From the
Heregulin is a ligand for the erbB3 and erbB4
receptors, with a region of high homology to epidermal growth factor
(EGF). Despite this homology, these ligands bind to their corresponding
receptors with great specificity. We report here the synthesis of
heregulin
Breast cancer cells express diverse growth factor receptors on
their surface that regulate cell proliferation and differentiation. The
epidermal growth factor receptor (EGFR)
Although there are multiple receptors for the HRGs, both EGF and HRG
bind to breast cancer cells with complete specificity, i.e. EGF or TGF-
Heregulin and Neu differentiation factor are
Alignment of the core regions of EGF and
HRG
Substitution of any single cysteine-bound region of EGF
or the EGF C terminus into HRG produced a chimera that retained full
affinity for the HRG receptor and ability to stimulate p185
phosphorylation in cells, but had no affinity for the EGF receptor
(, Fig. 3 A). The MDA-MB-453 cells examined
(Fig. 3 A) have a low level of basal tyrosine
phosphorylation of p185 ( lanes 1 and 2) that is
markedly increased (
Substitution of the HRG
amino acids 177-181 (SHLVK) for the N terminus of EGF produced a
chimera (XVII) which bound both receptors with high affinity
(). Specific binding of both EGF and HRG could be
completely blocked by this chimera (Fig. 4). Chimera XVII was as active as EGF itself in the activation of EGFR
autophosphorylation (Fig. 3 B, lanes 7 and 8)
and was highly active in the stimulation of p185 tyrosine
phosphorylation (, Fig. 3 A, lane 5). The
EGF chimera with substitution of the HRG sequence 191-195
(XIV) had moderate affinity for both receptors.
These data
indicate that amino acids 177-181 (SHLVK) of heregulin are
crucial for generating specific, high-affinity binding to its receptor,
with the region 191-195 also contributing to binding specificity.
Previous studies indicated that four N-terminal amino acids of
TGF-
The
solution structure of HRG
It is remarkable that substitutions of EGF sequences for HRG regions
bound by conserved cysteine residues or substitution of the EGF C
terminus for that of HRG have no effect on binding affinity for the HRG
receptor, although there are only six sites of identity and six sites
of similarity in the 38 amino acids comprising them. Several amino
acids in EGF shown to be important in EGFR binding
(18, 19, 20, 21, 22) , such as
Tyr
The features
of EGF and HRG, two growth factors that are highly specific for
distinct but homologous receptors, are combined in peptide XVII,
which is a potent agonist for both receptors and is therefore
designated ``biregulin.'' This chimeric peptide may be a
valuable tool for examining the physiological and pathological roles of
the EGF receptor family. Our findings also suggest that it may be
possible to design compounds that combine activity for the two
receptors at various ratios.
The
peptides indicated were evaluated for inhibition of
The
indicated peptides were evaluated as inhibitors of HRG and EGF binding
and for their ability to stimulate receptor phosphorylation. The
primary sequence of each refolded synthetic peptide is listed in Fig.
2.
We thank Ken Iwata (Oncogene Science Inc) for his gift
of horseradish peroxidase-labeled anti-phosphotyrosine antibody. We
also acknowledge helpful suggestions from our colleagues Catherine
DiOrio, Frank DiCapua, Penny Miller, Mike Morin, and Walt Massefski.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
177-241 and show that a region consisting of
amino acids 177-226 is sufficient both for binding and
stimulation of receptor phosphorylation. Studies of chimeric
EGF/heregulin peptides revealed that amino acids 177-181 of
heregulin provide the specificity for binding to the heregulin
receptor. The substitution of amino acids 177-181 of heregulin
for the N terminus of EGF produced a unique bifunctional agonist that
binds with high affinity to both the EGF receptor and the heregulin
receptor.
