©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neu Differentiation Factor Inhibits EGF Binding
A MODEL FOR TRANS-REGULATION WITHIN THE ErbB FAMILY OF RECEPTOR TYROSINE KINASES (*)

Devarajan Karunagaran (1), Eldad Tzahar (1), Naili Liu (2), Duanzhi Wen (2), Yosef Yarden (1)(§)

From the (1) Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel and the (2) Amgen Center, Thousand Oaks, California 91320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neu differentiation factor (NDF, or heregulin) and epidermal growth factor (EGF) are structurally related proteins that bind to distinct members of the ErbB family of receptor tyrosine kinases. Here we show that NDF inhibits EGF binding in a cell type-specific manner. The inhibitory effect is distinct from previously characterized mechanisms that involve protein kinase C and receptor internalization because it occurred at 4 °C and displayed reversibility. The extent of inhibition correlated with both receptor saturation and affinity of different NDF isoforms, and it was abolished upon overexpression of either EGF receptor or ErbB-2. Binding kinetics and equilibrium analyses indicated that NDF reduced the affinity, rather than the number, of EGF receptors, through an acceleration of the rate of ligand dissociation and deceleration of the association rate. On the basis of co-immunoprecipitation of EGF and NDF receptors, we attribute the inhibitory effect to the formation of receptor heterodimers. According to this model, EGF binding to NDF-occupied heterodimers is partially blocked. This model of negative trans-regulation within the ErbB family is relevant to other subgroups of receptor tyrosine kinases and may have physiological implications.


INTRODUCTION

Many growth factors, cytokines, and neurotrophic factors activate receptor tyrosine kinases. The activated receptors become phosphorylated on tyrosine residues, thereby initiating intracellular signaling, leading to cellular responses (1) . One of the subgroups of transmembrane tyrosine kinases includes the receptor for EGF() (ErbB-1), Neu/ErbB-2 (also called HER-2), ErbB-3/HER-3 (2, 3) , and the recently discovered ErbB-4/HER-4 (4) . In contrast with ErbB-1, for which many ligands have been characterized (5) , ligands that directly interact with other ErbB proteins were not known until recently. The observation in different cells of a secreted activity that stimulated phosphorylation of ErbB-2 on tyrosine residues eventually led to the isolation of cDNAs encoding novel EGF-related proteins (6, 7, 8) . The 44-kDa rat factor, named Neu differentiation factor (NDF), stimulated tyrosine phosphorylation of ErbB-2 and induced synthesis of milk components (casein and lipids) in certain breast carcinoma cell lines (7) . The homologous human factors, termed heregulins, were found to be mitogenic for other mammary tumor cells (8) . The NDF cDNA sequence predicted a transmembrane glycoprotein precursor (pro-NDF) containing an EGF-like domain, an immunoglobulin homology unit, and a variable length cytoplasmic domain. At least 10 isoforms of NDF exist and they fall into two groups, and , that differ in their EGF-like domains and in receptor binding affinity (9) . It has been previously suggested that NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions (9, 10) .

Although NDF activates ErbB-2/HER-2 in mammary and neuronal cells, no interaction occurs in ovarian and in fibroblastic cells that overexpress this receptor (11) . These and additional lines of evidence raised the possibility that the interaction between NDF and ErbB-2 involves another molecule that belongs to the family of EGF receptor. Phosphorylation of ErbB-4 by heregulin has been reported (12) . Moreover, by constructing soluble forms of ErbB proteins we found that isoforms of NDF specifically bind to the ErbB-3 and ErbB-4 receptors, but not to a soluble ErbB-2-alkaline phosphatase fusion protein (13) . In agreement with these in vitro studies, when ectopically expressed, the full-length ErbB-3 (13, 14, 15) and ErbB-4 (13) receptors conferred specific binding, as well as kinase activation, by the various isoforms of the ligand.

Unlike ErbB-3 and ErbB-4 that bind multiple isoforms of NDF ErbB-1 binds many different ligands (5) . Binding of EGF induces receptor dimerization and increases the intrinsic tyrosine kinase activity, which leads to receptor autophosphorylation and tyrosine phosphorylation of various cellular substrates (16) . In addition to this activation pathway, EGF receptor serves as a target for several negative regulatory mechanisms. EGF itself is responsible for receptor down-regulation because of an increase in the rate of receptor internalization and degradation. In addition, several growth factors induce a rapid decrease in high affinity EGF binding by two different pathways. The first one involves activation of protein kinase C (PKC) and is observed after treatment with bombesin and phorbol esters such as TPA (17) . The second pathway, induced by EGF, platelet-derived growth factor, fibroblast growth factor, interleukin 1, and tumor necrosis factor , appears to be independent of PKC activation (18) , but it nevertheless involves serine-threonine phosphorylation of the receptor (19) . Another mechanism of modulation of EGF binding involves heterodimerization with the ErbB-2 protein. Subsequent to EGF binding, ErbB-2 undergoes increased tyrosine phosphorylation (20, 21) that is due to complex formation of ErbB-2 with the ligand-occupied ErbB-1 (22, 23) . Heterodimer formation appears to be affected by receptor overexpression, and it leads to the appearance of EGF-receptors with very high affinity (23) .

