(Received for publication, August 29, 1994; and in revised form, November 4, 1994)
From the
We have exploited the differences in binding affinities of the
chicken epidermal growth factor (EGF) receptor for EGF and transforming
growth factor (TGF
) to study the role of the B-loop
-sheet of these ligands in receptor recognition and activation.
Although EGF and TGF
share similar secondary and tertiary
structures imposed by three highly conserved intramolecular disulfide
bonds, they have only 30-40% overall sequence identity. The
B-loop
-sheet is the major structural element in EGF and TGF
,
but sequence similarity in this region is low. To investigate its role
in receptor binding, we constructed two chimeric growth factors
(mEGF/hTGF
and
mEGF/hTGF
) composed of the murine EGF (mEGF)
amino acid sequence with residues 21-30 of the B-loop
-sheet
replaced by the equivalent residues of human TGF
(hTGF
); in
chimera mEGF/hTGF
, asparagine 32, which lies
at the boundary of the amino and carboxyl domains of mEGF, was also
replaced by its hTGF
counterpart (valine). In initial studies
using unpurified medium, it was found that the recombinant growth
factors exhibited differing mitogenic potencies
(mEGF/hTGF
>
mEGF/hTGF
> mEGF) when assayed on chicken
fibroblasts, even though they were equivalent in mitogenesis assays
using cells expressing the human EGF receptor. After purification,
mEGF/hTGF
was found to be 50 times more
potent than mEGF in the chick fibroblast mitogenesis assay and
exhibited a 10-fold increase in relative affinity for the chicken EGF
receptor; both growth factors still exhibited equivalent mitogenic and
receptor binding activity when tested on cells expressing human EGF
receptors. We conclude that the B-loop
-sheet of hTGF
is an
important determinant of EGF receptor binding affinity and biological
activity.
Epidermal growth factor (EGF) ()and transforming
growth factor
(TGF
) are mitogenic polypeptides of 53 and 50
amino acids, respectively. They belong to a family of EGF-like growth
factors (1) that act via the epidermal growth factor receptor
(EGF-R), a type 1 transmembrane growth factor receptor with intrinsic
tyrosine kinase activity(2) . EGF-R ligands are potent mitogens
for epithelial and mesenchymal cells, and abnormal regulation of
expression of these growth factors can lead to neoplastic
transformation(3) .
Ligands for the EGF-R show about 30%
overall sequence homology and are characterized by a three-looped
structure (designated A, B, and C), which is imposed by three highly
conserved intramolecular disulfide bonds(1, 4) . The
three-dimensional structures of EGF and TGF have been determined
by NMR; both have almost identical polypeptide chain folds and consist
of an amino-terminal domain (comprising residues up to the fifth
cysteine) and a carboxyl-terminal domain (comprising residues from the
fifth cysteine)(4) .
Using sequence information to identify
conserved residues together with knowledge of the three-dimensional
structure of EGF and TGF, it has been possible to identify key
structural residues such as the 6 cysteines and the 3 glycine residues
and to predict key receptor contact
residues(5, 6, 7, 8) .
Structure-activity relationships have unambiguously shown that Leu-47 (
)has a functional role in receptor
binding(9, 10) . Other studies indicate a similar role
for Arg-41(11) .
The segments of the B-loop between
cysteines 3 and 4 of EGF and TGF do not contain any conserved
residues, but they are folded into a double-stranded anti-parallel
-sheet linking residues 19-23 with residues
28-32(6, 7) . This is the dominant structural
element of each ligand. TGF
differs from EGF in that it possesses
a proline residue at position 30 in the B-loop, and its presence causes
slight distortion of the TGF
-sheet(4) . The role of
the B-loop in receptor binding is disputed. Based on structural
considerations, it has been proposed that this region of the molecule
is not involved directly in receptor binding but is only a scaffold on
which the recognition site is constructed(4, 12) . In
contrast, other studies have shown that non-conservative substitutions
of the hydrophobic residues, which predominate on one face of the
B-loop
-sheet of EGF, markedly reduce receptor binding
affinity(13) . Assuming that these mutations did not cause a
structural perturbation, they suggested that hydrophobic interactions
involving the B-loop
-sheet contributed to receptor binding.
