Ivan Budyak
The transmembrane + juxtamembrane Epidermal Growth Factor
Receptor (EGFR).
February 2006
I. Introduction
The epidermal growth factor receptor (EGFR) mediates many cellular responses in
normal biological processes and in pathological states. It regulates the intracellular effects of
ligands such as epidermal growth factor (EGF), transforming growth factor α (TGFα) and the
neuregulins. The stimulation of the EGFR results in activation of multiple cell signalling
pathways. It plays a critical role in the control of a host of cellular activities including cell
division, differentiation and migration. EGFR overexpression or disregulation is implicated
(directly and/or indirectly) in a variety of mammal cancers [1].
The EGFR subfamily consists of four closely related human receptors (the EGFR itself
(also referred to as ErbB1), HER2 (or ErbB2/Neu), HER3 (or ErbB3) and HER4 (or ErbB4))
and one each from D.melanogaster and C.elegans. They can interact with each other in a
ligand-dependent fashion forming heterodimers [2].
It is well established that ligand binding induces dimerization of the monomeric EGFR
[3]. Thus, receptor display, dynamics, affinity and competency play important regulatory roles
because the EGFR appears to exist in two different affinity classes at the cell surface [4].
The EGFR is an 1186-residue transmembrane protein with N-linked glycosylation. It
is synthesized from a 1210-resudue-precursor peptide; then the signal peptide is cleaved and
the protein goes into the cell membrane. The molecular mass of the EGFR is about 170 kDa
[5].
By convention the EGFR is subdivided into 8 domains (fig. 1.1). The EGFR
extracellular portion (or ectodomain, residues 1-621) consists of domains L1, CR1, L2 and
CR2.
Fig. 1.1. Schematic representation of domains of the EGFR. L1 and L2 - ligand-binding domains 1 and 2, CR1 and
CR2 - cysteine-rich domains 1 and 2, JM - juxtamembrane domain, CT - carboxy-terminal domain (taken from [1]).
The recently solved structure [6] shows that L1 and L2 domains responsible for ligand
binding consist of so-called β-solenoid or β-helix folds. CR1 and CR2 domains consist of a
number of small modules held together by disulfide bonds. Interestingly, a large loop of the
CR1 domain makes contact with the CR1 domain of the other receptor in dimer.
The intracellular part of the EGFR (687-1186) consists of kinase domain (that is a
tyrosine-kinase) and C-terminal domain. The structure of the EGFR tyrosine-kinase domain
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(residues 672-998) is mainly similar to other tyrosine kinases [7]; it has the ATP-binding site
where the γ-phosphate can be transferred to the acceptor tyrosine residues of exogenous
substrates and C-terminal domain of the EGFR (in a trans manner).
The carboxy-terminal domain of the EGFR contains Tyr residues where
phosphorilation modulates EGFR-mediated signal transduction. There are also some Ser/Thr
residues where phosphorilation has been inferred to be important for the receptor
downregulation process and sequences thought to be necessary for endocytosis. Moreover, the
residues 984-996 have been identified as a binding site for actin [8] and may be involved in
receptor oligomerization and clustering.
The residues 622-644 are assigned to the transmembrane region in accordance with
numerous prediction methods. The juxtamembrane domain (645-686) is considered to have a
number of regulatory functions (e.g. in downregulation and ligand-dependent receptor
internalization). The amphipathic sequence with its positive residue density is predicted to be
between 653 and 661 residues [9]. NMR studies of the peptide roughly corresponding to the
EGFR transmembrane domain (626-647) indicated that it is α-helical [10]. This suggests that
the α-helix continues into the juxtamembrane domain. The recent findings have shown that
the juxtamembrane domain is mainly α-helical in the membrane-bound state [11] and
disordered in solution [12].
II. Materials and methods
II.1. DNA manipulations
For all manipulations with DNA E.coli strain Top10 (Novagen, Germany) was used. All enzymes were
from New England Biolabs, USA.
PCR procedures
All constructs were amplified by standard PCR technique from the construct containing full-length
human egfr in pFastBac1 (received from Dr. G.Anderson).
Cloning into vectors
The newly designed and amplified egfr gene regions were ligated into pBlunt (Invitrogen, USA) vector
and then sequenced. pBlunt plasmids containing correct inserts were then cut by the corresponding restriction
enzymes and ligated into expression vector pET 27b+ (Novagen, Germany).
II.2. Transformation
Transformation of E.coli
A tube of frozen chemically competent cells was thawed on ice. The DNA solution (1-10 ng) was added
to the competent cells and incubated on ice for 30 min. Each transformation tube was heat-pulsed for 90 sec at
+42°C and placed on ice for 2 min. Room temperature S.O.C. ® (Invitrogen, USA) medium was added to each
tube to a final volume of 500-1000 μl and incubated at +37°C for 60 min. 50-500 μl of each transformation were
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spread onto LB-agar plate containing appropriate antibiotic (50 μg/ml kanamycin). Then the plates were
incubated overnight at +37°C.
II.3. Expression
Expression in E.coli
For bacterial expression E.coli BL21(DE3) Codon Plus RP (all strains from Navagen, Germany) cells
transformed by the corresponding plasmid containing the gene of interest were used.
For expression in flasks the cells were grown in dYT medium containing 50 μg/ml kanamycin at +37°C.
When OD600 reached 0.6-0.8, IPTG (0.5 mM) was added to induce protein expression. After 4 hours of
expression the cells were harvested by centrifugation (10 000g). The cell pellets were normally stored at -20°C.