In the present study we demonstrate a new pathway of trans-regulation of EGF receptors. We have found that NDF can reduce the binding of radiolabeled EGF in certain human tumor cells, but not in other cell lines, even when the cells are maintained at 4 °C. The inhibitory effect appears to be independent of PKC activation, it involves a decrease in affinity, rather than number, of EGF receptors, and it displays reversibility. On the basis of these and additional lines of evidence, we propose a model that attributes the trans-regulatory effect of NDF to heterodimerization of ErbB proteins.


EXPERIMENTAL PROCEDURES

MaterialsEGF (human, recombinant) was purchased from Boehringer Mannheim and recombinant NDF preparations were from Amgen (Thousand Oaks, CA). Iodogen and BSwere from Pierce. All other chemicals were purchased from Sigma unless otherwise indicated.

Cell Culture

T47D, A-431, MCF-7, and SKBR-3 cells were obtained from the American Type Culture Collection (Rockville, MD). MCF-7/ErbB-2, a transfected cell line, was established as described earlier (11) .

Establishment of MCF-7/ErbB-1 Cells

To establish MCF-7 cells that overexpress ErbB-1, the human EGF-receptor cDNA was digested with the restriction enzymes EcoRV and XhoI and inserted into the mammalian expression vector pcDNA3-NEO (Invitrogen, San Diego, CA). MCF-7 breast cancer cells were then transfected with the resulting pcDNA3-NEO/ErbB-1 plasmid by electroporation. Drug-resistant colonies were selected in medium containing 0.7 mg/ml gentamycin, grown as pools, and assayed for EGF receptor expression by I-EGF binding and Western blotting.

Antibodies

Polyclonal antibodies against the C-terminal portion of Neu/HER-2 (NCT) and against the cytoplasmic portion of the EGF receptor (RK2) were generated as described (24, 25) . Monoclonal antibodies against the extracellular part of EGF receptor (528, R1, and 2E9) have been previously described (26, 27, 28) .

Buffered Solutions

Binding buffer contained Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin. HNTG buffer contained 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol. Solubilization buffer contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1.5 mM EGTA, 1.5 mM MgCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, aprotinin (0.15 trypsin inhibitor unit/ml), and 10 µg/ml leupeptin.

Radiolabeling of Ligands

Human recombinant EGF and human recombinant NDF-1were labeled with Iodogen (Pierce) as follows: 5 µg of protein in PBS was mixed in an Iodogen-coated (1 µg of reagent) tube with Na-I (1 mCi). Following 10 min at 23 °C, tyrosine was added to a final concentration of 0.1 mg/ml, and the mixture was separated on a column of Excellulose GF-5 (Pierce). The range of specific activity varied between 2 and 5 10counts/min/ng.

Chemical Cross-linking

Monolayers (10cells) of T47D cells were incubated on ice for 2 h with either I-EGF (20 ng/ml) or I-NDF-1(10 ng/ml). The chemical cross-linking reagent BSwas then added (1 mM), and after 45 min at 22 °C cells were again kept on ice and washed with PBS. Cell lysates were prepared and analyzed by gel electrophoresis.

Lysate Preparation and Immunoprecipitation

For analysis of total cell lysates, gel sample buffer was directly added to cell monolayers. For other experiments, solubilization buffer was added to the monolayer of cells on ice. The proteins in the lysate supernatants were immunoprecipitated with aliquots of the protein A-Sepharose-antibody complex for 1 h at 4 °C. Immunoprecipitates were then washed three times with HNTG (1 ml each wash).

Ligand Binding Analysis

Monolayers of cells (1-2 10cells/well) in 24-well dishes were washed once with binding buffer and then incubated with 5 ng/ml of either I-NDF-1or I-EGF in the same buffer. The unlabeled ligand at different concentrations was coincubated with a radiolabeled ligand for 2 h at 4 °C, and the cells were washed three times with ice-cold binding buffer. Labeled cells were lysed in 0.5 ml of 0.1 N NaOH, 0.1% SDS for 15 min at 37 °C and the radioactivity was determined by using a -counter. Nonspecific binding was determined by the addition of 100-fold excess of the unlabeled ligand. Scatchard analysis was performed using the computerized program LIGAND (29) .

