(Received for publication, April 21, 1995; and in revised form, June 27, 1995)
From the
The finding that human epidermal growth factor (hEGF) and human
transforming growth factor (hTGF) bind with similar affinity to
the human EGF receptor but differ in their affinity for the chicken EGF
receptor was used as a model system to study ligand-receptor
interaction of EGF receptor agonists. We previously constructed
domain-exchange mutants of hEGF and hTGF
and found that the region
COOH-terminal of the sixth cysteine residue in hTGF
is important
for high affinity binding to the chicken EGF receptor (Kramer, R. H.,
Lenferink, A. E. G., Lammerts van Bueren-Koornneef, I., van der Meer,
A., van de Poll, M. L. M., and van Zoelen, E. J. J.(1994) J. Biol.
Chem. 269, 8708-8711). To analyze this domain in more
detail, we now constructed four additional chimeras in which either the
region between the sixth cysteine residue and the highly conserved
Leu-47 was exchanged or the region COOH-terminal of Leu-47. A mutant in
which the latter region in hEGF was replaced by hTGF
(designated
E6ET) showed intermediate binding affinity, whereas replacement of the
former region in hEGF by hTGF
was sufficient to generate a mutant
(designated E6TE) with a similar high affinity for the chicken EGF
receptor as wild type hTGF
. Furthermore, a deletion mutant of hEGF
lacking three COOH-terminal amino acids, EGF50, showed intermediate
binding affinity for the chicken EGF receptor similar to E6ET, but upon
additional deletions (EGF49 and EGF48), this initial gain in affinity
was lost. A systematic analysis of the region between the sixth
cysteine residue and Leu-47 showed that the low affinity of hEGF for
the chicken EGF receptor is mainly due to the presence of Arg-45.
Replacement of the positively charged Arg-45 by Ala, the corresponding
amino acid in hTGF
, was sufficient to generate a mutant growth
factor with high affinity for the chicken EGF receptor. This indicates
that in hEGF Arg-45 may play an important role in receptor binding. A
model is proposed in which positively charged amino acids close to or
within the receptor recognition site of hEGF prohibit high affinity
binding to the chicken EGF receptor due to electrostatic repulsion of
positively charged amino acids in the putative ligand binding domain of
the chicken EGF receptor.
Human epidermal growth factor (hEGF) ()and human
transforming growth factor (hTGF)
belong to the same family of
growth factors. They both bind with high affinity to the human EGF
receptor, but hEGF has a 10-50-fold lower affinity for the
chicken EGF receptor than hTGF
(1) . All members of the EGF
family are characterized by the presence of six identically spaced
cysteine residues, which form three intramolecular disulfide bridges.
Together with some highly conserved glycine residues they are essential
for the correct three-dimensional structure of the growth factor and
for high affinity binding to the EGF
receptor(2, 3, 4) . Several other amino acids
in hEGF like Leu-47 (Leu-48 in hTGF
) and Arg-41 (Arg-42 in
hTGF
), which are not involved in maintaining structural integrity,
have been shown to be crucial for high affinity binding to the EGF
receptor, which suggests that they form part of the binding
domain(5, 6, 7, 8, 9) . The
crystal structure of hEGF or hTGF
is not available, and most of
the information on the structure of these growth factors has come from
detailed
H NMR studies. Based on the observation that amino
acids surrounding the second cysteine residue are in close contact with
amino acids near the sixth cysteine residue, it has been postulated
that Tyr-13/Leu-15/His-16 together with Arg-41/Gln-43/Leu-47 form the
binding site in hEGF(10, 11, 12) . The exact
region involved in binding to the receptor is still not known, however,
and this has hampered the design of receptor antagonists.
To gain
more insight in the way hEGF and hTGF bind to their receptor, we
recently used the difference in binding affinity of these growth
factors for the chicken EGF receptor as a model system. A total of 10
hEGF/hTGF
chimeras were constructed in which regions bordered by
the highly conserved cysteine residues were exchanged, and their
relative binding affinity for the chicken EGF receptor was assessed (13) . Introduction of the region COOH-terminal of the sixth
cysteine residue of hTGF
into hEGF appeared to be sufficient to
confer high affinity binding characteristics to hEGF, and, in line with
this, an exchange of the same region in hTGF
with the
corresponding hEGF sequence caused hTGF
to lose its high affinity
for the chicken EGF receptor. These data indicate that the
COOH-terminal region in EGF receptor agonists plays an important role
in receptor binding. In a recent
H NMR study(14) ,
it has been shown that this region of hTGF
is flexible in the
unbound molecule but that its mobility is strongly reduced upon
receptor binding, which emphasizes again the role of the COOH-terminal
domain in receptor-ligand interaction.
