©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
HER4 Receptor Activation and Phosphorylation of Shc Proteins by Recombinant Heregulin-Fc Fusion Proteins (*)

Jean-Michel Culouscou (§) , Gary W. Carlton , Alejandro Aruffo

From the (1) Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heregulins (HRGs) are mosaic glycoproteins that bind to and induce the tyrosine phosphorylation of the HER4/p180 receptor. This work was aimed at studying the biological effects induced by recombinant epidermal growth factor (EGF)-like domains of HRGs as well as identifying intracellular molecules involved in HER4 signaling. To this end, we cloned the EGF-like domains of HRG-, -2, and -3 into a eukaryotic expression vector in frame with sequences encoding a thrombin cleavage site followed by the Fc portion of a human IgG1. These chimeric genes directed the expression of recombinant fusion proteins, rHRGs-T-Fc, which specifically stimulated the phosphorylation of HER4/p180. We also show that rHRG--T-Fc bound to human breast cancer cells that express HER4 receptors and induced the expression of intercellular adhesion molecule-1. After thrombin protease cleavage of rHRGs-T-Fc, their EGF-like domains were purified and shown to stimulate protein phosphorylation in HER4-expressing cells. Moreover, the rHRG-2 EGF-like domain markedly induced the phosphorylation of Shc proteins on tyrosine, suggesting a role for these adaptor molecules in HRG-mediated signaling.


INTRODUCTION

Heregulins (HRGs)()(1) , neu differentiation factor (NDF) (2, 3, 4) , glial growth factors (5) , and acetylcholine receptor-inducing activity (6) are homologous multifunctional proteins. The HRG isoforms originate from a single gene by alternative RNA splicing. HRG cDNAs encode large transmembrane precursors with multiple domains, including an immunoglobulin-like domain, a spacer domain with several glycosylation sites, an EGF-like domain, a juxtamembrane domain of variable length, a transmembrane region, and a cytoplasmic domain. Soluble mature HRGs are most probably released from the cell surface by proteolytic cleavage. Glial growth factors (5) and acetylcholine receptor-inducing activity (6) , which were isolated from brain tissues, also contain a kringle-like domain that is absent in HRGs and NDFs. HRG - and -isoforms display sequence differences in the third loop of the EGF-like domain and in the juxtamembrane domain. The EGF-like domain of HRGs contains six cysteine residues that are characteristic of the EGF family of growth factors, including EGF (7) , transforming growth factor- (8) , vaccinia virus growth factor (9) , amphiregulin (10) , heparin-binding EGF-like growth factor (11) , and betacellulin (12) .

Although HRGs contain an EGF-like motif, they do not bind to EGFR/p170(1) . In fact, HRGs bind to HER4/p180, a recently isolated member of the epidermal growth factor receptor family (13, 14, 15) . The HER3/p180 receptor, another member of this family, has also been reported to be a receptor for HRGs (16) . HRGs do not directly interact with the HER2/p185 receptor, as originally proposed (1, 2) . However, it appears that HER2/p185 indirectly participates in HRG-mediated signaling through transphosphorylation or receptor heterodimerization with HER4 and/or HER3 (15, 16) .

As a consequence of HRG/NDF binding to its receptor(s), human mammary tumor cells have been shown to differentiate (2, 17) and up-regulate their expression of intercellular adhesion molecule-1 (ICAM-1) (17) . The biological effects of HRGs are mediated through receptors that possess an intrinsic tyrosine kinase activity and are autophosphorylated upon HRG binding (15) . Studies on receptor tyrosine kinases such as the epidermal growth factor receptor, the platelet-derived growth factor receptor, and the insulin receptor have demonstrated a crucial role for receptor autophosphorylation in intracellular signal transduction following ligand binding (18, 19) . It has been demonstrated that specific autophosphorylation sites on receptor tyrosine kinases serve as recognition structures for target molecules containing Src homology 2 (SH2) domains. SH2 domains are conserved noncatalytic sequences of 100 amino acids found in various signaling molecules and oncogenic proteins (20, 21) . SH2 domain-containing proteins bind with high affinity to phosphotyrosine residues in the context of specific flanking amino acids. For example, the p85 subunit of phosphatidylinositol 3`-kinase, the p21 GTPase-activating protein, and phospholipase C- have been shown to contain SH2 domains. More recently, SH2 domain-containing proteins that lack an apparent catalytic domain and that seem to function as adaptors linking proteins involved in signal transduction have been described (22-27). One of them, Shc, was identified and cloned based on its homology to SH2 sequences from the human c-fes gene (23) . The Shc cDNA is predicted to encode two proteins of 46 and 52 kDa that contain a single C-terminal SH2 domain and a collagen homologous region that is rich in glycine and proline. No catalytic domain was identified in Shc. Anti-Shc antibodies have been shown to recognize three proteins of 46, 52, and 66 kDa in a wide range of mammalian cells. A variety of growth factors and cytokines have been shown to induce the phosphorylation of Shc proteins (28, 29, 30, 31, 32, 33) . Furthermore, overexpression of Shc proteins is associated with a transformed phenotype in fibroblasts (23) and neuronal differentiation of PC12 cells (34) , strongly suggesting that Shc is involved in cell growth regulation.