Analysis of Ligand Association

T47D cells in 24-well dishes were first preincubated for 1 h at 4 °C with NDF-1(25 ng/ml) or medium alone, and then the association of radiolabeled EGF was examined at 4 °C by incubating the cells for 0.5-10 min with 5 ng/ml of I-EGF. The cells were rapidly washed thrice with binding buffer, dissolved in 0.1 M NaOH, 0.1% SDS, and the radioactivity was determined. The association data were analyzed by determination of the ratio between the amount of ligand bound at full saturation ( B) and that at time t ( B). Based on published theories of ligand association (30) , the term ln (1- B/ B), which is a measure of the association rate, was calculated and presented as a function of time.

Analysis of Ligand Dissociation

Confluent monolayers of T47D cells in 24-well dishes were incubated for 2 h at 4 °C with 5 ng/ml I-EGF and then washed three times. Dissociation was monitored by using two different protocols. According to the first one, the cells were incubated in binding buffer, with or without NDF-1(25 ng/ml), for various periods of time at 4 °C, and the amounts of released and cell-associated radioactive ligand were determined. Alternatively, after washing unbound radiolabeled EGF, the cells were incubated for 5 min at 4 °C with high concentration of EGF (100 ng/ml), and then dissociation was assayed in the presence or absence of NDF as described above.


RESULTS

and Isoforms of NDF Reduce EGF Binding to Mammary Tumor Cells

In order to investigate the functional consequences of possible cross-talk between ErbB proteins, we used T47D human mammary tumor cells, which express relatively high levels of erbB-4 (4) , and analyzed binding of radiolabeled NDF and EGF. Preincubation of cells with EGF at 37 °C exerted only a limited effect on NDF-1 binding that was measured at 4 °C after removal of EGF. By contrast, the reciprocal experiment indicated that NDF-1inhibited, in a concentration-dependent manner, binding of I-EGF (Fig. 1A). To exclude the possibility that the observed partial inhibitory effect was due to co-internalization of EGF receptors together with NDF receptors, we performed the experiment also at 4 °C. The results presented in Fig. 1 B implied that the effect of NDF on EGF binding was temperature-independent. Similar to NDF-1, which contained only the EGF-like domain, the full-length form of this ligand inhibited EGF binding to T47D cells (Fig. 1 C). More important, an isoform of NDF was also inhibitory, but less effectively than the isoform (Fig. 1 C). Presumably, this difference was due to an 8-10-fold reduced affinity of NDF- in comparison with isoforms (9) .


Figure 1: Binding of EGF and NDF to ligand-treated T47D cells. A, monolayers of T47D cells were preincubated at 37 °C with the indicated concentrations of either unlabeled EGF or unlabeled NDF-1. After 2 h the cells were washed three times with binding buffer and incubated at 4 °C with the radiolabeled form of the other ligand (each at 5 ng/ml). The amount of specifically bound ligand was determined by washing the monolayers 2 h later and subtracting the radioactivity that bound in the presence of 100-fold excess of the respective unlabeled ligand. B, T47D cells were incubated at 4 °C with different concentrations of either NDF-1 or EGF. The incubation was performed for 2 h in the presence of the other ligand in its radiolabeled form, namely I-EGF (5 ng/ml) or I-NDF-1(5 ng/ml). The monolayers were then washed three times with binding buffer and specific ligand binding was determined after subtraction of nonspecific binding that was measured in the presence of 100-fold excess of the corresponding unlabeled ligand. C, varying concentrations of three isoforms of NDF, namely NDF-1( open circles), NDF-1( filled circles), and NDF-1( open squares) were individually incubated with 5 ng/ml I-EGF for 2 h at 4 °C, and the cell-bound radioactivity was determined as described above. Means of triplicate determinations and the corresponding standard errors ( bars) are shown in all panels. The binding analyses were repeated three times.



The Inhibitory Effect of NDF Is Independent of Protein Kinase C

Because activation of PKC by tumor promoting phorbol diesters causes down-regulation of EGF binding (31) , we examined the involvement of PKC in the effect of NDF. Two lines of evidence supported lack of mediation by PKC. First, the inhibitory effect of TPA (approximately a 60% reduction in EGF binding after a 2-h long incubation at 37 °C with 100 ng/ml TPA; data not shown), but not the effect of NDF, was abolished when incubation was performed at 4 °C (Fig. 2 A). Second, depletion of PKC by performing a long incubation with a relatively high concentration of TPA (32) significantly reduced EGF binding, but did not abolish the inhibitory effect of NDF (Fig. 2 B).