Studies using synthetic peptides have also implicated the B-loop
segment of EGF in receptor binding. Indeed, only peptides incorporating
the B-loop of EGF have been shown by independent researchers to possess
biological activity(14, 15) ; however, it should be
noted that these peptides were significantly less active
(1/10) than EGF. Similarly, using a solid phase peptide
mapping technique, we have previously identified sequences within the
B-loop, as well as in the C-loop and carboxyl-terminal tail of
TGF
, which bind to hEGF-R(16) . Identification of these
peptides in the solution structure of TGF
revealed a possible
receptor binding cavity comprising the B-loop
-sheet and the
carboxyl domain of the growth factor. This receptor binding cavity
included known receptor contact residues such as Leu-48 and Arg-42 and
also included the side chain of Phe-15. This led us to propose a
multidomain model for the interaction of TGF
with the EGF-R, and
we postulated that correct orientation of the two domains on EGF-R is
mediated by modification of the main chain torsion angles of the single
residue that lies between cysteines 4 and 5 and links the amino and
carboxyl domains. Although this residue is not conserved, all known
TGF
sequences have a valine at this position (Val-33), whereas all
EGF sequences have an asparagine (Asn-32). Site-directed mutagenesis
studies of Asn-32 in hEGF have indicated that this residue comprises a
part of the receptor binding epitope(17) .
The chicken EGF-R
(cEGF-R) is a convenient tool for studying ligand/receptor
interactions, as it has an affinity for EGF 2 orders of magnitude lower
than that for TGF(18) . In view of the low level of
sequence similarity between EGF and TGF
in the B-loop
-sheet
segment, we hypothesized that this may account for the differential
binding affinities of these ligands for the cEGF-R. To test this, we
generated murine EGF (mEGF)/human TGF
(hTGF
) chimeras (see Fig. 1) and assessed their activity in receptor binding and
mitogenesis assays utilizing cEGF-R as well as hEGF-R. During the
course of this work, Kramer et al.(19) reported use
of a similar approach to identify the carboxyl-terminal tail of
hTGF
as an important site for high affinity binding to the cEGF-R.
In this study, we identify the B-loop
-sheet of hTGF
as a
further important region that enables the cEGF-R to distinguish between
mEGF and hTGF
.
Figure 1:
Schematic representation of the loop
structure and sequence of the chimeric mEGF/hTGF peptides. The
sequence shown in whitecircles corresponds to mEGF,
and the graycircles indicate those residues that
correspond to TGF
. Residue 32 was asparagine in
mEGF/hTGF
and valine in
mEGF/hTGF
. The boldcircles show the disulfide bond cysteine
pairings.
To create pWYG9/mEGF/hTGF, the above
steps were repeated except that pUC/mEGF/hTGF
was used initially instead of pUC/mEGF/1, and a single amino acid
change, N33V, was made in the site-directed mutagenesis step.
The precipitated protein was resuspended in PBS
and affinity purified using sheep anti-mEGF-Affi-Gel affinity
chromatography. After washing to remove unbound material, mEGF and
mEGF/hTGF were eluted from the column with
0.1 M glycine HCl, pH 2.5. The growth factors were
subsequently purified by MonoQ anion exchange chromatography using a
linear gradient of 0-0.5 M NaCl in 25 mM Tris,
pH 7.4, over 20 ml and/or Pep-RPC C
reverse phase
chromatography using a linear gradient of 0-70% acetonitrile in
40 mM trifluoroacetic acid/H
O over 30 ml (MonoQ
ion exchange chromatography was employed for one purification involving
the recombinant mEGF, but as the reverse phase step was found to be
more effective in separating variously processed forms of the growth
factors, this was omitted from subsequent purifications). Samples were
lyophilized and redissolved in water; their absorbance at 280 nm (A
) was determined. Protein concentrations
were calculated from the A
readings using the
extinction coefficients for tryptophan and tyrosine and the molecular
masses of the growth factors, which were determined by laser desorption
mass analysis. For mEGF 1-51 (see ``Results'' section),
the A
of a 1 mg/ml solution was 3.1, whereas
for mEGF/hTGF
, which possessed one less
tyrosine, the corresponding value was 2.9.
The cEGF-R has been previously cloned and characterized and
shown to possess an affinity for hTGF that is approximately 100
greater than that of mEGF(18) . Fig. 2A shows a comparison of the mitogenic activity of mEGF and hTGF
on CEF and confirms the differential activity of these growth factors
on the cEGF-R. For comparison, the same mEGF and hTGF
standards
were shown to be equivalent when tested for their ability to induce DNA
synthesis in cells bearing hEGF-R (Fig. 2B).