(
)
family
of tyrosine kinase-linked receptors, i.e. EGFR,
erbB2/ neu, erbB3, and erbB4, are
frequently overexpressed in breast cancer and may modulate tumor cell
proliferation
(1) . Although multiple growth factors that
activate EGFR (EGF, TGF-
, amphiregulin, and heparin binding EGF)
are known, much less is known about the role of ligands that regulate
the other members of this receptor family. The heregulins (HRGs, also
termed neu differentiation factors) are a family of proteins, generated
by splice variants from a single gene, that stimulate the tyrosine
phosphorylation of p185 and have a region of high homology to EGF
(2, 3, 4, 5) . HRGs have also been
identified as an acetylcholine receptor inducing activity
(6) and as glial growth factors
(5) . These factors may
play important roles both in the regulation of cancer cell
proliferation and in neuronal function. Although HRG was initially
proposed to act as a ligand for p185, subsequent studies revealed that
binding of HRG did not correlate with p185 expression
(7) and
that expression of p185 is not sufficient for binding of HRG
(7, 8) . Further studies have demonstrated that
erbB4
(8, 9) , erbB3
(9, 10, 11, 12, 13) , and
erbB3/ erbB2 heterodimers
(12, 13) are
receptors for HRG. These receptors and EGFR have substantial homology
both in the intracellular and extracellular domains
(1) and
therefore may bind ligands that also share common structural features.
do not block HRG binding and HRG does not block
EGF binding
(2, 14) . This specificity is obtained
despite topological similarity generated by three conserved disulfide
bonds in their core domains (Ref. 15, Fig. 1). A 65-amino acid
EGF-like domain of heregulin
1, amino acids 177-241, was
shown to be sufficient for high affinity binding and stimulation of
p185B2 phosphorylation
(2) . We have synthesized the receptor
binding domain of heregulin
(HRG
177-241, I)
and heregulin
(HRG
177-239, IV) and utilized a
series of truncated synthetic HRGs and EGF/HRG chimeric peptides (Fig.
2) to determine the essential structural elements of HRG receptor
binding.
Figure 1:
The primary sequences
and disulfide bond patterns of heregulin (177-226) and
human EGF 1-48.
Peptide Synthesis
Peptides were synthesized on
an Applied Biosystems (ABI) 430A peptide synthesizer using standard
tert-butyloxycarbonyl ( t-Boc) chemistry protocols as
provided (version 1.40;
N-methylpyrrolidone/hydroxybenzotriazole). Acetic anhydride
capping was employed after each activated ester coupling. The peptides
were assembled on phenylacetamidomethyl polystyrene resin using
standard side chain protection except for the use of t-Boc
Glu( O-cyclohexyl) and t-Boc
Asp( O-cyclohexyl). The peptides were deprotected using the
``Low-High'' hydrofluoric acid (HF) method of Tam et al. (23) In each case crude HF product was purified by reverse
phase HPLC (C-18 Vydac, 22250 mm), diluted without drying into
folding buffer (1
M urea, 100 m
M Tris, pH 8.0, 1.5
m
M oxidized glutathione, 0.75 m
M reduced glutathione,
10 m
M Met), and stirred for 48 h at 4 °C. Folded, fully
oxidized peptides were purified from the folding mixture by reverse
phase HPLC and characterized by electrospray mass spectroscopy;
quantities were determined by amino acid analysis. The heregulins and
heregulin chimeras were analyzed for disulfide bonding in the following
manner: first the peptide was cleaved with cyanogen bromide (CNBr),
which opened up the peptide for further digestion. After removal of the
CNBr, the peptide was digested sequentially with trypsin and
endoproteinase Glu-C in order to obtain cleavage between the cysteines.
Samples were analyzed using capillary liquid chromatography coupled
with electrospray ionization mass spectrometry, and the disulfide
bonding pattern was determined using the molecular weights of the
fragmented peptides and shown to be the expected C1-C3,
C2-C4, C5-C6 bonding pattern. Complete disulfide bonding
patterns could not be determined for EGF and EGF chimera by this
method, but the C5-C6 bond was confirmed. The primary sequence of
each peptide is given in Fig. 2.
Figure 2:
The primary sequence of chimeras used in
these studies. Truncated HRGs are in A, HRG with
substituted regions from EGF in B, and EGF with substituted
regions from HRG
in C. Gaps in the aligned sequence are
indicated by dashes; dots represent sequence
identities with the top sequence. Boxed elements are conserved
cysteine residues common to the EGF-like ligand
family.