Figure 2: EGF binding after treatment with TPA or NDF. A, monolayers of T47D cells were incubated for 2 h at 4 °C with radiolabeled EGF (5 ng/ml) in binding buffer ( CONTROL), or in buffer that contained NDF-1(25 ng/ml) or TPA (100 ng/ml). The cells were then washed, and the specifically bound radioactivity was determined as described under ``Experimental Procedures.'' Each data point represents the average and range ( bars) of a duplicate determination. B, T47D cells were treated with TPA (600 nM) at 37 °C for 20 h in order to down-regulate PKC. The control and TPA-treated cells were then washed extensively and incubated for 1 h at 4 °C in the presence of buffer alone or NDF-1(25 ng/ml). Specific binding of radiolabeled EGF was then determined as described under ``Experimental Procedures.'' Averages and ranges ( bars) of duplicate determinations are shown. The experiments were repeated twice with similar results.



In analogy with NDF, the platelet-derived growth factor also can inhibit EGF binding in PKC-depleted cells (18) . However, the effect of platelet-derived growth factor appears to be mediated by an amplification mechanism because an almost maximal effect was observed with platelet-derived growth factor concentrations as low as those needed for 5% of maximal receptor occupancy (33) . To examine this aspect with NDF as a ligand, we compared the inhibitory effect on EGF binding with the extent of receptor saturation by NDF. Evidently, the concentration dependences of these two parameters displayed parallelism at NDF concentrations below 1 nM (approximately 50% receptor saturation), but they displayed disparity at higher ligand concentrations (Fig. 3).


Figure 3: Concentration dependences of NDF receptor saturation and inhibition of EGF binding. NDF displacement was measured by a 2-h long incubation at 4 °C of T47D cells with radio-labeled NDF-1(5 ng/ml) in the presence of increasing concentrations of unlabeled ligand. Likewise, binding of radiolabeled EGF (5 ng/ml) was performed in the presence of increasing concentrations of NDF-1. This was followed by extensive washing of the cell monolayers and determination of bound radioactivity. The results are expressed as means ± S.E. of triplicate determinations. The experiment was repeated three times with similar results.



Reversibility and Cell Type Specificity of the NDF Effect

The observed partial correlation between receptor occupancy and inhibition of EGF binding (Fig. 3), together with the lack of dependence on temperature (Fig. 1), raised the possibility that the mechanism of inhibition involved reversible interactions at the cell surface. To address this mechanism we attempted to remove surface-bound NDF by washing with a low pH solution (34) and then assaying EGF binding. Fig. 4presents the results of this experiment. Evidently, washing NDF-treated cells under low pH conditions significantly reduced the inhibitory effect.


Figure 4: Acid sensitivity of the inhibitory effect of NDF on EGF binding. T47D cells were incubated with 25 ng/ml NDF-1at 4 °C for the indicated periods of time. The cell monolayers were then washed extensively with binding buffer and incubated at 4 °C for 5 min with 1 ml of either binding buffer ( open squares) or 0.1% acetic acid solution that contained 150 mM NaCl and 0.1% bovine serum albumin ( filled squares). These solutions were removed, and the specific binding of I-EGF was determined at 4 °C as described in the legend to Fig. 3. Averages of triplicate determinations and their standard errors (bars) are shown. The experiment was repeated twice with similar results.



The reversible nature of the effect of NDF implied that different cells may display a variable response due to differences in the number and repertoire of their surface receptors. This paradigm was examined in a series of human tumor cell lines. Unlike T47D and MCF-7 mammary tumor cells, A-431 cells, which overexpress the EGF receptor (ErbB-1), and SKBR-3 cells, which overexpress ErbB-2, displayed limited or no effect of NDF on EGF binding (Fig. 5 A). Similarly, in N87 human gastric cells that overexpress ErbB-2, we observed no inhibitory effect of NDF (data not shown). The presumed dependence on the relative levels of ErbB proteins was examined by establishment of two sub-lines of MCF-7 cells that overexpress either ErbB-2 or ErbB-1 as a result of cDNA transfection. Transfection of these cells with the corresponding expression vector resulted in >10-fold increase in the expression of ErbB-1 and ErbB-2, as revealed by Western blot analysis (Fig. 5, B and C). As predicted, overexpression of either ErbB protein in MCF-7 cells led to significantly smaller NDF inhibitory effect in comparison with the parental cell line (Fig. 5, B and C).


