 2002). The dimer model shown uses the coordinates of the
EGF•sEGFR dimer from Ogiso et al. (2002), to which we have added domain IV (by maintaining the domains III/IV relationship seen in [A]).
While domains I, III, and IV are unaltered in structure and orientation in going from (C) to (D), the conformation of domain II is altered significantly
(see text). For clarity, domain II on the right-hand sEGFR molecule in the dimer is colored black.

chains, likely to be protonated with a pH profile similar
to that presented in Figure 3. As suggested by Garrett
et al. (2002) from the TGF-␣•sEGFR complex structure,
protonation of these histidines is likely to disrupt the
interface between the ligand and domain III, and pro-
vides an explanation for the absence of domain III inter-
actions in our complex at pH 5. The data in Figure 3
also argue that EGF and TGF-␣ bind much more strongly
to domain III of sEGFR than to domain I. Whereas iso-
lated domain III binds these ligands with KD values of
400 nM (EGF) and 1 ␮M (TGF-␣) (Kohda et al., 1993;
Lemmon et al., 1997), the residual domain I-mediated
EGF binding to sEGFR (when domain III interactions
are abolished at low pH) can barely be detected in our
BIAcore studies. Since domain I occupancy must be
significant at the protein concentrations used for crystal-
lization (120 ␮M) for us to see this interaction in our
crystals, we estimate that the KD for monovalent EGF
binding to domain I is in the range of 50–200 ␮M.
It is likely that EGF binding to domain III (but not
domain I) is disrupted in vivo when the activated EGF
receptor is exposed to the low pH environment of endo-
somal compartments following its internalization. We
suggest that our structure represents the situation im-
mediately following internalization of activated EGFR,
after domain III interactions have been disrupted and
the two protomers in the dimer have dissociated from
one another (deactivating the receptor). The EGF bound
only weakly to domain I of this structure would be ex-
pected to dissociate rapidly from the monomeric recep-
tor in vivo.
An EGF-Induced Domain Rearrangement Relieves
Autoinhibition of sEGFR Dimerization
The structure of sEGFR with EGF bound only to domain
I provides a unique opportunity to model the conforma-
tional changes that must accompany ligand-induced di-
merization of the EGF receptor. A key requirement is
that the dimerization arm, responsible for the majority
of receptor•receptor interactions in the dimer (Garrett
Molecular Cell
512

et al., 2002; Ogiso et al., 2002), is removed from the

intramolecular tether shown in Figure 1 and becomes
exposed. Studies of ligand-induced changes in trypto-
phan fluorescence have suggested conformational
changes consistent with such a domain rearrangement
(Greenfield et al., 1989).
In our autoinhibited sEGFR structure (Figures 2 and
4A) the ligand binding site on domain III is distant from
the domain I-bound EGF molecule and faces the wrong
direction. For example, two critical EGF side chains
(from R41 and L47; labeled iii in Figure 4A) are over 30 Å
away from the sites on domain III with which they interact
in the EGF•sEGFR dimer (circles labeled i and ii in Figure
4A). With a simple but substantial domain rearrange-
ment the domain I-bound EGF molecule can be docked
onto domain III so as to satisfy all ligand•domain III
interactions observed in the dimer structures (Garrett
et al., 2002; Ogiso et al., 2002). To approximate this
conformational change, we treated the N-terminal half
of sEGFR (containing domains I and II plus EGF) as one
rigid body and the C-terminal half (containing domains
III and IV) as a second rigid body. The two rigid bodies Figure 5. Mutations that Disrupt Intramolecular Domain II/IV Inter-
were allowed to move with respect to one another about actions Increase EGF Binding Affinity
a pivot close to the domain II/III junction. This treatment EGF binding to wild-type and mutated sEGFR was measured using
surface plasmon resonance (BIAcore). As reported previously (Fer-
is justified by a comparison of domain relationships in
guson et al., 2000), wild-type sEGFR (black curve) binds to EGF with
the sEGFR, sErbB3, sErbB2, and IGF-1 receptor struc- a KD of 132 ⫾ 9.9 nM. Mutation of K585 to alanine increased binding
tures (Cho and Leahy, 2002; Cho et al., 2003; Garrett et affinity to give a KD of 54.3 ⫾ 8.3 nM (blue curve), mutation of both
al., 1998), which suggests that the structural relationship D563 and H566 to alanine gave a KD of 58.8 ⫾ 5.5nM (green curve),
between a ␤ helix domain (domain I or III) and its and a triple D563A/H566A/K585A mutant binds with KD ⫽ 50.7 ⫾
6.3 nM (red curve). Mean results from at least three independent
C-terminal cysteine-rich domain (domain II or IV) is rela-
experiments are plotted, with errors shown as standard deviations.
tively fixed.
