©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Structural Basis for the Specificity of Epidermal Growth Factor and Heregulin Binding (*)

Elsa G. Barbacci , Bradley C. Guarino , Justin G. Stroh , David H. Singleton , Kenneth J. Rosnack , James D. Moyer (§) , Glenn C. Andrews

From the (1) From Pfizer Central Research, Groton, Connecticut 06340

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Breast cancer cells express diverse growth factor receptors on their surface that regulate cell proliferation and differentiation. The epidermal growth factor receptor (EGFR)() 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.

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- 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.




EXPERIMENTAL PROCEDURES

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 Kof 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. ICvalues 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.


RESULTS AND DISCUSSION

Heregulin and Neu differentiation factor are 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 HRG1 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.

Alignment of the core regions of EGF and HRG (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.

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 (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).

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.


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 ICof 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.

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- 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.

The solution structure of HRG 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.

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, 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.

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.

  
Table: Minimum requirements for HRG activity

The peptides indicated were evaluated for inhibition of 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

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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 203-441-4629; Fax: 203-441-1290.

The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HRG, heregulin; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.