This report describes the generation of versatile recombinant HRGs and the study of some of the biological functions and intracellular signaling pathways that these proteins trigger following receptor activation. Because the EGF-like domain of HRG is sufficient for receptor binding (1, 16) , we cloned the cDNA fragments encoding the EGF-like domain of HRG-, -2, or -3 into a eukaryotic expression vector containing sequences encoding a thrombin cleavage site followed by the Fc portion of a human IgG. The recombinant fusion proteins generated, referred to as rHRGs-T-Fc, were used either as chimeric proteins or as EGF-like domains (reHRGs) after thrombin cleavage and removal of the Fc portion of the molecule. Herein, we demonstrate that recombinant HRGs, in either form, bind to and activate the HER4 receptor and show that the Shc proteins are tyrosine-phosphorylated following HRG stimulation.


EXPERIMENTAL PROCEDURES

Antibodies

RC20 recombinant anti-phosphotyrosine antibody (Transduction Laboratories) and PY20 anti-phosphotyrosine antibody (ICN Biomedicals, Inc.) were used in Western blotting studies. Polyclonal anti-Shc antibodies were purchased from Upstate Biotechnology, Inc., and the monoclonal anti-Shc antibody was from Transduction Laboratories. BBA 3, an anti-human ICAM-1 monoclonal antibody, was from R& Systems.

Cell Lines

MDA-MB-453 human breast cancer cells were obtained from the American Type Culture Collection. CHO/EGFR cells were generated by Dr. B. Thorne (Bristol-Myers Squibb, Seattle, WA) as follows. The complete recombinant human EGF receptor coding sequence was inserted into a CDM8 expression vector containing the neomycin resistance gene. The resulting construct was transfected into CHO-K1 cells. G418-resistant clones were analyzed for EGFR expression. Levels of expression of functional EGFR in CHO/EGFR stable cells were assessed by stimulating the cells with EGF, immunoprecipitating the EGFR, and determining its phosphorylation level by phosphotyrosine Western blotting as reported (13) . CHO/HER4 cells expressing high levels of recombinant human HER4 have previously been described (13, 14, 15) .

Construction of HRG-T-Fc Expression Plasmids

DNA fragments encoding part of the spacer domain of human HRGs, the EGF-like domains, the transmembrane domain, and a few residues of the cytoplasmic domain were amplified by reverse transcription-PCR from total RNA isolated from HepG2 cells. The oligonucleotide primers were designed based on the sequence of human HRG- (1) . The PCR primers used were 5`-GTGTCTTCAGAGTCTCCCATTAGA-3` (forward) and 5`-CTTGGTTTTGCAGTAGGCCAC-3` (reverse). Amplification was performed with Taq DNA polymerase (Perkin-Elmer) using 35 cycles, with each cycle being composed of a denaturing step for 1 min at 95 °C, an annealing step for 1 min at 65 °C, and an extension step for 30 s at 72 °C. The PCR products were blunt-ended using the Klenow fragment of Escherichia coli DNA polymerase I and subcloned into an SmaI-digested pBluescript II vector (Stratagene), and the nucleotide sequences of individual clones were determined by the dideoxy-mediated chain termination reaction.