To generate a structure in which domain I-bound EGF
also interacts appropriately with domain III, it is neces-
Mutations that Disrupt the Intramolecular Domain II/IV
sary to rotate the domain I/II rigid body (with bound
Interaction Increase EGF Binding Affinity
EGF) by 130⬚ about an axis close to the R310 ␣ carbon
Autoinhibition in sEGFR resembles that in Src, WASP
(approximately perpendicular to the plane of the page
(Pufall and Graves, 2002), and the kinase domains of
in Figure 4) and to translate it along this axis (into the
RTKs (Hubbard et al., 1998), in that intramolecular inter-
page) by approximately 20 Å. This manipulation converts
actions mask a key activity. However, activation of
our structure (Figure 4A) into an extended configuration
sEGFR (or relief of autoinhibition) differs from the other
that resembles the protomers in the ligand-induced examples in being truly allosteric. EGF does not directly
sEGFR dimer (Figures 4B–4D). The autoinhibitory intra- contact either of the sites involved in intramolecular
molecular domain II/IV interaction is broken, and the autoinhibitory interactions but instead influences them
domain II dimerization arm is exposed for participation only indirectly. By contrast, in each of the other cases
in receptor•receptor interactions. This modeled confor- listed above, activation requires direct interference with
mational change also positions the equivalent loop from one arm of the autoinhibitory interaction, either by bind-
the domain IV C1IVd module (marked with an asterisk in ing an activator or by enzymatic modification.
Figure 4) ideally to make contacts across the dimer There appear to be two mutually exclusive sEGFR
interface, as previously suggested (Berezov et al., 2002; configurations that must compete with one another. In
Schlessinger, 2002). The fact that domain IV was disor- the autoinhibited configuration (Figure 4A), intramolecu-
dered in crystals of the EGF•sEGFR complex obtained lar domain II/IV interactions pull domains I and III apart,
by Ogiso et al. (2002) argues that interreceptor domain preventing EGF from binding simultaneously to both
IV interactions may not be strong and well defined. How- domains. Alternatively, in the extended configuration
ever, peptides corresponding to this module have been (Figure 4C), bivalent EGF binding to both domains I and
shown to interact with ErbB receptors in vitro and in vivo III pulls domains II and IV apart, preventing their interac-
(Berezov et al., 2002), providing experimental support for tion with one another. In other words, the intramolecular
a domain IV role in dimerization. It therefore appears domain II/IV interaction occurs at the cost of high-affinity
that both arms of the intramolecular autoinhibitory inter- (bivalent) ligand binding, and vice versa. According to
action seen in Figure 1 (primarily modules C1IIb and C1IVd) this model, mutations that disrupt the intramolecular
may cooperate to drive EGF receptor homodimerization domain II/IV interaction should promote high-affinity li-
following a ligand-stabilized domain rearrangement of gand binding. Indeed, Elleman et al. (2001) showed that
the sort presented in Figure 4. deletion of domain IV from sEGFR increases its EGF
Autoinhibition of EGF Receptor Dimerization
513
binding affinity. Furthermore, as shown in Figure 5, indi-
vidual mutations that disrupt the domain II/IV interaction
increase EGF binding affinity. When all side chain-medi-
ated hydrogen bonds between domains II and IV (Figure
1B) are disrupted, in a D563A/H566A/K585A triple mu-
tant, EGF binds with a KD (measured with BIAcore) of
50 nM (⫾ 6 nM), similar to the value measured (⬇30
nM) when domain IV was deleted (Elleman et al., 2001).