Figure 5: Cell type specificity of the effect of NDF on EGF binding. A, varying concentrations of NDF-1were incubated with I-EGF (5 ng/ml) for 2 h at 4 °C with various human cancer cell lines. The specific binding of labeled EGF was then determined as described under ``Experimental Procedures.'' The following cell types were used for binding analyses: A431 human epidermoid carcinoma cells ( filled circles), and the human breast cancer cell lines SKBR-3 ( open squares), T47D ( open circles), and MCF-7 ( triangles). B and C, the above experiment was performed with MCF-7 cells and the derivative cell lines, denoted MCF-7/ErbB-2 and MCF-7/ErbB-1, that were selected for overexpression of ErbB-2 or ErbB-1, respectively. Averages of triplicate determinations are shown, and the bars represent the standard errors. Insets show the relative levels of ErbB-2 ( B) and ErbB-1 ( C) expression in MCF-7 cells and the derived cell lines, as determined by Western blotting of cell lysates with polyclonal antibodies to ErbB-2 (NCT) or ErbB-1 (RK2). The experiments were repeated twice with similar results.



The Number of Surface-exposed EGF Receptors Undergoes No Change by NDF

Since the inhibitory effect of NDF occurred also at 4 °C (Fig. 1 B) it was conceivable that endocytosis and proteolytic degradation of EGF receptors were not involved. To confirm this proposition we determined the level of surface-exposed EGF receptors by using a binding assay of a monoclonal anti receptor antibody to T47D cells. As control we incubated the cells in the presence of EGF at either 4 or 37 °C and observed extensive receptor down-regulation only at the elevated temperature (). By contrast, the monoclonal antibody that we used detected no change in surface expression of EGF receptors after treatment with NDF.

To visualize surface-exposed EGF receptors we employed an alternative assay that made use of covalent cross-linking of radiolabeled EGF to its membrane receptor (35) . Under normal conditions this assay detected two protein bands upon resolving whole cell lysates by gel electrophoresis (Fig. 6). Presumably, these bands represent monomers and dimers of the ligand-receptor complexes because they disappeared in the presence of high concentrations of unlabeled EGF (Fig. 6), and they were immunoprecipitable by antibodies to human EGF receptor (data not shown). However, the identitification of the upper band as a receptor dimer remains open. Exposure to NDF prior to the cross-linking step reduced labeling of both receptor species, with no detectable change in their electrophoretic mobilities (Fig. 6). Together with the data presented in , our results implied that NDF did not affect the number of EGF receptors, but reduced their binding affinities.


Figure 6: NDF effect on covalent cross-linking of I-EGF to its receptor. Monolayers (10cells) of T47D cells were incubated at 4 °C for 2 h with I-EGF (20 ng/ml) in the presence or absence of NDF-1or 1 µg/ml unlabeled EGF ( Ex EGF), as indicated. Covalent cross-linking was then performed by using the bivalent reagent BS(1 mM), and cell lysates were prepared and subjected to gel electrophoresis. The resulting autoradiogram is shown, and the location of a molecular weight standard protein (myosin) is indicated. The experiment was repeated twice.



NDF Reduces Binding Affinity of EGF Receptors by Accelerating Ligand Dissociation Rate and Decelerating Its Association

In order to further investigate the mechanism by which NDF affected EGF receptors, we performed equilibrium I-EGF binding assays in the presence or absence of NDF. Fig. 7 shows that T47D cells exhibited saturable binding that corresponded to a single population of ligand-binding sites, as reflected in a linear Scatchard curve (36) . Co-incubation with NDF-1 resulted in reduction in EGF binding, due to an increase in the apparent dissociation constant with little effect on the number of EGF receptors. Thus, the calculated number of receptors in the absence of NDF was 5 10molecules/cell, and they displayed a dissociation constant ( K) of 1.8 nM, whereas in the presence of NDF these parameters were 5.2 10and 5.0, respectively.


Figure 7: Scatchard analysis of I-EGF binding to NDF-treated T47D cells. Monolayers of T47D cells were incubated for 2 h at 4 °C with different concentrations of I-EGF in the presence ( filled symbols) or absence ( open symbols) of NDF-1(25 ng/ml). Binding results were analyzed by the Scatchard method and also by plotting saturation curves ( inset). Triplicate determinations of total binding were performed, and their means are given. Nonspecific binding that was determined in the presence of 1 µg/ml of unlabeled ligand was subtracted. Similar results were obtained in three other experiments.