The EGF-like domains of HRG-, -2, and -3 were generated by PCR using HRG- or -2 template plasmids generated as described above. The oligonucleotide primers described below were designed to place an SpeI site at the 5`-end and a BamHI site at the 3`-end of the amplified products for cloning purposes. The epidermal growth factor-like domain of human HRG- was amplified using the following primers: 5`-GAGACTAGTAGCCATCTTGTAAAATGTGCG-3` (forward) and 5`-CCGTGGATCCTTCTGGTACAGCTCCTCCGC-3` (reverse). PCR conditions consisted of 40 cycles of 30 s at 94 °C, 1 min at 55 °C, and 2 min at 72 °C using Pfu polymerase and reagents recommended by the vendor (Stratagene). The PCR product encoded complementary sequences corresponding to residues 177-241 of HRG-. The epidermal growth factor-like domains of human HRG-2 and -3 were amplified using an HRG-2 clone as a template. The forward primer was described above. The HRG-2 reverse primer had the sequence 5`-CCGTGGATCCTTCTGGTACAGCTCCTCCGCCTT-3`. Amplification was performed with Pfu polymerase using the same temperature program as that used for HRG-. This PCR product encoded sequences corresponding to residues 177-238 of HRG-2. The HRG-3 reverse primer contained a silent point mutation introducing a HindIII site for diagnostic purposes and had the following sequence: 5`-CCGTGGATCCTCAGGCAAGCTTAGAAAGGGAGTGGACGTACTGTAGAAGC-TGGCCATTAC-3`. PCR conditions consisted of 40 cycles of 1 min at 94 °C, 2 min at 50 °C, and 3 min at 72 °C using Pfu polymerase. The PCR product encoded sequences corresponding to residues 177-241 of HRG-3. All PCR products were digested with BamHI and SpeI and ligated to a BamHI-SpeI-cut CDM7-derived vector containing cDNA sequences coding for the CD5 signal peptide 5` of the cloning site for proper secretion of the expressed proteins as well as cDNA sequences encoding a thrombin cleavage site (amino acid sequence: DPGGGGGRLVPRGFGTG) and cDNA sequences encoding the hinge and constant regions of a human IgG1 3` of the cloning site.() All constructs were sequenced by the dideoxy-mediated chain termination reaction to confirm the sequence of the EGF-like domains as well as to verify that their sequences were in frame with the thrombin and Fc coding sequences. Constructs were transfected into COS cells as described previously (35) , and the resulting fusion proteins were recovered from culture supernatants using protein A-Sepharose (Repligen). Purified proteins were visualized on 8% SDS-polyacrylamide gel under reducing and nonreducing conditions. Protein concentrations were determined using a protein assay kit (Bio-Rad).

Thrombin Cleavage

Fusion proteins were incubated for 30 min at room temperature with human thrombin (Sigma) at a 1:50 (w/w) thrombin/fusion protein ratio. Cleaved proteins were then loaded on a protein A-Sepharose column. Column flow-through fractions containing the recombinant EGF-like domains of HRGs were stored at -20 °C until further use.

Detection of Tyrosine-phosphorylated Proteins by Western Blotting

CHO/HER4 cells (5 10), CHO/EGFR cells (2 10), and MDA-MB-453 cells (4 10) were seeded in 48-well plates. 24 h later, cells were serum-starved for 8 h and then stimulated with various samples for 10 min at 37 °C. Supernatants were discarded, and cells were lysed by adding boiling electrophoresis sample buffer. Lysates were subjected to SDS-PAGE on 8% polyacrylamide gels (Novex) and then electroblotted onto nitrocellulose. PY20 monoclonal anti-phosphotyrosine antibody and horseradish peroxidase-conjugated goat anti-mouse IgG F(ab`) (Cappel) were used as primary and secondary probing reagents, respectively. Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Corp.).

Immunoprecipitation

CHO/HER4 cells were seeded in 100-mm dishes. 80-90% confluent monolayers were washed and incubated with various recombinant HRGs for 10 min at 37 °C. Monolayers were washed with ice-cold PBS and solubilized for 10 min on ice in PBSTDS lysis buffer (10 mM sodium phosphate, pH 7.3, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM NaVO, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 20 µg/ml pepstatin. The protein concentrations of the clarified extracts were determined using a bicinchoninic acid protein assay kit (Pierce). Lysates (1 mg/immunoprecipitation) were incubated overnight at 4 °C with a rabbit anti-Shc antibody (Upstate Biotechnology, Inc.). Immune complexes were precipitated by adding protein G Plus-/protein A-agarose (Oncogene Science Inc.) to the suspensions. After 1 h of incubation at 4 °C, the immunoprecipitates were washed three times with PBSTDS lysis buffer and then resolved on 8% polyacrylamide gels under reducing conditions. Proteins were electroblotted onto nitrocellulose and probed with RC20 recombinant anti-phosphotyrosine antibody or a monoclonal anti-Shc antibody. Immunoreactive bands were visualized using enhanced chemiluminescence.