Figure 2:
Mitogenic activity of mEGF () and
hTGF
(
) standards on CEF (A) or NR6/HER (B) cells. Activity was measured by stimulation of
incorporation of [
I]iododeoxyuridine into
acid-insoluble material as described under ``Experimental
Procedures.'' Points represent the mean ± S.E. (n = 3).
In
initial experiments, the activities of mEGF and the two chimeras,
mEGF/hTGF and
mEGF/hTGF
, secreted by the recombinant yeast
cells were assessed by assay of the crude yeast medium. Western blot
analysis of the media showed that the yeast cells had efficiently
expressed the chimeric growth factors and that the level of expression
of each growth factor was similar. Consistent with this result, it was
found that the crude media containing mEGF,
mEGF/hTGF
, or mEGF/hTGF
were of similar potency in mitogenesis assays using NR6/HER
cells, indicating that the chimeric growth factors were functionally
active on hEGF-R and that formation of the chimera had not affected the
ability of the growth factor to be correctly folded by the yeast
expression system. However, when tested on chick fibroblasts, the media
exhibited differing potencies; the mEGF/hTGF
containing medium was about 6 times more potent than that
containing mEGF/hTGF
and 20 times more
potent than that containing mEGF (data not shown). The former chimera
was therefore chosen for purification and comparison with mEGF.
Murine EGF and mEGF/hTGF secreted by the
recombinant yeast cells were purified by anti-mEGF-Affi-Gel affinity
chromatography and reverse phase C18 chromatography. This revealed that
the yeast cells had secreted three major forms of the growth factors.
Laser desorption mass analysis showed that this had resulted from
carboxyl-terminal truncations of the growth factors, as has been
previously reported for mEGF using the yeast
-factor expression
system(20) . No full-length (i.e. 1-53) growth
factor was detected, and the forms of the growth factor used in the
following studies lacked either 1 or 2 carboxyl-terminal residues.
Previous studies have shown that loss of these residues has no
significant effect on receptor binding(25) . The concentration
of the two purified growth factors was determined from their UV
absorbance, and this was used as the basis for all subsequent assays.
When tested in mitogenesis assays, purified mEGF and
mEGF/hTGF were found to be of similar
potency on NR6/HER cells (Fig. 3A) with the EC
for mEGF being 140 pM and that for
mEGF/hTGF
being 85 pM. In
mitogenesis assays utilizing CEF, the mEGF/hTGF
chimera (EC
, 80 pM) was found to be 50
times more potent than mEGF (EC
, 4 nM); however,
the activity of the chimera was still about 5 times less than that of
the commercial hTGF
standard (EC
, 15 pM) (Fig. 3B).
Figure 3:
Mitogenic activity of purified recombinant
protein when tested on NR6/HER cells (A) or CEF (B). Symbols correspond to mEGF () and
mEGF/hTGF
(
) and represent the mean
± S.E. (n = 3). EC
values were
determined after correction for base line
[
I]iododeoxyuridine incorporation. In B, results obtained with a TGF
standard are also shown
(
).
A similar pattern was observed when the
receptor binding activity of the purified proteins was determined in a
competitive binding assay utilizing I-labeled mEGF. Thus,
the IC
values for binding of mEGF and
mEGF/hTGF
to the hEGF-R were 1.4 and 0.9
nM, respectively (Fig. 4A). As found in the
CEF mitogenesis assays, mEGF/hTGF
was more
potent than mEGF for competing with
I-labeled mEGF
binding to cEGF-R (IC
values, 1.1 and 10 nM,
respectively) (Fig. 4B); however, it was still less
effective than the TGF
standard used in the same assays.
Figure 4:
Binding of I-labeled mEGF to
NR6/HER (A) or CEF (B) in the presence of purified
recombinant mEGF (
) or mEGF/hTGF
(
). The competitive binding assay was performed as
described under ``Experimental Procedures.'' Points are the mean ± S.D. of two determinations. In B,
results obtained with a TGF
standard are also shown (
) and
are the mean of two observations.
