©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Contribution of the Transforming Growth Factor B-loop -Sheet to Binding and Activation of the Epidermal Growth Factor Receptor (*)

(Received for publication, August 29, 1994; and in revised form, November 4, 1994)

Audrey Richter Douglas R. Drummond (§) Jennie MacGarvie Sarah M. Puddicombe Stephen G. Chamberlin Donna E. Davies (¶)

From the Cancer Research Campaign Medical Oncology Unit, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGFalpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGFalpha 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 beta-sheet is the major structural element in EGF and TGFalpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGFalpha and mEGF/hTGFalpha) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGFalpha (hTGFalpha); in chimera mEGF/hTGFalpha, asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGFalpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGFalpha > mEGF/hTGFalpha > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor. After purification, mEGF/hTGFalpha 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 beta-sheet of hTGFalpha is an important determinant of EGF receptor binding affinity and biological activity.


INTRODUCTION

Epidermal growth factor (EGF) (^1)and transforming growth factor alpha (TGFalpha) 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 TGFalpha 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 TGFalpha, 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 (^2)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 TGFalpha do not contain any conserved residues, but they are folded into a double-stranded anti-parallel beta-sheet linking residues 19-23 with residues 28-32(6, 7) . This is the dominant structural element of each ligand. TGFalpha 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 TGFalpha beta-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 beta-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 beta-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 (1/10^5) 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 TGFalpha, which bind to hEGF-R(16) . Identification of these peptides in the solution structure of TGFalpha revealed a possible receptor binding cavity comprising the B-loop beta-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 TGFalpha 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 TGFalpha 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 TGFalpha(18) . In view of the low level of sequence similarity between EGF and TGFalpha in the B-loop beta-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 TGFalpha (hTGFalpha) 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 hTGFalpha as an important site for high affinity binding to the cEGF-R. In this study, we identify the B-loop beta-sheet of hTGFalpha as a further important region that enables the cEGF-R to distinguish between mEGF and hTGFalpha.


Figure 1: Schematic representation of the loop structure and sequence of the chimeric mEGF/hTGFalpha peptides. The sequence shown in whitecircles corresponds to mEGF, and the graycircles indicate those residues that correspond to TGFalpha. Residue 32 was asparagine in mEGF/hTGFalpha and valine in mEGF/hTGFalpha. The boldcircles show the disulfide bond cysteine pairings.




EXPERIMENTAL PROCEDURES

Materials

Standard mEGF was purified from murine submaxillary glands according to the method of Savage and Cohen (30) and was a gift from Dr. P Moores (Commonwealth Scientific and Industrial Research Organization, Australia). This was also used as immunogen for production of a polyclonal sheep anti-mEGF antiserum. An IgG fraction was prepared from the immune serum by ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography; the purified IgG fraction (approximately 20 mg/ml gel) was coupled to Affi-Gel 10 (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom) according to the manufacturer's instructions. I-Labeled mEGF was prepared using Iodo-beads (Pierce, UK) according to manufacturer's instructions. Recombinant hTGFalpha was purchased from British Biotechnology. NR6/HER cells (NR6 cells transfected with hEGF-R) were a gift from Dr. G. Panayatou (Ludwig Institute for Cancer Research, London, W1P 8BT). The yeast alpha factor secretion vector encoding murine EGF, pWYG9EGF, was a gift from Dr J. J. Clare (Wellcome Research Laboratories, Beckenham, Kent).

Preparation of mEGF/hTGFalpha Expression Vectors

The yeast expression vector, pWYG9/EGF(20) , was used for production of wild type mEGF. To generate EGF/hTGFalpha, the 715-bp AatII-BclI fragment from pWYG9/EGF, which contained the yeast gal7 promoter and the alpha-factor prepro leader sequence fused to mEGF, was inserted into the AatII and BamHI sites of puc18 to form pUC/mEGF/1. The 167-bp EcoRI-SmaI fragment that encodes the mature mEGF sequence was then taken from this plasmid and inserted into the EcoRI-SmaI sites of M13 mp19 (21) to create M13/mEGF/1. This construct was then used to perform site-directed mutagenesis(22) , such that the 10 amino acids between Cys-20 and Cys-31 that form part of the B-loop of mEGF (MHIESLDSYT) were replaced by the 10 amino acids from the analogous region of hTGFalpha (RFLVQEDKPA) creating M13/mEGF/hTGFalpha (see Fig. 1). Codons for the replacement amino acids ED were selected to create a unique BbsI restriction site (gAA gAC). After creating an intermediate vector, pUC/alpha/2, containing the yeast gal7 promoter and alphafactor prepro sequence using the 555-bp AatII-EcoRI fragment from pUC/mEGF/1 and cloning into the AatII-EcoRI sites of pUC19, the 204-bp EcoRI-HindIII fragment from M13/mEGF/hTGFalpha was inserted into the EcoRI-HindIII sites of pUC/alpha/2 to generate pUC/mEGF/hTGFalpha. The yeast expression construct, pWYG9/mEGF/hTGFalpha, was then formed by taking the 719-bp AatII-BamHI fragment from pUC/mEGF/hTGFalpha and inserting it into the AatII-BclI sites of pWYG9/EGF. Plasmid pWYG9/mEGF/hTGFalpha contained extra BbsI and SmaI sites not present in pwyg9/EGF; these were used to ensure correct replacement of mEGF in this construct.