Binding Studies
Binding studies were done with
SKBr3 cells because these cells express both EGF and HRG receptors.
SKBr3 cells (ATCC, Rockville, MD) in 24-well tissue culture plates at
200,000 cells/well were changed to serum-free McCoy's medium
1-2 h prior to the binding assay. Heregulin
177-241
was labeled with
I using chloramine T to a specific
activity of
200 µCi/µg. The binding assay was initiated by
aspiration of the medium and addition of 1 ml of 0.05 µCi/ml (33
p
M)
I-heregulin
177-241 and the
competing peptides in ice-cold McCoy's medium. The cells were
incubated at 4 °C for 2 h with rocking. Binding was terminated by
washing cells twice with 2 ml of ice-cold medium. The cells with bound
heregulin were lysed in 200 µl of 0.1
N NaOH, 0.1% SDS.
This lysate was then transferred to a scintillation vial, LSC fluid
added (Ready Safe, Beckman Instruments), and
I determined
by scintillation counting. Specific binding is calculated by
subtraction of nonspecific binding in the presence of 200 ng/ml of
unlabeled HRG
1 177-241; typical values for controls were
3400 dpm specific binding and 220 dpm nonspecific. Scatchard analysis
of the binding indicated a K
of 150
p
M for binding of
I-heregulin to SKBr3 cells, in
good agreement with previous reports
(2) . EGF binding was
measured by the same method, but with
I-EGF (DuPont NEN,
150-200 µCi/µg). Typical values for controls were 11,000
dpm specific binding and 400 dpm nonspecific binding. IC
values were determined graphically from titrations over a range
of four or five concentrations and are representative of two or more
independent titrations.
Tyrosine Phosphorylation Assays
MCF-7, MDA-MB-468,
or MDA-MB-453 cells as indicated (ATCC) were seeded at 100,000
cells/well in 24-well plates in 1 ml of McCoy's medium with 10%
fetal bovine serum and used the next day. The indicated peptide was
added at 50 ng/ml and incubated 5 min at 37 °C. The medium was
removed by aspiration and the cells washed with TNK buffer (50
m
M Tris HCl, pH 7.4, 140 m
M NaCl, 3.3 m
M
KCl, 0.5 m
M sodium orthovanadate) and lysed with 0.1 ml of
boiling Laemmli sample buffer
(24) . The lysate was placed in a
boiling water bath for 10 min and then stored at -20 °C for
analysis the next day. Proteins in the extracts were separated by
electrophoresis on 4-20% SDS-PAGE gel (84 70 mm,
Integrated Separation Systems, Natick, MA), transferred to Immobilon-P
membranes (Millipore, Bedford, MA), and probed with horseradish
peroxidase-conjugated anti-phosphotyrosine antibody (PY-20 (ICN, Costa
Mesa, CA) or PY-54 (Oncogene Science, Uniondale, NY).
Phosphotyrosine-containing proteins were visualized by ECL reagents
(Amersham Corp). For studies of p185 tyrosine phosphorylation in
response to heregulin chimeras, we used MCF7 and MDA-MB-456 cells as
reported previously
(2) . There was no detectable
``basal'' level of p185 autophosphorylation in MCF7 cells and
HRG
177-241 gave at least a 500-fold increase as determined
by densitometry. MDA-MB-453 cells, which overexpress erbB2
(4) , respond to HRG
177-241 with a 5.8-fold
increase over the low basal (unstimulated) level. Autophosphorylation
of EGFR was examined in MDA-MB-468 cells, which overexpress EGFR
(4) . EGF gave a 16-fold increase in EGFR autophosphorylation in
MDA-MB-468 cells over the unstimulated level as quantitated by
densitometry.