In the present study, we
investigated in more detail which amino acids in the COOH-terminal
domain are responsible for high affinity binding to the chicken EGF
receptor. To do so, the binding characteristics of an additional 7
hEGF/hTGF exchange mutants and 3 COOH-terminal truncated forms of
hEGF were investigated. A single amino acid exchange, Arg-45 to Ala,
was found to be sufficient to generate an hEGF mutant with high
affinity for the chicken EGF receptor.
For the construction of T6TE and T6ET
(for definition, see Fig. 1. and ``Results''), the
gene coding for hTGF was cleaved at the sixth cysteine codon by DraIII and at the 3`-end by SalI. The gap was filled
in using synthetic double-stranded oligonucleotides. For the
construction of E6TE, hEGF was digested at the fourth cysteine codon by SphI, and T6TE was cleaved at the sixth cysteine codon by DraIII. The fragments were ligated to a double-stranded
oligonucleotide spanning the region between the SphI site and
the DraIII site. To generate E6ET, the SphI site and
the SalI site of the hEGF construct were used, and the gap was
filled in using two double-stranded oligonucleotides. DNA constructs
that code for hEGF mutants truncated at the COOH-terminal end, EGF50,
EGF49, and EGF48, were made by polymerase chain reaction using
pEZZ/FX/EGF as a template. Three different antisense primers were
designed such that the generated polymerase chain reaction products
contained either the codon for Trp-50, Trp-49, or Lys-48 at their
3`-end followed by a stop codon and the SalI recognition site.
For all constructs, the same sense primer was used, which annealed 5`
of the EcoRI site of pEZZ/FX/EGF. Polymerase chain reaction
products of the correct length were isolated and cloned into the
modified EcoRV site of pT7blue T (Novagen Inc., Madison, WI)
and subsequently transferred to pEZZ18 by EcoRI-SalI
digestion. Point mutations in the hEGF gene were generated using the
Altered Sites
II in vitro mutagenesis system
(Promega). All pEZZ18 mutant constructs were verified by DNA
sequencing.
Figure 1:
Schematic representation of
mutant growth factors. Amino acids COOH-terminal of the sixth cysteine
residue are indicated: circles, hEGF-derived amino acids; boxes, hTGF-derived amino acids; diamonds, the
conserved sixth cysteine residue and Leu-47 (Leu-48 in hTGF
). The
sequence NH
-terminal of the sixth cysteine residue is
either hEGF (E) or hTGF
(T).
Figure 2: Identification of ZZ/FX/growth factor fusion proteins by SDS-polyacrylamide gel electrophoresis and Western blotting. Aliquots of 10 µl of unpurified periplasm were run on a 12.5% SDS-polyacrylamide gel under non-reducing (A) or reducing (B) conditions. Proteins were transferred to nitrocellulose, and the Western blots were probed with rat-anti-goat antibody linked to horseradish peroxidase: wild type hEGF, lanes1 and 6; Q43E, lanes2 and 7; Y44H, lanes3 and 8; R45A, lanes4 and 9; control periplasm (pEZZ18 without insert), lane5.
Figure 3:
RP-HPLC chromatogram of the hEGF point
mutant Q43E (A) and biological activity in the RP-HPLC
fractions (B). Elution was carried out with a linear gradient
of CHCN in 0.1% trifluoroacetate at a flow rate of 1
ml/min. Biological activity present in the column fractions was
determined in a binding competition assay with
I-mEGF.
To determine the differential binding characteristics of the
mutant growth factors for the human and the chicken EGF receptors, all
recombinant proteins were eventually calibrated to give the same 50%
competition of I-mEGF binding to HER-14 cells as wild
type mEGF. An example of this is shown in Fig. 4for the EGF
point mutants Q43E, Y44H and R45A.
Figure 4:
Inhibition of binding of I-mEGF to HER-14. The binding activity of the mutant
growth factors was calibrated to give the same competition of binding
of
I-mEGF to HER-14 as wild type mEGF. Representative
curves of the hEGF point mutants Q43E, Y44H, and R45A are shown after
the final calibration. Experiments were repeated at least three
times.