Immunohistochemical Staining

MDA-MB-453 cells were plated on 8-well borosilicate-chambered slides (Lab-Tek, Nunc). For receptor binding visualization, after a 48-h culture period, the cells were placed on ice for 10 min, washed twice with ice-cold binding buffer (Dulbecco's modified Eagle's medium supplemented with 44 mM sodium bicarbonate, 50 mM Bes, pH 7.0, 0.1% bovine serum albumin), and then incubated on ice for 2 h with rHRG--T-Fc or, as a negative control, an irrelevant fusion protein consisting of the extracellular domain of the Tek receptor (36) fused to the thrombin cleavage site followed by the Fc region of an IgG1, as in rHRGs-T-Fc.() The reagents, cloning vector, and mammalian cells used to construct and to generate the Tek-Fc fusion protein were identical to the ones used to make rHRGs-T-Fc. The cells were washed twice and incubated for 45 min on ice with a fluorescein-conjugated goat anti-human IgG F(ab`) fragment (Tago, Inc.). The cells were rinsed twice with PBS and fixed for 20 min in PBS, 2% formaldehyde. For ICAM-1 expression studies, after a 24-h culture period, the cells were incubated for 3 days with 50 ng/ml rHRG--T-Fc, p45 (14) , Tek-Fc fusion protein as a negative control, or culture medium alone. Staining was then performed on live cells. The cells were washed and incubated for 1 h on ice with an anti-ICAM-1 antibody diluted 1:500 in binding buffer. The cells were washed and incubated for 45 min on ice with a fluorescein-conjugated goat anti-mouse IgG F(ab`) fragment (Tago, Inc.). The cells were rinsed and fixed as described above. The levels of receptor staining and ICAM-1 expression were analyzed using a Leica confocal microscope.


RESULTS AND DISCUSSION

Construction of rHRGs-T-Fc

Our objective was to generate versatile recombinant HRGs to study various aspects of the biology of the HER4/HRG receptor/ligand pair. Because the EGF-like domain of HRG-1 had previously been shown to be sufficient for receptor binding (1) , we constructed three chimeric genes that encode soluble proteins consisting of the EGF-like domain of HRG-, -2, or -3 linked to a thrombin cleavage site followed by the hinge, CH2, and CH3 regions of a human IgG1 antibody, with secretion of the proteins directed by the signal sequence of CD5 (see ``Experimental Procedures'' for details). The EGF-like domain of HRG- corresponded to residues 177-241 of the mature protein, while those of HRG-2 and -3 corresponded to residues 177-238 and residues 177-241, respectively. The three fusion proteins (rHRGs-T-Fc) were prepared by transient expression in COS cells, purified from culture supernatants on protein A-Sepharose, and gave yields in the range of 350-1900 µg/liter. The CD5 signal peptide allowed efficient processing and secretion of rHRGs-T-Fc. All three fusion proteins were secreted as disulfide-linked homodimers similar to immunoglobulins and therefore were each capable of presenting two HRG EGF-like domains. To establish that rHRGs-T-Fc were able to bind and to activate the HER4 receptor, we examined their potential to induce the phosphorylation of HER4 as well as morphological changes and up-regulation of ICAM-1 expression.