45-kDa
glycoproteins found in conditioned medium
(2, 3) . They
are produced by cleavage of a transmembrane glycoprotein with multiple
domains, including a domain that is highly homologous to EGF
(3) . The biological activity of heregulin appears to reside in
its EGF-like domain, because a recombinant protein consisting of amino
acids 177-241 of HRG
1 was shown to be active in both binding
assays and in the stimulation of p185 tyrosine phosphorylation
(2) . We have further delineated the crucial domain of both
HRG
and HRG
and find that a 50-amino acid region
(177-226) was fully active both for binding to cellular receptors
and for stimulation of p185 phosphorylation (see , peptides II and V). Further truncation by a single amino acid
(HRG
177-225, III) or two amino acids (HRG
177-224, VI) reduced both the receptor binding and
phosphorylation by > 100-fold. Interestingly, the 50-amino acid
peptide (II) was somewhat more potent than the 65 amino acid
heregulin fragment (I). Thus the critical regions for receptor
binding and activation are contained within a 50-amino acid portion of
the heregulins, beginning at amino acid 177 and ending at amino acid
226. It is possible however that the other domains of heregulin
influence binding, receptor activation, and specificity in
physiological situations.
(Fig. 2 B) shows the extensive homology between
these factors. On the basis of the results with the truncated
heregulins, we examined chimeras of EGF 1-48 and the core
50-amino acid region of HRG
to determine the essential structural
elements of HRG receptor binding and the basis for receptor binding
selectivity. In HRG
each of four separate intracysteine regions
(amino acids 183-189, 191-195, 197-209, and
213-220) and the C terminus (amino acids 222-226) was
replaced by the corresponding sequence from EGF
(Fig. 2 B, proteins VIII-XII). In
EGF, the corresponding intracysteine regions (amino acids 7-13,
15-19, 21-30, and 34-41) and the N terminus (amino
acids 1-5) were replaced by the corresponding HRG sequences
(Fig. 2 C, proteins XIII-XVII). These
chimeras as well as HRG
177-241 and EGF were then evaluated
in four distinct assays: 1) inhibition of
I-EGF binding
to SKBr3 cells, 2) inhibition of
I-HRG binding to SKBr3
cells, 3) stimulation of EGFR autophosphorylation in MDA-MB-468 Cells,
and 4) stimulation of p185 tyrosine phosphorylation in MDA-MB-453 and
MCF7 cells.
15-fold) on addition of HRG
(peptide II, lane 3), but unaffected by addition of EGF
( lane 4). All the HRG chimeras were potently active
(VIII, X, XI, and XII are shown in
lanes 6-9). Surprisingly, even the region comprising
amino acids 197-209 of HRG, postulated to be involved in receptor
binding
(15) , can be replaced with the smaller corresponding
region of EGF (amino acids 21-30) without loss of HRG binding
affinity. These results indicate that even with the substantial amino
acid differences between EGF and HRG found in the regions between
cysteine residues, neither these regions, nor the C terminus,
contribute to the specificity of binding for EGFR versus HRG
receptors.
Figure 3:
Stimulation of tyrosine phosphorylation by
EGF/HRG chimeras. MDA-MB-453 ( A) or MDA-MB-468 ( B)
human breast cancer cells were stimulated with chimeric EGF/HRG
peptides (50 ng/ml) for 5 min. For A, the peptides were:
HRG (177-226) II ( lane 3), murine EGF ( lane
4), XVII ( lane 5), VIII ( lane 6), X ( lane
7), XI ( lane 8), and XII ( lane 9) (buffer
(controls) ( lanes 1 and 2)). For B, the
peptides added were: XIII ( lane 3), XV ( lane 4), XVI
( lane 5), XIV ( lane 6), XVII ( lane 7), and
murine EGF (Sigma) ( lane 8) (buffer (controls) ( lanes 1 and 2)). Proteins containing phosphotyrosine were
separated and visualized as described under ``Experimental
Procedures.'' The position of molecular weight markers are
indicated and the positions of p185 ( A) and EGFR ( B)
are indicated by an arrow. This result is representative of
two independent experiments. A similar stimulation of p185 tyrosine
phosphorylation was observed with this set of peptides, but not with
EGF, when added to MCF7 breast cancer cells (not
shown).