Disruption of domain II/IV interactions increases EGF
binding affinity by almost 3-fold compared with our Sf9
cell-derived wild-type sEGFR (KD ⫽ 135 ⫾ 9.9 nM), and
by 9-fold compared with the KD (⬇400 nM) that we and
others have measured for sEGFR from mammalian cells
(Elleman et al., 2001; Lemmon et al., 1997). These data
support the hypothesis that the intramolecular domain
II/IV interaction in sEGFR inhibits ligand binding by re-
straining the positions of domains I and III.
The effect on EGF binding of disrupting domain II/IV
interactions is less dramatic than expected from a static
view of the autoinhibited sEGFR structure. The intramo-
lecular domain II/IV interaction does not reduce ⌬G for
ligand binding by more than 1 to 2 kcal/mole (corre-
sponding to a 5- to 30-fold enhancement of ligand bind-
ing upon domain IV deletion). If this is taken as an esti-
mate of ⌬G for the domain II/IV interaction, it can be
argued that 80%–97% of unliganded sEGFR molecules
will be in the autoinhibited state at equilibrium and 3%–
20% will be in the extended configuration. Each sEGFR
molecule will dynamically sample both configurations.
When EGF is added, it will bind preferentially to the
3%–20% of sEGFR molecules in the extended configu-
ration (Figure 4C), where EGF can contact domains I
and III simultaneously and therefore binds with higher
affinity. EGF binding will trap this extended configuration
and drive the equilibrium so that more unliganded, au-
toinhibited, EGFR molecules undergo the domain re-
arrangement schematized in Figure 4. Since the ex-
tended form of sEGFR (with the dimerization arm
exposed) is the form that dimerizes, its accumulation in
the presence of EGF will serve to promote dimerization.
Thus, EGF does not need to induce specific conforma-
tional changes in the domain II/IV contact site but in-
stead may drive EGFR dimerization simply by biasing
the conformational exploration of a dynamic, flexible,
multidomain structure.
Additional Ligand-Induced Structural Changes Are
Required to Drive Dimerization and Define
Dimerization Specificity
Exposure of the domain II dimerization arm is required
for sEGFR to dimerize but is not sufficient. A form of
sEGFR in which the dimerization arm is constitutively
exposed (by domain IV deletion) still does not dimerize
unless EGF is added (Elleman et al., 2001). Similarly,
mutations that disrupt the domain II/IV interface do not
diminish the ligand dependence of sEGFR dimerization
in vitro or EGF receptor activation in vivo (data not
shown).
These observations argue that EGF binding must also
induce other more subtle structural alterations to pro-
mote receptor•receptor interactions. Garrett et al. (2002)
identified two regions of domain II in addition to the
dimerization arm that interact across the dimer interface.
One is close to the N terminus of domain II, and the
other is immediately C-terminal to the dimerization arm
(Figure 4D). Efficient sEGFR dimerization presumably
requires the correct spatial relationship between these
individual contact points to allow them to cooperate
at the dimer interface. EGF binding (or dimerization)
induces a significant alteration in the domain II confor-
mation to achieve this, as can be seen by comparing
Figure 4C (which represents our sEGFR structure follow-
ing the rigid-body rearrangement described above) and
Figure 4D (where coordinates of the activated dimer
structure [Ogiso et al., 2002] were used for domains I,
II and III). The change in domain II appears to involve
an outward (multiply articulated) bend that is spread
over the five C1 modules of domain II (C1IIa to C1IIe). This
bend rotates the dimerization arm (in module C1IIb) into
a horizontal position (in Figure 4D) and rotates the most
C-terminal C1 modules of domain II toward the dimer
interface (away from domain III).
The structures of sErbB2 (Cho et al., 2003) and sErbB3
(Cho and Leahy, 2002) also argue that exposure of the
dimerization arm is insufficient for receptor dimerization.
The dimerization arm is constitutively exposed in sErbB2
(there is no known ErbB2 ligand), yet sErbB2 remains
monomeric under all conditions studied (Ferguson et
al., 2000; Horan et al., 1995; Landgraf and Eisenberg,
2000) and did not crystallize as a dimer (Cho et al., 2003).
Furthermore, the structure of autoinhibited sErbB3 (Cho
and Leahy, 2002) argues that neuregulin binding should
expose its dimerization arm, yet numerous studies show
that ligand-bound sErbB3 does not homodimerize (Cho
and Leahy, 2002; Ferguson et al., 2000; Horan et al.,
1995; Landgraf and Eisenberg, 2000; Tzahar et al., 1997).