Activation of the HER4 Receptor by rHRGs-T-Fc

For these experiments, we used CHO/HER4 cells, which express high levels of recombinant human HER4/p180 and have previously been shown to respond to HRG (13, 14) . rHRG--T-Fc, -2-T-Fc, and -3-T-Fc were added to CHO/HER4 cells at 50 and 200 ng/ml for 10 min at 37 °C. Cells were lysed, and then the pattern of tyrosine-phosphorylated proteins was analyzed by anti-phosphotyrosine Western blotting as compared with untreated cells. As shown in Fig. 1A, all three rHRGs-T-Fc induced the hyperphosphorylation of the HER4 receptor. Ligand activation not only resulted in receptor autophosphorylation, but also in the tyrosine phosphorylation of several substrates, including an M 100,000 band, yet to be identified (Fig. 1A). When tested on CHO/EGFR cells, which express high levels of recombinant human EGFR, rHRGs-T-Fc (200 ng/ml) failed to activate the EGFR (Fig. 1B). As expected, EGF (200 ng/ml) markedly induced the phosphorylation of the EGFR in CHO/EGFR cells (Fig. 1B, lane2). These experiments indicate that rHRGs-T-Fc are active molecules and are able to specifically induce HER4 tyrosine phosphorylation.


Figure 1: Tyrosine autophosphorylation of the HER4 receptor following rHRGs-T-Fc stimulation. A, CHO/HER4 cells were incubated in the absence (lane1) or presence of rHRG--T-Fc (lanes2 and 3), rHRG-2-T-Fc (lanes4 and 5), and rHRG-3-T-Fc (lanes6 and 7) at 50 ng/ml (lanes2, 4, and 6) or 200 ng/ml (lanes3, 5, and 7). Cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine antibodies. Horseradish peroxidase-conjugated goat anti-mouse IgG antibodies and chemiluminescence reagents were used to visualize the bound antibodies. B, CHO/EGFR cells were incubated in the absence (control (c); lane1) or presence of EGF (lane2), rHRG--T-Fc (lane3), rHRG-2-T-Fc (lane4), and rHRG-3-T-Fc (lane5) at 200 ng/ml. Cells lysates were processed as described for A. The positions of HER4 and EGFR are indicated.



Binding of rHRG--T-Fc to HER4-expressing Cells

After demonstrating that the three fusion proteins are activators of HER4, we further characterized rHRG--T-Fc and checked its ability to bind to HER4-expressing cells. MDA-MB-453 cells, which are known to express the HER4 receptor (13) as well as the related receptors HER2 and HER3 (37, 38) , were used in this assay. These cells were incubated on ice with 1 or 10 µg/ml rHRG--T-Fc. Bound fusion proteins were detected by adding fluorescein-conjugated anti-human IgG antibodies, which recognized the human Fc portion of rHRG--T-Fc. As shown in Fig. 2, rHRG--T-Fc bound to MDA-MB-453 cells (Fig. 2, B, 10 µg/ml; and D, 1 µg/ml). Fluorescence, as analyzed by confocal microscopy, was localized at the periphery of the cells, which is consistent with the fact that the staining was performed on live cells kept on ice. When no fusion protein was added, the fluorescein-conjugated anti-human IgG showed no detectable binding to the MDA-MB-453 cells (Fig. 2A). Minimal background staining was observed when an irrelevant Tek-Fc fusion protein was used at 10 µg/ml (Fig. 2C). This experiment indicates that rHRG--T-Fc binds to HER4-expressing cells and can be used to detect cells expressing HRG-binding proteins in a manner similar to monoclonal antibodies. Thus, rHRGs-T-Fc might represent an interesting alternative to antibodies for cell staining.


Figure 2: Binding of rHRG--T-Fc to MDA-MB-453 cells. Cells were plated on 8-well Lab-Tek chamber slides at 2 10 cells/well. After 2 days, the cells were placed on ice and stained with rHRG--T-Fc at 10 µg/ml (B) and 1 µg/ml (D) or with an irrelevant fusion protein used at 10 µg/ml (C). No fusion proteins were added in the experiment shown in A. Fluorescein-labeled goat anti-human Fc antibodies were used to visualize the bound fusion proteins. Fluorescent staining was analyzed by confocal microscopy.