Figure 5:
Inhibition of binding of I-mEGF to CER-109 by hEGF/hTGF
mutants. The binding
activity of the mutant growth factors was calibrated based on their
ability to give 50% competition of
I-mEGF binding to
HER-14 (see Fig. 4). Their relative affinity for the chicken EGF
receptor was subsequently measured in a binding competition assay on
CER-109. The concentrations of the growth factors are thus expressed as
ng mEGF equivalents/ml. Representative curves of at least three
experiments are shown.
Because the binding affinity of all recombinant proteins was
calibrated based on their ability to give 50% competition of I-mEGF binding to HER-14 cells, any difference in
relative mitogenic activity is most easily detected on the same cells.
In Fig. 6is shown that all growth factors were biologically
active when tested for their ability to stimulate
[
H]thymidine incorporation into serum-starved
HER-14 cells. Most of the mutant growth factors were similarly active
as wild type hEGF or hTGF
, but one mutant, EGF48, induced a
slightly higher mitogenic response. The absolute affinity of EGF48,
however, was calculated to be
2-fold lower than the affinity of
the wild type growth factors (Table 1). We therefore expect that
EGF48 will induce a similar mitogenic response as wild type hEGF when
assayed on a protein basis. Loss of binding affinity without a
concomitant decrease in mitogenic activity has been reported before by
Walker et al.(5) for mEGF in which Leu-47 was
mutated to Ala. In data to be published elsewhere, we will show that,
even on a protein basis, some of the hEGF/hTGF
chimeras
constructed previously by us (13) are truly superagonistic for
HER-14 when compared with wild type hEGF or hTGF
.
Figure 6:
Mitogenic response of HER-14. The relative
mitogenic activity of the mutant growth factors for HER-14 was assessed
by measuring [H]thymidine incorporation into
serum-starved cells 24 h after growth factor addition. Radioactivity
incorporated in the presence of 10% NCS was 197,600 ± 6,000 cpm
in A and 217,200 ± 3,000 cpm in B.
Radioactivity incorporated in control cells (without growth factor
addition) was 26,300 ± 1,800 cpm in A and 61,500
± 4,700 cpm in B. Representative curves of at least
three experiments are shown.
Mammalian EGF and TGF bind with similar high affinity to
the human EGF receptor, but their affinity toward the chicken EGF
receptor differs substantially(1) . Human EGF has a
10-50-fold lower affinity for the chicken EGF receptor than human
TGF
, and the affinity of mouse EGF is
5-fold lower than of
human EGF(1, 13) . We have previously used the
differential binding characteristics of hEGF and hTGF
as a model
to study ligand-receptor interactions(13) . A total of 10
chimeras of hEGF and hTGF
were constructed, and it was found that
chimeras with hTGF
sequences COOH-terminal of the sixth cysteine
residue all had a similar high affinity for the avian EGF receptor as
wild type hTGF
, whereas those having hEGF sequences in this region
showed EGF-like binding characteristics. This indicates the importance
of the COOH-terminal domain in discriminating between hEGF and
hTGF
.
To identify amino acids involved in high affinity binding
to the EGF receptor, a detailed analysis of the COOH-terminal domain of
hEGF was made in the present study. Here, we show that the low affinity
of human EGF for the avian EGF receptor is mainly due to the presence
of arginine on position 45. Replacement of the positively charged
Arg-45 for alanine, the corresponding amino acid in hTGF, was
sufficient to generate a hEGF mutant with high affinity for the chicken
EGF receptor. Thus far, point mutation studies of the carboxyl-terminal
region of hEGF and hTGF
have focused mainly on the highly
conserved Asp-46 and Leu-47 (Asp-47 and Leu-48 in hTGF
). Leu-47
and (less stringently) Asp-46 have been shown to be crucial for
receptor binding and
activation(5, 7, 9, 12) . By using a
domain exchange strategy, however, a systematic survey of the
importance of non-conserved amino acids can be made. The present
finding that Arg-45 discriminates between hEGF and hTGF
with
respect to their affinity for the chicken EGF receptor, implicates that
this amino acid lies close to or forms part of the receptor recognition
site.