Affinity constants obtained under equilibrium conditions reflect both the rates of ligand association ( k) and dissociation ( k). It was, therefore, necessary to determine if NDF preferentially affected one of these processes. The kinetics of EGF association with T47D cells in the presence or absence of NDF-1 (25 ng/ml) were determined at 4 °C in order to minimize involvement of enzymatic processes. Short incubation periods with radiolabeled EGF, in the presence or absence of NDF, revealed that the rate of EGF binding was decreased in the presence of NDF (Fig. 8 A). Ligand dissociation analyses were also performed at 4 °C. The cells were first saturated with the radiolabeled ligand, then unbound EGF was removed and a short incubation step with unlabeled EGF was performed in order to prevent ligand reassociation. Ligand release was then followed in the presence or absence of NDF. The dissociation data obtained were analyzed by plotting the natural logarithm of fractional receptor occupancy, B/ B, as a function of time, where Bis the amount of ligand bound at time t and Bis the amount of ligand bound before starting dissociation. The negative value of the slope of such plots should indicate the dissociation constant. This experiment revealed that NDF slightly accelerated the rate of EGF release (Fig. 8 B). However, in both NDF-treated and -untreated cells the plots displayed biphasic dissociation with an initial rapid release followed by a major component with slower dissociation rate (Fig. 8 B). Apparently, NDF affected only the major component by increasing the off rate ( k= 0.22 10sin the absence of NDF and 0.31 10sin its presence). Taken together, NDF appeared to reduce EGF binding affinity through a dual effect on both on and off rates. This possibility was examined by using a modified protocol of the dissociation experiment, in which we omitted the incubation step with unlabeled EGF, in order to allow reassociation of the radiolabeled ligand. The results of this experiment are presented in Fig. 8 C. Comparison with the plots shown in Fig. 8B revealed that NDF induced a remarkable effect on the combined kinetics of EGF dissociation and reassociation.


Figure 8: Kinetics of EGF binding in the presence of NDF. Association ( A) and dissociation ( B and C) kinetics were analyzed on confluent monolayers of T47D cells in 24-well dishes. A, the cells were preincubated with binding buffer ( closed circles) or NDF-1(25 ng/ml) ( open circles) at 4 °C for 1 h, and the incubation was continued for the indicated periods of time at 4 °C after adding I-EGF (5 ng/ml). The amount of EGF that was specifically bound at each time point ( B) was determined after a brief washing step. Maximal EGF binding ( B) was separately determined by incubating the cells for 2 h with I-EGF at 4 °C. Each point represents the mean of a triplicate determination ± S.E. B, the cells were first incubated with 5 ng/ml I-EGF for 2 h at 4 °C and then washed three times. Ligand reassociation was prevented by incubating the cells for 5 min with unlabeled EGF (100 ng/ml), and this was followed by ligand dissociation in the presence ( open circles) or absence ( closed circles) of NDF-1(25 ng/ml). The radioactivity released into the medium, as well as cell-associated I-EGF, was determined and analyzed by plotting the natural logarithm of B/ B versus time. ( B is the concentration of ligand bound at time t and Bis the concentration of ligand bound at the starting time of dissociation). Averages of triplicate determinations and their standard errors ( bars) are shown. C, the experiment was performed as in B, except that no unlabeled EGF was added to the dissociation medium. Each of the three experiments was repeated twice.



EGF and NDF Receptors Form Complexes in Living Cells

The characteristics of NDF-induced inhibition of EGF binding raised the possibility that the inhibitory effect was mediated by direct interactions between EGF and NDF receptors. In an attempt to demonstrate the existence of such complexes, we labeled NDF receptors by covalent cross-linking of the radioactive ligand and tried to immunoprecipitate the presumed complex with EGF receptor by using various antibodies to the latter protein. When whole lysates of T47D cells were electrophoresed after cross-linking of I-NDF two labeled protein bands, that presumably correspond to monomers (major species) and dimers of receptors, were observed in polyacrylamide gels, and they both disappeared in the presence of high concentration of unlabeled ligand (Fig. 9, upper left panel). Immunoprecipitation of EGF receptors from such lysates recovered both monomers and dimers of NDF receptors (Fig. 9). Control immunoprecipitation analyses that were performed with either a preimmune antiserum (PI), an unrelated monoclonal antibody (G63), or a polyclonal antibody to Kit/stem cell factor receptor (Ab 212) did not recover the labeled proteins. In addition, similar experiments, that were performed with radiolabeled stem cell factor, showed that co-immunoprecipitation by the RK2 antibody was specific to surface-linked I-NDF. Replacement of the EGF receptor-specific antibodies with a monoclonal antibody to human ErbB-2, namely antibody N24 (37) , also resulted in co-immunoprecipitation signals of both monomers and dimers, which were abolished in the presence of unlabeled NDF (Fig. 9, lower panel). One possible interpretation of these results is that ligand-occupied NDF receptors form heterodimers with both EGF receptors and ErbB-2, in analogy to EGF-induced heterodimers between ErbB-1 and ErbB-2 (22, 23) . According to this explanation, antibodies that are directed to either ErbB-1 or ErbB-2 preferentially co-precipitate the heterodimeric form of NDF receptors and, therefore, this molecular species is more abundant in immunoprecipitates than it is in whole cell lysates (compare the ratio between monomers and dimers in the upper panels of Fig. 9). When combined with other biochemical characteristics of the inhibitory effect of NDF, the existence of molecular complexes between NDF and EGF receptors may provide a structural basis for the observed receptor cross-talk.