Induction of ICAM-1

NDF, the rat homologue of HRG, has been shown to induce morphological changes in AU565 mammary tumor cells (2) as well as the expression of ICAM-1 (17) . We have previously used the human breast cancer cell line MDA-MB-453 in differentiation assays to monitor the purification of an HRG isoform, p45 (14) . These cells can also be induced to differentiate when treated with rHRG--T-Fc, -2-T-Fc, and -3-T-Fc at similar concentrations (data not shown). To examine the ability of rHRG--T-Fc to induce the expression of ICAM-1 at the surface of MDA-MB-453 cells, these cells were treated for 3 days with a 50 ng/ml concentration of the fusion protein and stained with an anti-ICAM-1 antibody. Bound anti-ICAM-1 antibodies were detected using a fluorescein-conjugated anti-human IgG antibody. As shown in Fig. 3B, rHRG--T-Fc induced a clear up-regulation of ICAM-1 expression in MDA-MB-453 cells as compared with untreated cells (Fig. 3A) and with cells treated with an irrelevant Tek-Fc fusion protein (Fig. 3C). p45, an HRG isoform purified from conditioned medium from HepG2 cells (14), was used at 50 ng/ml as a positive control and as expected induced up-regulation of ICAM-1 (Fig. 3D). In conclusion, rHRGs-T-Fc elicited biological responses similar to those elicited by natural HRGs in breast carcinoma cells expressing the HER4 receptor and thus can be used to study the biological consequences of HRG binding to such cells.


Figure 3: Induction of ICAM-1 expression in response to rHRG--T-Fc. MDA-MB-453 cells were cultured for 24 h on 8-well Lab-Tek chamber slides. Cells were treated with 50 ng/ml rHRG--T-Fc (B), irrelevant fusion protein (C), or p45 (14) (D) or were left untreated (A). Following an additional 3 days of incubation, the cells were stained with an anti-ICAM-1 monoclonal antibody. Fluorescein-labeled goat anti-mouse Fc antibodies were used to visualize the bound anti-ICAM-1 antibodies. Staining was analyzed by confocal microscopy.



Release of EGF-like Domains of HRGs after Thrombin Cleavage of Fusion Proteins

We have constructed CDM7-derived vectors containing sequences encoding a thrombin cleavage site upstream and in frame with the Fc portion of a human IgG1 antibody. The addition of a thrombin cleavage site in the expression vector was based on a system developed by Hakes and Dixon (39) for recombinant protein expression in bacteria. The presence of a thrombin cleavage site in rHRGs-T-Fc allowed for separation of the two functional domains of the fusion proteins. Following thrombin cleavage, the purified EGF-like domains would be recovered as monomeric proteins since the thrombin site is located upstream of the hinge region of the Fc domain of the fusion proteins. rHRG-2-T-Fc and -3-T-Fc were incubated with human thrombin. reHRGs were then separated from the Fc portion of the molecules by protein A-Sepharose chromatography and recovered in the column flow-through fractions. Fc portions were recovered from protein A-Sepharose by acid elution. Fig. 4 (lane1) shows a silver-stained polyacrylamide gel of rHRG-3-T-Fc before thrombin cleavage. The intact fusion protein displays an apparent molecular mass of 40 kDa under reducing conditions, corresponding to its monomeric form. After cleavage but before protein A-Sepharose chromatography (lane2), a 34-kDa band, corresponding to the Fc portion of the fusion protein, and a 6-kDa band, corresponding to the EGF-like domain of HRG-3, were identified. The two fragments were separated by protein A-Sepharose chromatography. The 6-kDa EGF-like domain of HRG-3 (reHRG-3) was recovered in the column flow-through fraction (lane3), and the 34-kDa Fc domain of the fusion protein was acid-eluted from the column (lane4).


Figure 4: Thrombin cleavage of rHRG-3-T-Fc. The fusion protein was incubated with human thrombin at room temperature for 30 min and loaded on a protein A-Sepharose column. reHRG-3 was recovered in the column flow-through fraction, while the Fc portion of the fusion protein was eluted from the column. The resulting products were analyzed by SDS-PAGE and silver-stained. Lane1, untreated rHRG-3-T-Fc; lane2, rHRG-3-T-Fc after thrombin cleavage; lane3, protein A-Sepharose column flow-through fraction (reHRG-3); lane4, protein A-Sepharose column eluate (Fc portion of the fusion protein).



Stimulation of Protein Phosphorylation in Response to reHRGs

The purified reHRG-2, reHRG-3, and Fc domains of rHRG-2-T-Fc and -3-T-Fc were tested for their ability to stimulate protein phosphorylation in MDA-MB-453 cells. As expected, rHRG-2-T-Fc and -3-T-Fc (Fig. 5, lanes3 and 6, respectively) were potent stimulators of the tyrosine phosphorylation of a 180-kDa protein as compared with background levels of phosphorylation observed in the absence of treatment (lane1) or following EGF treatment (lane2). reHRG-2 (lane4) and reHRG-3 (lane7) elicited an increase in the phosphorylation level of the 180-kDa protein similar to that obtained with rHRGs--T-Fc, whereas the Fc domains of rHRG-2-T-Fc and -3-T-Fc failed to induce protein phosphorylation (lanes5 and 8, respectively).