Lax et al.(21) analyzed the differential
binding characteristics of the human and chicken EGF receptor using a
domain-exchange strategy, and they demonstrated that domain III of the
EGF receptor extracellular domain is most important for ligand
recognition. Within this region the sequence between amino acids 351
and 364 was found to be the epitope recognized by ligand-competitive
monoclonal antibodies(22) . The participation of this epitope
in the formation of a ligand binding site, however, was recently
questioned since exchange of this region in the human EGF receptor for
the corresponding sequence in the chicken EGF receptor did not alter
the affinity of mouse EGF or human TGF for the
receptor(23) . On the other hand, one might expect that the
epitope recognized by an antibody that competes with the natural ligand
for binding to the receptor lies close to the ligand binding site of
the receptor. Immediately COOH-terminal of the epitope two lysine
residues are found in the chicken EGF receptor that are not conserved
in the human EGF receptor (24) . For one of these lysine
residues, a conservative replacement (Arg) is found in the murine EGF
receptor; this amino acid is, therefore, less likely to discriminate
between the chicken and the mammalian EGF receptor. The second lysine
residue, however, is only found in the chicken EGF receptor, whereas in
the human EGF receptor this positively charged amino acid is replaced
by a negatively charged glutamate. We hypothesize that the lysine
residue on position 367 in the chicken EGF receptor lies close to or
forms part of the ligand binding domain and that the positively charged
Arg-45 in the putative receptor recognition site of hEGF prohibits the
interaction of hEGF with the chicken EGF receptor due to electrostatic
repulsion.
Other positively charged amino acids in hEGF that might
interfere with ligand-receptor interaction, are Lys-48 and Arg-53 in
the carboxyl-terminal tail of hEGF. Replacement of this region in hEGF
(KWWELR) for the corresponding uncharged sequence in hTGF (LA),
making E6ET, caused an increase in binding affinity. A similar
improvement of binding affinity was found upon truncation of the
carboxyl-terminal tripeptide ELR (EGF50) removing Arg-53 as the
COOH-terminal amino acid. Additional deletion of Trp-50, however,
resulted in a decrease in affinity for the chicken EGF receptor.
Besides a difference in charge distribution, there is also a difference
between hEGF and hTGF
in conformation of the COOH-terminal tail.
In hEGF, this region adopts an
-helix conformation involving
Leu-47-Glu-51, whereas in hTGF
the COOH-terminal dipeptide
is flexible in solution and lacks a defined
structure(10, 14, 25) . The
-helix in
hEGF has an amphipatic character with Lys-48 and Glu-51 on the
hydrophilic site and Leu-47/Trp-50 and Trp-49/Leu-52 on the hydrophobic
site. In addition, Trp-50 interacts with other hydrophobic amino acids
in the protein such as Val-34 and Tyr-37(10) . One might
speculate that in EGF50 the carboxyl-terminal tail can still adopt an
-helix conformation, which is stabilized by hydrogen bond
formation between Leu-47 and Trp-50 as well as by VanderWaals
interactions between Trp-50 and other hydrophobic side chains. In
contrast to EGF50, no
-helix structure will be formed in EGF49 or
EGF48. The relatively high binding affinity of EGF50 for the chicken
EGF receptor suggests that
-helix formation of the
carboxyl-terminal tail of hEGF will prevent the positively charged
Lys48 from interfering with ligand-receptor interaction, whereas it
does interfere in the case of EGF49 and EGF48.
In conclusion, we
propose a model in which positively charged amino acids close to or
within the putative receptor recognition site of hEGF (Arg-45 and to a
lesser extent Lys-48 and Arg-53) prohibit high affinity binding to the
chicken EGF receptor due to electrostatic repulsion of positive charges
in or near the putative binding domain of this receptor. Comparing the
carboxyl-terminal sequences of EGF receptor agonists of different
origin, the proposed model would predict that EGF derived from human,
mouse, or rat will have a low affinity for the avian EGF receptor,
whereas human and rat TGF but also EGF from guinea pig will have a
high affinity for the avian EGF receptor.
In addition to amino acids
in the COOH-terminal domain, residues in other domains are thought to
form part of the binding domain in EGF and TGF.
H NMR
studies have shown, for instance, that amino acids near the sixth
cysteine residue are in close contact with residues surrounding the
second cysteine residue(10) , and in a recent study Richter et al.(26) hypothesized that amino acids in the
B-loop
-sheet determine the difference in binding affinity between
human EGF and mouse EGF for the chicken EGF receptor. Perhaps EGF
receptor agonists contain two distinct binding domains that each can
bind one receptor monomer similar as seen for the interaction of human
growth hormone with its receptor(28) . Data in favor of this
model have been discussed previously by Gullick(29) .
Additional studies will be necessary, however, to increase our
understanding of the way EGF and TGF
interact with their receptor
and to make the design of receptor antagonists feasible.