To create pWYG9/mEGF/hTGFalpha, the above steps were repeated except that pUC/mEGF/hTGFalpha was used initially instead of pUC/mEGF/1, and a single amino acid change, N33V, was made in the site-directed mutagenesis step.

Expression and Purification

mEGF and mEGF/hTGFalpha, fused to the alpha-factor secretion signal of vector pWYG9, were expressed in Saccharomyces cerevisiae S150-2B as previously described(20) . The vector allowed for selection by resistance to G418, and expression was regulated by the promoter from the gal7 gene. Yeast cells were grown in YP medium with 2% raffinose at 30 °C to mid-exponential phase and then induced with 2% galactose for 48 h (20) . The culture was then centrifuged at 9000 times g, 4 °C, for 20 min to remove yeast cells, and recombinant protein was precipitated from the supernatant by the addition of ammonium sulfate (0.39 g/ml) at 4 °C and pelleted by centrifugation at 9500 times g, 4 °C, for 45 min.

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/hTGFalpha 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(18) reverse phase chromatography using a linear gradient of 0-70% acetonitrile in 40 mM trifluoroacetic acid/H(2)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/hTGFalpha, which possessed one less tyrosine, the corresponding value was 2.9.

Laser Desorption Mass Spectrometry

The molecular masses of the purified growth factors were determined using a Vestec Lasertec Benchtop Laser Desorption Time of Flight mass spectrometer with alpha-cyano-4-hydroxycinnamic acid as matrix and bovine heart cytochrome c and insulin as standards. The determined molecular masses were within 0.01-0.08% of predicted masses.

Western Blotting

Yeast medium was passed through a 0.2 µM filter, and recombinant protein was separated by SDS-polyacrylamide gel electrophoresis (23) on a 20% acrylamide gel. Proteins were transferred to a nitrocellulose membrane (Hybond C, Amersham, UK) by electrophoresis. Membranes were incubated with a polyclonal anti-mEGF antibody (10 µg/ml) in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% (w/v) BSA, 0.5% Tween 20 for 2 h, followed by a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1/1000) in the same buffer as above. Bound antibody was detected using enhanced chemiluminescence and autoradiography according to the manufacturer's instructions (Amersham, UK).

Cell Culture

Chick embryo fibroblasts (CEF) were allowed to grow out from fragments of epidermal tissue dissected from 14-day-old chick embryos. Establishment of the primary culture took about 7 days. At this point, tissue debris was removed by washing, and then the fibroblastic cells were detached by trypsinization and passaged into fresh medium. If necessary, any residual debris present in the cell suspension was removed by passing through a cell strainer (70-µm pore size, Becton Dickinson, UK). Cells were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, 2 mM glutamine, non-essential amino acids, 10 units/ml penicillin, and 10 µg/ml streptomycin at 37 °C in a humidified atmosphere of air containing 5% CO(2). CEF cells were routinely used between passages 5 and 10.

Measurement of Mitogenic Activity

Mitogenic activity was measured using [I]iododeoxyuridine as previously described(24) , except that NR6/HER or CEF cells were used in place of human foreskin fibroblasts, and the CEF cells were pulsed with the labeled thymidine analogue for 4 h instead of the usual 2 h.

I-Labeled mEGF Competitive Binding Assay

NR6/HER or CEF cells were trypsinized and resuspended in 10% (v/v) fetal bovine serum/Dulbecco's modified Eagle's medium. The cells were diluted to 5 times 10^4 cells/ml and 500 µl/well aliquoted into sterile 24-well trays. The trays were incubated at 37 °C, 5% CO(2) for 18 h. The adherent cells were then washed twice with 500 µl/well of PBS and preincubated with 500 µl/well of PBS, 1% (w/v) BSA at 22 °C. After 30 min, the buffer was removed and replaced with 250 µl/well of PBS, 1% (w/v) BSA containing 0.02% (w/v) sodium azide (to inhibit EGF-R internalization), and growth factors as appropriate. 250 µl/well of I-labeled mEGF (20 ng/ml, 3.7 mBq/µg) in PBS, 1% (w/v) BSA, 0.02% (w/v) sodium azide was added to all wells, and the cells incubated at 22 °C for 4 h. After this time, buffer containing unbound material was removed from the trays by pipette, and the cells were washed with PBS, 0.1% BSA. The trays were left to dry, after which the cells were solubilized with 200 µl/well of 0.5 M NaOH, and bound radioactivity was determined by counting. Nonspecific binding, determined in the presence of an excess of unlabeled mEGF, was less than 5 (NR6/HER) and 20% (CEF) of total bound ligand.