Figure 5: Stimulation of protein phosphorylation in response to reHRGs. MDA-MB-453 cells were incubated in the absence (lane1) or presence of EGF (lane2), rHRG-2-T-Fc (lane3), reHRG-2 (lane4), the Fc portion of rHRG-2-T-Fc (lane5), rHRG-3-T-Fc (lane6), reHRG-3 (lane7), and the Fc portion of rHRG-3-T-Fc (lane8) at 200 ng/ml. Cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and blotted with an anti-phosphotyrosine antibody. Immunoreactive bands were visualized with enhanced chemiluminescence reagents.



Following cleavage of the fusion proteins, several amino acid residues from the glycine-rich region of the thrombin cleavage site remain at the carboxyl terminus of reHRGs. Cleavage occurs at the Pro-Arg recognition sequence of the thrombin cleavage site (see Ref. 39 for details). These additional residues did not affect the properties of reHRGs, as demonstrated above. In fact, among the multiple HRG/NDF isoforms, the region proximal to the EGF domain (referred to as the juxtamembrane region in the HRG/NDF precursor forms) can be absent, e.g. HRG-2/NDF-2, or comprise up to 26 amino acids, e.g. NDF-4 (1, 4) . A truncated form of NDF- that lacks the juxtamembrane region displays the same receptor binding affinity as the full-length NDF -isoform, implying that this region proximal to the EGF domain is not involved in receptor binding (4) . We conclude that, after thrombin cleavage, reHRGs retain the activity displayed by rHRGs-T-Fc.

Because MDA-MB-453 cells express HER3, HER4, and also high levels of HER2, the exact identity of the 180-kDa phosphorylated band is unknown. HER4 and HER3 have both been identified as HRG receptors (13, 14, 15, 16) , making them obvious candidates. However, since the samples were electrophoresed under reducing conditions, the phosphorylated species might correspond to a combination of monomeric forms of all three receptors, including HER2. In fact, in response to HRG stimulation, HER2 is stimulated indirectly through receptor transphosphorylation (15). In addition, a recent study has indicated that a high affinity binding site for the EGF-like domain of HRG-1 can be reconstituted by coexpression of HER2 and HER3 in COS-7 cells and that binding of HRG results in the tyrosine phosphorylation of both HER2 and HER3 (40) .

The experiment described above clearly demonstrates that the EGF-like domains of rHRGs-T-Fc mediate the observed biological effects and that these effects cannot be attributed to the Fc portion of the fusion proteins.