Figure 9: Co-immunoprecipitation of surface-linked NDF with EGF receptors. Radiolabeled NDF-1(10 ng/ml) or human stem cell factor (SCF, 20 ng/ml) were incubated for 2 h at 4 °C with 10T47D cells. After a brief wash, BSwas added to a final concentration of 1 mM in PBS, and incubation was continued at 22 °C for 45 min. Cells were lysed at 4 °C, and the lysates were either electrophoresed directly ( upper left panel) or first subjected to immunoprecipitation with the indicated antibodies, and then the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis. Note that the gels shown in the upper two panels were run under slightly different conditions, so that they differ in separation of the two labeled bands. The following antibodies were used: anti-EGF receptor antibodies: RK2, a rabbit antiserum directed to a synthetic peptide derived from the cytoplasmic portion of the receptor, and monoclonal antibodies 528 and 2E9 that recognize the extracellular domain, the N24 anti-ErbB-2 monoclonal antibody, and three control antibodies: PI, a preimmune rabbit antiserum, Ab212, a rabbit antibody to the stem cell factor receptor, and G63, a monoclonal antibody to a rat mast cell antigen. Immunocomplexes were separated on SDS-polyacrylamide gel electrophoresis (7.5% acrylamide), dried, and exposed to an x-ray film for 72 h at 70 °C. The resulting autoradiogram is shown, and the location of the uppermost standard marker protein is indicated. The lower right panel shows a control immunoprecipitation that was performed after cells were incubated with both I-NDF and 1 µg/ml unlabeled NDF (labeled Ex cold). The experiments were repeated three times with similar results.




Discussion

Most mammalian receptor tyrosine kinases belong to subgroups of highly related transmembrane proteins. This multiplicity is not found in insects, suggesting that it has been developed in order to provide solutions for more complex physiological requirements in mammals (38) . The ErbB subgroup of receptors is one of the best studied families, not only because it is widely expressed in mesenchymal, neuronal, and epithelial cells, but also because members of this family were implicated in the development of human cancer (39) . As expected, the biological activities of ErbB proteins display variation; whereas ErbB-1 delivers mitogenic signals, that are strictly ligand-dependent, ErbB-2 delivers growth signals even in the absence of a ligand (40) , and NDF, which binds to ErbB-3 and ErbB-4, can promote cellular differentiation (12) . Despite this functional heterogeneity, several lines of evidence indicate that ErbB proteins retained an ability to cross-talk and thereby trans-modulate incoming signals (41) . In the present study we identified a new trans-modulatory effect between NDF and EGF receptors and discuss the potential biological significance of this molecular interaction.

Before dealing with the mechanism by which NDF inhibits EGF binding, it is relevant to list the biochemical characteristics of this process.

1) Inhibition is partial and never exceeds 90% of maximal EGF binding (Fig. 1).

2) The effect of NDF is cell type specific and appears to be affected by the relative expression levels of ErbB proteins (Fig. 5).

3) The extent of inhibition is proportional to saturation of NDF receptors and correlates with ligand affinity of different isoforms (Figs. 3 and 1 C).

4) The inhibitory effect is independent of temperature (Figs. 1, A and B) and requires no preincubation (data not shown).

5) Simultaneous occupation of NDF receptors is essential for display of reduced EGF receptor binding (Fig. 4).

6) Decreased EGF binding is due to reduction in affinity (Fig. 7), rather than number () of EGF receptors.

Because direct interaction between NDF and EGF receptors was undetectable (6, 8) the inhibitory effect of NDF cannot be caused by direct competition on the ligand-binding site of EGF receptor. On the basis of specific recovery of radiolabeled NDF in immunoprecipitates of EGF receptors (Fig. 9) and the existence of heterodimeric complexes between EGF receptors and either ErbB-2 (22, 23) or NDF receptors (13) , we favor the following model of trans-regulation of ErbB-1 by NDF receptors. Depending on the relative levels of expression of different ErbB proteins, NDF binding to its own receptors, namely ErbB-3 and ErbB-4, induces their homodimerization, as well as heterodimerization with ErbB-1 and ErbB-2. In the case of ErbB-1, EGF binding to the predimerized receptor, which already exists in a complex with a ligand-occupied NDF receptor, is partially inhibited. Apparently, this is due to acceleration of the rate of EGF dissociation, while the on rate of the heterodimerized receptor for the second ligand binding event is reduced in comparison with that of monomeric or homodimeric receptor species.