The multiple receptor contact points equivalent to those
that cooperate in sEGFR dimerization may not be ar-
ranged in sErbB2 and sErbB3 in a way that can promote
efficient homodimerization. Instead, it can be specu-
lated that the arrangements of interreceptor interaction
sites in sErbB2 and in neuregulin-bound sErbB3 might
complement one another, so that formation of Erb-
B2•ErbB3 heterodimers is promoted to the exclusion of
either homodimer. Indeed, ErbB2•ErbB3 heterodimers
have been reported to occur in preference to either ho-
modimer both in vivo (Yarden and Sliwkowski, 2001) and
in vitro (Ferguson et al., 2000). The relative orientations
of potential receptor•receptor interactions sites could
thus contribute to ErbB receptor heterodimerization
specificity.
This possibility is illustrated in principle by differences
between the structures of autoinhibited sEGFR and
sErbB3 (Figure 6). Domain I and the N-terminal 65 amino
acids of domain II have significantly different orienta-
tions in the sEGFR and sErbB3 extracellular regions.
Following ligand-stabilized extension of these struc-
tures, according to the scheme in Figure 4, the spatial
relationship between interreceptor contact points in do-
main II and between domain II and domain IV could be
significantly different for sErbB3 and sEGFR, and this
might be reflected in their homodimerization properties.
Molecular Cell
514

Figure 6. Different Domain Orientations in Autoinhibited Structures of sEGFR and sErbB3

The structures of autoinhibited sEGFR and sErbB3 (Cho and Leahy, 2002) were overlaid by superimposition of domains III and IV.
(A) The sEGFR/sErbB3 overlay is shown in the orientation used for sEGFR in Figure 1A. sEGFR is gray; sErbB3 is magenta.
(B) The same sEGFR/sErbB3 overlay is shown in an orthogonal view to illustrate the different orientation of the domain I/II fragment in the
two structures. The difference is approximated by a 60⬚ rotation about the axis marked with a black circle.

A Possible Inactive EGF Receptor Dimer
The structure described here represents an inactive, or
autoinhibited, form of the EGFR extracellular region, and
only one sEGFR molecule is found in the asymmetric
unit. However, we do observe an interesting crystallo-
graphic dimer that is physiologically feasible and could
possibly represent a preformed inactive dimer of the
EGF receptor, of the sort reported by several in vivo
studies (Gadella and Jovin, 1995; Moriki et al., 2001;
Sako et al., 2000)—although it does not form in solution.
This dimer juxtaposes domain IV of two adjacent sEGFR
molecules (Figure 7). However, interreceptor interac-
tions occur almost exclusively between domain II of one
receptor molecule and domain IV of its dimeric partner,
burying 1170 Å2 of solvent-accessible surface (585 Å2
on each molecule) in an interface with a relatively low
shape complementarity parameter (Lawrence and Col-
man, 1993) of 0.55. The region of domain II that mediates
these contacts is the same ␤-hairpin/loop responsible
for intramolecular autoinhibitory interactions (Figure 1)
and for intermolecular interactions in the activated re-
ceptor dimer (Figure 4D). Specifically, the side chains
of N247 and D254 from the domain II dimerization loop of
one molecule make hydrogen bonds with the backbone
carbonyl of residue 528 and the ⫺NH of residue 526,
respectively, in domain IV of the adjacent molecule in
the dimer (Figure 7). If a dimer of the sort shown in
Figure 7 does occur at the cell surface, the so-called
dimerization arm in domain II (Ogiso et al., 2002) would
play a role in all different states of the receptor: stabiliz-
ing autoinhibited monomers, activated dimers, and inac-
tive dimers.
Implications for the Design of EGF
Receptor Antagonists
Finally, the structure presented in Figure 2 suggests
approaches for designing EGF receptor antagonists that
could be of significant therapeutic value. Ligands that
compete effectively with EGF for EGFR binding, but
which fail to promote the domain rearrangement de-
picted in Figure 4, would be potent antagonists. A mono-
valent ligand that binds with very high affinity to only
domain I or domain III (but not both) would fulfill these
criteria. This may be how potato carboxypeptidase in-
hibitor (PCI), which structurally resembles a mini EGF,
antagonizes EGFR activation (Blanco-Aparicio et al.,
1998). Argos, the secreted antagonistic ligand of the
Drosophila EGF receptor (Jin et al., 2000), could function
similarly. A domain I-targeted monovalent antagonist of
this sort would form a complex similar to that seen in
Figure 2.