RESULTS

The cEGF-R has been previously cloned and characterized and shown to possess an affinity for hTGFalpha that is approximately 100 times greater than that of mEGF(18) . Fig. 2A shows a comparison of the mitogenic activity of mEGF and hTGFalpha on CEF and confirms the differential activity of these growth factors on the cEGF-R. For comparison, the same mEGF and hTGFalpha 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 (bullet) and hTGFalpha () 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/hTGFalpha and mEGF/hTGFalpha, 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/hTGFalpha, or mEGF/hTGFalpha 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/hTGFalpha containing medium was about 6 times more potent than that containing mEGF/hTGFalpha 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/hTGFalpha 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 alpha-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/hTGFalpha 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/hTGFalpha being 85 pM. In mitogenesis assays utilizing CEF, the mEGF/hTGFalpha 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 hTGFalpha 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 (bullet) and mEGF/hTGFalpha () 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 TGFalpha 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/hTGFalpha to the hEGF-R were 1.4 and 0.9 nM, respectively (Fig. 4A). As found in the CEF mitogenesis assays, mEGF/hTGFalpha 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 TGFalpha 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 (bullet) or mEGF/hTGFalpha (). 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 TGFalpha standard are also shown () and are the mean of two observations.




DISCUSSION

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 hTGFalpha 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 beta-sheet were replaced by the equivalent residues of hTGFalpha 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 hTGFalpha 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/hTGFalpha chimeras and found that chimeras containing the COOH-terminal tail sequence of hTGFalpha had an affinity for the cEGF-R comparable with that of native hTGFalpha 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 hTGFalpha 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 hTGFalpha 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 beta-sheet segment with the equivalent residues of hTGFalpha 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 hTGFalpha-like by substitution of the hTGFalpha COOH-terminal tail.

In light of this, we propose that there are two sets of determinants that distinguish the EGFs and hTGFalpha, and each set contributes 1 order of magnitude to the binding affinities of the growth factors to the chicken receptor. Thus, hTGFalpha 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 hTGFalpha and either hEGF or mEGF, while the second set lies in the B-loop segment, enabling the receptor to distinguish between mEGF and either hTGFalpha or hEGF. This is consistent with our previous studies that indicated that the interaction of hTGFalpha with the hEGF-R is a multidomain process(16) .



By comparing the sequences of hTGFalpha, hEGF, and mEGF in the B-loop beta-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 hTGFalpha are phenylalanine and lysine, it is possible that these represent key distinguishing residues between mEGF and either hEGF or hTGFalpha. 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 beta-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/hTGFalpha did not just depend on the presence of His-22 and Lys-28, as the other chimera, mEGF/hTGFalpha, was intermediate in activity between mEGF and mEGF/hTGFalpha. 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 hTGFalpha 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.


FOOTNOTES

*
This work was supported by the Cancer Research Campaign, UK. 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.

§
Present address: School of Biological Sciences, University of Manchester, Stopford Bldg., Oxford Rd., Manchester M13 9PT, UK.

To whom correspondence should be addressed. Tel.: 703-798795; Fax: 703-783839.

(^1)
The abbreviations used are: EGF, epidermal growth factor (h or m prefix refers specifically to the human or murine sequence, respectively); TGFalpha, transforming growth factor alpha (h prefix refers specifically to the human sequence); EGF-R, epidermal growth factor receptor (h or c prefix refers specifically to the human or chicken receptor, respectively); CEF, chick embryo fibroblasts; PBS, phosphate-buffered saline; BSA, bovine serum albumin; bp, base pair(s).

(^2)
Unless referring to TGFalpha specifically, the numbers cited refer to the EGF sequence.


ACKNOWLEDGEMENTS

We thank I. Davidson of the Dept. of Molecular and Cell Biology, University of Aberdeen, for performing the Laser Desorption mass analyses and Dr J. J. Clare of Wellcome Research Laboratories, Beckenham, Kent, UK, for provision of the yeast pWYG9/EGF expression vector.


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