For expression in 5L fermenter the cells were grown in minimal medium containing 50 μg/ml
kanamycin at +37°C. When OD600 reached 3-4, IPTG (0.5 mM) was added to induce protein expression and the
temperature was lowered to +28°C. After 24 hours of expression the cells were harvested by centrifugation (10
000g). The cell pellets were normally stored at -20°C.
II.3. Purification
The cell pellet was resuspended in 300 mM NaCl, 50 mM Tris-HCl pH 8.5. The suspension was passed
through a French press (SLM Aminco) and the soluble and insoluble fractions were separated by centrifugation
(100 000 g, 1 h, +4°C). The pellet was resuspended in 3000 mM NaCl, 50 mM Tris-HCl pH 8.5, 4% (w/v) OG
and the suspension was incubated for about 16 h at +4°C. Another centrifugation step was carried out to isolate
solubilised membranes from cell debris (100 000 g, 1 h, +4°C). The supernatant was applied to a Chelating
Sepharose (Amersham Biosciences) pre-loaded with Cu2+ ions and pre-equilibrated with 300 mM NaCl, 50 mM
Tris-HCl pH 8.5, 0.88% (w/v) OG. The column was extensively washed with the same buffer + 50 mM
imidazole, and the protein was eluted with the same buffer + 500 mM imidazole. The eluate was applied to a
Strep-Tactin Sepharose (IBA, Germany) pre-equilibrated with 300 mM NaCl, 50 mM Tris-HCl pH 8.5, 0.88%
(w/v) OG and immediately eluted with the same buffer + 2.5 mM D-desthiobiotin. All fractions were analyzed
by SDS-PAGE and Western-blot.
III. Results and discussion (the transmembrane EGFR (615-686 a.a.))
The construct was designed to contain the transmembrane domain itself (residues 626644) and the amphipathic sequence of the juxtamembrane (residues 653-661) of the EGFR as
well as to carry an N-terminal 7His-tag (HHHHHHH) and C-terminal Strep-tag
(WSHPQFEK). It was amplified by standard PCR, cloned into pET 27b+ vector for bacterial
expression and sequenced. The expression was detected on Western-blotting for E.coli strain
BL21(DE3) Codon Plus RP. The expression conditions were further optimized to obtain the
highest protein yield (Fig. 3.1).
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Fig. 3.1 His-blotting (right) and Strep-blotting (left) of crude E.coli lysates before induction
(0), 4h, 16h and 24h after induction (correspondingly). The expression temperature was
+37C (RED) or +28C (BLUE).
The purification procedure included Me-affinity chromatography in OG as the first
step immediately followed by Strep-Tactin chromatography, and then reverse phase in TFA
and acetonitrile gradient (Fig. 3.2).
Fig. 3.2. Purified tj-EGFR: 1 – SDS-PAGE, 2 – Hisblotting, 3 – Strep-blotting
CD in detergents in lipids (Fig. 3.3) and preliminary NMR spectra in OG and SDS
(data not shown) were recorded. The secondary structure prediction is in the good agreement
with the presence of the transmembrane helix and random coil conformation of the
juxtamembrane domain in detergent micelles. Moreover, it was possible reconstitute tj-EGFR
into liposomes (data not shown), which is important for further studies of this fragment in
native lipid environment.
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Fig. 3.3. CD spectra of the tj-EGFR in water / 95% (v/v) TFE / 50 mM NaP pH 6.0, 100 mM
detergent
IV. References
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growth factor receptor: mechanisms of activation and signalling. Exp.Cell Res.
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2. Olayioye MA, Neve RM, Lane HA, Hynes NE: The ErbB signaling network:
receptor heterodimerization in development and cancer. EMBO J 2000, 19:31593167.
3. Schlessinger J: Cell signaling by receptor tyrosine kinases. Cell 2000, 103:211-225.
4. Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase
activity. Cell 1990, 61:203-212.
5. Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y,
Libermann TA, Schlessinger J, .: Human epidermal growth factor receptor cDNA
sequence and aberrant expression of the amplified gene in A431 epidermoid
carcinoma cells. Nature 1984, 309:418-425.
6. Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, Kim JH, Saito K, Sakamoto A,
Inoue M, Shirouzu M, Yokoyama S: Crystal structure of the complex of human
epidermal growth factor and receptor extracellular domains. Cell 2002, 110:775787.
7. Stamos J, Sliwkowski MX, Eigenbrot C: Structure of the epidermal growth factor
receptor kinase domain alone and in complex with a 4-anilinoquinazoline
inhibitor. J Biol Chem 2002, 277:46265-46272.
8. den Hartigh JC, van Bergen en Henegouwen PM, Verkleij AJ, Boonstra J: The EGF
receptor is an actin-binding protein. J Cell Biol 1992, 119:349-355.
9. Seligman L, Manoil C: An amphipathic sequence determinant of membrane
protein topology. J Biol Chem 1994, 269:19888-19896.
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10. Rigby AC, Grant CW, Shaw GS: Solution and solid state conformation of the
human EGF receptor transmembrane region. Biochim Biophys Acta 1998,
1371:241-253.
11. Choowongkomon K, Carlin CR, Sonnichsen FD: A structural model for the
membrane-bound form of the juxtamembrane domain of the epidermal growth
factor receptor. J Biol.Chem 2005, 280:24043-24052.
12. Choowongkomon K, Hobert ME, He C, Carlin CR, Sonnichsen FD: Aqueous and
micelle-bound structural characterization of the epidermal growth factor
receptor juxtamembrane domain containing basolateral sorting motifs. J
Biomol.Struct.Dyn. 2004, 21:813-826.
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