An alternative to this approach would be to generate
bivalent EGFR antagonists that bind to both domain I
and domain III, but utilizing interfaces that lock the re-
ceptor into the autoinhibited configuration shown in Fig-
ures 1A and 2. A ligand that binds simultaneously to the
EGF binding site of domain I and to the N-terminal end
of the domain III ␤-helix (filling in the space between
EGF and domain III in Figure 2) could achieve this.
Conclusions
The structure of an EGF•sEGFR complex at pH 5 repre-
sents the inactive, monomeric form of the EGF receptor
extracellular region, with EGF bound weakly to only one
of its two binding sites. In this state, the second ligand
binding site (domain III) is held at a significant distance
from monovalently bound EGF by an intramolecular do-
main II/IV tether that also occludes the domain II dimer-
ization arm. Simultaneous binding of ligand to both bind-
ing sites in sEGFR requires that the intramolecular
domain II/IV tether be disrupted—thus exposing the di-
merization arm. Similarly, exposure of the sEGFR dimer-
ization arm, by breaking intramolecular domain II/IV in-
teractions, increases EGF binding affinity. We propose
a model in which the unliganded EGF receptor extracel-
lular region constantly samples autoinhibited (dimeriza-
Autoinhibition of EGF Receptor Dimerization
515

Figure 7. Crystallographic Dimer of Inactive sEGFR

(A) Stereo view of the crystallographic dimer. One protomer (shaded gray, with domain IV black) is behind, while the other (with domains
colored as in Figure 1A) is in front. EGF is colored magenta in both protomers. N and C termini and individual domains are all labeled. Three
domain II side chains are shown: Y246, which is involved in the intramolecular domain II/IV interaction, plus N247 and D254, which make
hydrogen bonds to domain IV of the adjacent molecule in this dimer (involving the carbonyl oxygen of N528 and amide nitrogen of V526,
respectively, shown in ball and stick representation). If this dimer exists in vivo, the plane of the membrane would pass under the structure,
perpendicular to the page.
(B) The dimer shown in (A) has been rotated by 90⬚ about the horizontal axis shown to give a view facing up from the presumed membrane
plane. The C termini project out of the page. Regions of intermolecular interaction between N247 and D256 on domain II and the backbone
of domain IV are circled.

tion-incompetent) and extended (dimerization-compe- Experimental Procedures

tent) configurations, existing primarily (⬎80%–97%) in
the autoinhibited form at equilibrium. EGF will bind se-
lectively to the extended form of the receptor by con-
tacting both domains I and III, and will shift the equilib-
rium so that an increasing proportion of the receptor
becomes dimerization competent. EGF receptor dimers
are then stabilized by interactions involving the domain
II dimerization arm, as well as additional contacts pro-
moted by EGF binding. This proposed mechanism of
ligand-induced receptor dimerization differs dramati-
cally from the more common RTK-activation mechanism
described in the Introduction, where bound growth fac-
tor makes bivalent contacts across the dimerization in-
terface.
The recent structures of sErbB3 (Cho and Leahy, 2002)
and sErbB2 (Cho et al., 2003), as well as sEGFR in both
active (Garrett et al., 2002; Ogiso et al., 2002) and in-
active (this study) forms, also suggest new (entirely
receptor-mediated) hypotheses for ErbB receptor het-
erodimerization that can now be tested rationally. Fur-
thermore, building on existing competitive inhibitors of
EGF receptor activation, the insight provided by these
structures into how EGF and related growth factors acti-
vate their receptors should provide a sound basis for
future design of ErbB receptor antagonists that will be
clinically important.
Protein Expression and Purification
sEGFR was produced and purified from baculovirus-infected Sf9
cells exactly as described (Ferguson et al., 2000) and was used
without modification of its glycosylation state. Recombinant human
EGF was purchased from Intergen Inc. and used without further
purification.