Substitution of the intracysteine HRG sequences
197-209 and 213-220 into EGF resulted in peptides
(XV, XVI) that bound neither EGFR nor the HRG receptor
with high affinity (). EGF chimeras with substitution of
the HRG sequences 183-189 (XIII) or 191-195
(XIV) retained some affinity for the EGF receptor, but had
ICvalues about 1000-fold higher than EGF itself. EGF
chimeras with substitutions of HRG
sequences 197-209
(XV) and 213-220 (XVI) were unable to stimulate
EGFR autophosphorylation, whereas EGF with substitution of HRG
sequences 183-189 (XIII) or 191-195 (XIV)
were active, but much less active than EGF (,
Fig. 3B). These EGF chimeras incorporating HRG
intracysteine regions (XIII-XVI) were also
incapable of stimulating p185 phosphorylation in MDA-MB-453 cells
() or MCF7 cells (not shown).
Figure 4:
Inhibition of I-HRG binding
and
I-EGF binding to SKBR3 cells by EGF/HRG chimeras. The
chimeras were examined for inhibition of
I-HRG binding
( A) or
I-EGF binding ( B) at the
indicated concentrations of peptide as described under
``Experimental Procedures.'' The peptides are: murine EGF
( open squares), HRG
177-226 (II) ( closed
squares), EGF with HRG 191-195 region (XIV) ( closed
triangles); EGF with HRG 177-181 region (XVII) ( open
circles). Human recombinant EGF (Collaborative Biomedical
Products, Bedford, MA) was also evaluated and inhibited
I-EGF binding to SKBR3 cells with an IC
of
0.16 n
M, but had no effect on
I-HRG binding,
even at 16 n
M. Similarly, human TGF-
and rat TGF-
(Sigma) completely blocked
I-EGF binding to SKBR3 cells,
but only minimally reduced
I-HRG binding (<25%) at 300
n
M (data not shown).
These results
stimulated further investigation of the N-terminal sequence of
heregulin/EGF chimera XVII. Individual nonconservative
substitutions in this chimera at amino acids 181 (K to E, chimera XVIII), 180 (V to S, chimera XIX), or 179 (L to D,
chimera XX) with the aligned amino acid from EGF had little
effect on affinity for the heregulin receptor (Table II). However,
removal of amino acids 177 and 178 reduced affinity for the heregulin
receptor by > 10-fold (chimera XXI, ) without a
corresponding effect on affinity for the EGF receptor. This supports
the conclusion that amino acids 177-181 are of specific
importance for binding to the heregulin receptor.
or EGF could be removed without substantial reduction of
binding affinity, suggesting that the N terminus of neither EGF nor
TGF-
is critical for binding to EGFR
(16, 17, 18) . While the N terminus of EGF
(NSDSE) is quite different from the corresponding region of the
heregulins, the corresponding sequence (SHFNK) in rat TGF-
is
remarkably similar in composition, but does not confer on rat TGF-
affinity to the heregulin receptor. Thus while the region comprising
residues 177-181 (SHLVK) of HRG is the most important element for
binding specificity, additional regions are also necessary for high
affinity binding, most notably, loss of the C-terminal residues at
225-226 ablated activity (). The pentapeptide SHLVK
(XXII) was unable to compete for HRG binding even at
170,000-fold excess (), further supporting a role of
multiple regions of interaction of ligand with receptor.
179-225, deduced by
two-dimensional NMR, indicated that residues 179-181 of the
N-terminal
strand is well defined in HRG, but disordered in EGF
(15) . On the basis of this structure, Nagata et al.
(15) suggested that a hydrophobic patch, including
Leu
, and an ionic cluster, including Lys
,
were important for specific binding of HRG. Our findings demonstrate
directly that this region is important for binding to the HRG receptor.
, Tyr
, and Arg
, are either
conserved or replaced with a conservative substitution (Tyr to Phe) in
the HRGs. The conserved residues in the intracysteine regions of EGF
and HRG together with the unique HRG N terminus are therefore the
essential elements of HRG binding and the specificity for the HRG
receptor is entirely generated by the HRG N terminus.
Table:
Minimum requirements for HRG activity
I-heregulin
177-241 (I) binding to SKBr3
cells and for stimulation of p185 phosphorylation in MCF-7 cells. The
primary sequence of each refolded synthetic peptide is listed in Fig 1.
Table:
Chimeric EGF/heregulin
peptides: an analysis of the structural basis for selectivity
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.