Phosphorylation of Shc upon HER4 Receptor Activation

Activation of receptor tyrosine kinases, such as the EGF receptor, the insulin receptor, and the platelet-derived growth factor receptor, results in the phosphorylation of a number of intracellular signaling molecules (18, 19) . In a preliminary attempt to analyze the molecules that might be involved in HRG signaling, we stimulated MDA-MB-453 cells with or without 200 ng/ml reHRG-2. Cell lysates were immunoprecipitated with the following antibodies: anti-GTPase-activating protein, anti-phospholipase C-1, anti-phosphatidylinositol 3`-kinase, and anti-Shc. Precipitated proteins were separated by SDS-PAGE and then immunoblotted with anti-phosphotyrosine antibodies. Shc and, to a lesser degree, phosphatidylinositol 3`-kinase immunoprecipitates displayed enhanced patterns of protein phosphorylation following HRG stimulation (data not shown). We decided to further analyze Shc phosphorylation. Shc proteins are ubiquitously expressed proteins containing a single SH2 domain. Three structurally related Shc proteins, p46, p52, and p66, have been described as adaptor molecules that are implicated in Ras activation (23, 34) . CHO/HER4 and MDA-MB-453 cells were exposed to reHRG-2 and lysed. Equivalent amounts of cell lysates were immunoprecipitated with an anti-Shc antibody and blotted with either anti-Shc (Fig. 6A) or anti-phosphotyrosine (Fig. 6B) antibodies. Fig. 6A shows that equal amounts of proteins from stimulated and unstimulated cell lysates were loaded per lane and that MDA-MB-453 cells (lanes1 and 2) express only p46 and p52 (p66 was not detected in our assay), whereas CHO/HER4 cells (lanes3 and 4) express all three Shc isoforms. p66 is translated from a different transcript than the other two Shc isoforms and is not expressed in every cell type; for example, it is absent in human hematopoietic cell lines (23) . As seen in Fig. 6B, reHRG-2 induced the hyperphosphorylation of Shc in both cell types. In MDA-MB-453 cells, reHRG-2 stimulation resulted in the tyrosine phosphorylation of both p46 and p52 (lanes1 and 2). Following reHRG-2 stimulation, the phosphorylation of p52 was markedly increased in CHO/HER4 cells (lanes3 and 4). p46 appeared to display a relatively high endogenous level of phosphorylation in those cells and was only marginally affected following HRG treatment. A longer exposure time of the blot shown in Fig. 6B (lanes3 and 4) resulted in a loss of resolution between the p46 and p52 bands, but revealed that p66 was phosphorylated in response to reHRG-2 (lane6) as compared with unstimulated cells (lane5). We were not able to show, by immunoprecipitation, ligand-induced association of Shc with HER4 in CHO/HER4 cells (data not shown), an interaction that has been reported to take place between Shc and the EGFR (23) , the HER2/p185 receptor (41) , or the platelet-derived growth factor receptor (29) . Further studies will be required to assess the ability of HER4 to recruit Shc. The possibility remains that Shc might indirectly bind to HER4 via another adaptor molecule, such as Grb2 (22, 24, 25, 26, 27) .


Figure 6: Tyrosine phosphorylation of Shc proteins upon HER4 activation. MDA-MB-453 cells (lanes1 and 2) and CHO/HER4 cells (lanes 3-6) were treated with (+) or without (-) 200 ng/ml reHRG-2 for 10 min at 37 °C and solubilized. Cell lysates containing equal amounts of protein (1 mg) were precipitated with a polyclonal rabbit anti-Shc antibody. Immune complexes were washed, separated by SDS-PAGE, and transferred to nitrocellulose. A, Shc proteins were detected by immunoblotting using a monoclonal anti-Shc antibody; B, the tyrosine phosphorylation of Shc proteins was analyzed by immunoblotting using anti-phosphotyrosine antibodies (Anti-Ptyr). The positions of the three Shc isoforms are indicated. IP, immunoprecipitation.



In summary, we have generated recombinant EGF-like domains of HRG-, -2, and -3 fused to a thrombin cleavage site followed by the Fc domain of a human IgG1. These reagents can be used in in vitro assays as fusion proteins or as a source of truncated recombinant HRGs. We have demonstrated in this study that both forms can activate the HER4 receptor and elicit known HRG biological responses. We have shown for the first time that, following HRG stimulation, Shc proteins, which have been implicated in the Ras activation pathway, are phosphorylated on tyrosine. The availability of recombinant HRGs will allow us to further dissect the mechanism of HRG receptor signaling as well as to compare the HER4 substrates with those of other members of the EGFR family of tyrosine kinases.


FOOTNOTES

*
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§
To whom correspondence should be addressed: Bristol-Myers Squibb Pharmaceutical Research Inst., 3005 First Ave., Seattle, WA 98121. Tel.: 206-727-3641; Fax: 206-727-3602.

The abbreviations used are: HRGs, heregulins (the prefix ``r'' stands for recombinant); reHRGs, EGF-like domains of recombinant HRGs; NDF, neu differentiation factor; EGF, epidermal growth factor; EGFR, EGF receptor; HER, human EGF receptor; ICAM-1, intercellular adhesion molecule-1; SH2, Src homology 2; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Bes, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.

A. Aruffo and D. Hollenbaugh, unpublished data.

J.-M. Culouscou, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Dr. S. Myrdal and T. Bailey for expert assistance with confocal microscopy studies. We thank M. Neubauer and the members of the DNA/peptide chemistry group for DNA sequencing and oligonucleotide synthesis. We also thank Dr. D. Hollenbaugh for helpful discussions and critical reading of the manuscript.


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