The approach of generating chimeric growth factors to study
the ligand receptor interaction has been successful in studies of
several growth factors (26, 27) and
cytokines(28) . As the chicken and human EGF-Rs are
structurally homologous, it is likely that their interaction with
ligand is similar. Thus, exploitation of the differential affinity of
the chicken receptor for the EGFs and hTGF may enable
identification of regions of the ligands that interact with
the receptor. However, it should be noted that the extent of binding
will depend on interactions between side chains in the ligand and the
receptor and that some of these will be specific to the nature of the
chicken receptor.
In the present study, mEGF and a chimera of this
growth factor in which residues 21-32 of the B-loop -sheet
were replaced by the equivalent residues of hTGF
were expressed as
recombinant proteins in yeast and their activities assessed after
purification and characterization. Whereas these two growth factors
were found to be similar when tested in both receptor binding and
mitogenesis assays using cells expressing the hEGF-R, the chimera was
10-50 times more potent than wild type mEGF when tested on cells
expressing the cEGF-R. These data indicate that residues contained in
the B-loop segment of hTGF
contributed to binding and activation
of the cEGF-R.
During the course of this work, Kramer et
al. (19) reported a similar approach using cEGF-R to test
the activity of hEGF/hTGF chimeras and found that chimeras
containing the COOH-terminal tail sequence of hTGF
had an affinity
for the cEGF-R comparable with that of native hTGF
and concluded
that this peptide segment is responsible for conferring high affinity
binding of the ligand to the chicken receptor. Although these results
are apparently at odds with our own, it should be noted that Kramer and
co-workers compared the activity of chimeras of hTGF
with human EGF, rather than the murine EGF used in our
studies. As Kramer and colleagues determined the relative affinities of
hEGF and mEGF for cEGF-R and showed that they differed by 1 order of
magnitude (i.e. hEGF was intermediate in affinity between
hTGF
and mEGF), it cannot be assumed that mEGF and hEGF bind
equivalently to the chicken receptor as has been previously
supposed(29) . In the present studies, exchanging the mEGF
B-loop
-sheet segment with the equivalent residues of hTGF
caused its affinity for the cEGF-R to become more like that of hEGF. In
the studies of Kramer et al.(19) , hEGF that already
had an intermediate binding affinity became hTGF
-like by
substitution of the hTGF
COOH-terminal tail.
In light of this,
we propose that there are two sets of determinants that distinguish the
EGFs and hTGF, and each set contributes 1 order of magnitude to
the binding affinities of the growth factors to the chicken receptor.
Thus, hTGF
possesses both sets of determinants, hEGF possesses one
set, and mEGF possesses none (Table 1). Based on our results and
those of Kramer et al.(19) , it seems likely that one
set of determinants lies in the flexible COOH-terminal tail and enables
the chicken EGF-R to distinguish between hTGF
and either hEGF or
mEGF, while the second set lies in the B-loop segment, enabling the
receptor to distinguish between mEGF and either hTGF
or hEGF. This
is consistent with our previous studies that indicated that the
interaction of hTGF
with the hEGF-R is a multidomain
process(16) .
By comparing the sequences of hTGF, hEGF,
and mEGF in the B-loop
-sheet region (Table 1), it can be
seen that whereas hEGF and mEGF have 9 out of 13 identical residues,
mEGF differs most significantly from hEGF at residues 22 and 28 with
tyrosine to histidine and lysine to serine substitutions, respectively.
As the equivalent residues in hTGF
are phenylalanine and lysine,
it is possible that these represent key distinguishing residues between
mEGF and either hEGF or hTGF
. However, it should be noted that as
the hEGF-R does not distinguish between mEGF and hEGF, it is likely
that the requirement for one or both of these residues in the B-loop
-sheet is a feature peculiar to the cEGF-R and the nature of its
ligand binding site. It should also be noted that the enhanced
mitogenic potential of the chimeric mEGF/hTGF
did not just depend on the presence of His-22 and Lys-28, as the
other chimera, mEGF/hTGF
, was intermediate
in activity between mEGF and mEGF/hTGF
. This
suggested that Val-32 also contributed to the binding or activation
process either directly as a receptor contact residue or indirectly,
perhaps by interaction with hTGF
B-loop residues. We are now
systematically modifying the B-loop residues to determine their
contribution to the overall process of receptor binding and activation
of DNA synthesis. We are also studying the effect of substitutions in
the carboxyl tail of mEGF to test the hypothesis that domains in both
the B-loop and the flexible carboxyl-tail are required to convert it
into an high affinity ligand for the cEGF-R.