Crystallization and Data Collection
sEGFR was buffer exchanged into 25 mM HEPES (pH 8.0), con-
taining 50 mM NaCl, and crystallized by the hanging drop method
from a drop containing equal parts of a 240 ␮M sEGFR solution
(containing a 1.2-fold molar excess of EGF) and reservoir solution.
Crystals (Table 1) grew in 4 to 6 weeks from 12% PEG3400, 200
mM (NH4)2SO4, 50 mM MgSO4, and 50 mM sodium citrate, at pH 5.0,
to approximate dimensions 0.08 ⫻ 0.1 ⫻ 0.8 mm. Silver-stained
SDS-PAGE gels of extensively washed crystals confirmed the pres-
ence of both full-length sEGFR and EGF. Crystals were stabilized
by soaking for 12 hr in 20% PEG3400, 12% glycerol, containing 200
mM (NH4)2SO4, 50 mM MgSO4, and 50 mM sodium citrate, at pH 5.0,
and were flash frozen in liquid nitrogen directly from this stabilization
solution. Data were collected at CHESS beamline F1, using an ADSC
CCD detector, and were processed using MOSFLM and SCALA
(CCP4, 1994).
Structure Determination and Refinement
To generate search models for molecular replacement, the sErbB3
coordinates (Cho and Leahy, 2002) were modified to remove all
nonidentical side chains. A solution was found using the sErbB3
domain III/IV pair with AMORE (CCP4, 1994). With the positions of
Molecular Cell
516
these domains fixed, domain I was found similarly. Neither domain
II nor EGF could be found by molecular replacement. Following
several rounds of model building using O (Jones et al., 1991) and
refinement using CNS (Brunger et al., 1998), interpretable density
for the remaining portions of sEGFR and EGF could be seen in
composite simulated-annealing omit-maps (calculated with CNS).
The final stages of refinement employed TLS refinement (Winn et
al., 2001) with anisotropic motion tensors refined for each of the
four domains plus EGF, using REFMAC5 (CCP4, 1994). Significant
uninterpretable density, presumed to represent oligosaccharide (our
protein was fully glycosylated), is not accounted for by the model.
Figures were generated using GRASP (Nicholls et al., 1991), MOL-
SCRIPT (Kraulis, 1991), and Raster3D.
BIAcore Studies of EGF Binding by sEGFR
Surface plasmon resonance binding experiments, performed using
a BIAcoreX instrument, were performed (unless stated otherwise)
in 10 mM HEPES buffer (pH 8.0), containing 150 mM NaCl, 3 mM
EDTA, and 0.005% Tween 20 at 25⬚C. EGF was immobilized on a
CM5 BIAcore chip, and binding studies were performed and ana-
lyzed exactly as described (Ferguson et al., 2000). For pH titrations,
sEGFR protein was injected on to the EGF surface at 150 nM in 50
mM NaCl, 3 mM EDTA, containing 100 mM of the relevant buffer.
Buffers used at different pH values were Tris-HCl (pH 7.0, 8.5),
HEPES (pH 8.0, 7.5), MES (pH 6.5), cacodylate (pH 6.0), and acetate
(pH 5.5, 5.0).
Acknowledgments
We thank T. Garrett, T. Burgess, S. Yokoyama, and their colleagues
for discussions and for generously sharing information prior to publi-
cation; J. Schlessinger, G. Van Duyne, and members of the Lemmon
laboratory for valuable discussions and comments on the manu-
script; M. Becker for assistance with beamline X-25 at the National
Synchrotron Light Source, Brookhaven; and the staff of beamline
F1 at the Cornell High-Energy Synchrotron Source (CHESS), which
is supported by the NSF and the NIH. This work was supported by
National Cancer Institute grants R01-CA79992 and R21-CA87182
(to M.A.L.), K01-CA092246 (to K.M.F.), R01-CA090466 (to D.J.L.);
fellowships from the U.S. Army Breast Cancer Research Program
to K.M.F. (DAMD17-98-1-8228) and M.B.B. (DAMD17-01-1-0363),
and from the American Cancer Society (PF-00-174-01-TBE) to
J.M.M.; as well as funds from the Damon Runyon Cancer Research
Fund (M.A.L.), the Howard Hughes Medical Institute (D.J.L.), and
the Burroughs Wellcome Foundation (K.M.F.).
Received: October 2, 2002
Revised: December 18, 2002
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