Although a direct proof to this model cannot be obtained with the currently available methods, it can explain all of the above described characteristics, and it is consistent with observations that were made in related experimental systems. For example, the proposed mechanism attributes lack of effect of NDF on EGF binding in certain cell types to the quantitative relationships between homo- and heterodimers of ErbB-1. Although alternative explanations to the cell type specificity, such as different expression levels of ErbB-3 and ErbB-4, cannot be excluded, it is worth noting that receptor density was identified as a major determinant of dimer formation in a theoretical model (42) , and in experimental systems involving homodimers of ErbB-2 (43) or heterodimers of ErbB-1 (44) . Similarly, the model we suggest can explain two characteristics of the inhibitory phenomenon, sensitivity to low pH (Fig. 4) and lack of temperature dependence (Fig. 1); both are landmarks of receptor dimerization (45, 46) ,

The major feature of the proposed model refers to the kinetics of binding of a second ligand to predimerized receptors. We speculate that conformational changes that take place within heterodimers of ErbB-1 result in partial blocking of EGF receptors. It is interesting that according to a recent study, EGF binding to a receptor in a dimer having one receptor already bound occurs with lower affinity than the initial binding event (47) . Moreover, the lower affinity of binding to the second binding site was proposed to be the reason for negatively curved Scatchard plots that are often, but not always, observed in the case of EGF. Independent of the exact molecular mechanism of inhibition of EGF binding by NDF, this effect and especially its possible sensitivity to the expression profile of ErbB proteins may have important physiological implications. Potentially, this mechanism may act as a filter of incoming signals; by blocking binding of a second growth factor, a cell may avoid excessive, or even opposing, growth regulatory signals. Furthermore, if indeed this process is mediated by heterodimer formation, then it may shed new light on the frequent overexpression of ErbB proteins in certain human adenocarcinomas (39, 48) . Overexpression of a particular ErbB receptor is expected to force heterodimerization of the overexpressed receptor with other ErbB molecules, and thereby favor binding of one ligand over others. This model may be relevant to the growth advantage of ErbB-2 overexpressing clones of human adenocarcinomas (48) , but it leaves many open questions. For example, it is unclear whether heterodimer formation is a random, rather than a hierarchial process, that prefers certain ErbB heterodimers over others. Another open question relates to the relationships between homodimers and heterodimers and their relative ligand binding affinities. Lastly, it will be interesting to examine the relevance of our observations with ErbB proteins to other sub-groups of receptors, such as the and types of the platelet-derived growth factor receptor or the different Trk receptors for neurotrophic factors, that may be functionally interlinked by trans-modulation of heterologous ligand binding.

  
Table: Changes in the level of EGF receptor following treatment with either NDF or EGF

Confluent monolayers of T47D cells in 24-well dishes were incubated with 25 ng/ml of the indicated ligands. Following 2 h at 4 or at 37 °C, the monolayers were washed and further incubated for 90 min at 4 °C with 5 µg/ml of a monoclonal antibody to EGF receptor (R1) and subsequently washed. The amount of bound antibody, reflecting the level of surface-exposed EGF receptors, was determined by incubating the cells at 4 °C for 90 min with radiolabeled rabbit antibodies to mouse immunoglobulin G, and determination of the bound radioactivity. Control monolayers were incubated with a first antibody to Kit/stem cell factor receptor and their background binding was subtracted. Averages of triplicate determinations and their standard errors are shown. The experiment was repeated three times with the same results.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA51712 and The Wolfson Foundation administered by The Israel Academy of Sciences and Humanities. 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.

§
Recipient of a Career Research Development Award from The Israel Cancer Research Fund. To whom correspondence should be addressed. Tel.: 972-8-343974; Fax: 972-8-344141.

The abbreviations used are: EGF, epidermal growth factor; BS, bis(sulfosuccinimidyl) suberate; NDF, Neu differentiation factor; PBS, phosphate-buffered saline; PKC, protein kinase C; TPA, 12- O-tetradecanoyl phorbol 13-acetate.


ACKNOWLEDGEMENTS

We thank L. Defize, J. Mendelsohn, M. Waterfield, M. Guthman, and J. Blechman for monoclonal antibodies and E. Peles, S. Lavi, Daniel Schindler, and N. Ben-Baruch for help and advice.


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