Structural Determinants of the Interaction between the erbB2 Receptor and the Src Homology 2 Domain of Grb7*

(Received for publication, October 30, 1996, and in revised form, January 16, 1997)

Peter W. Janes Dagger , Martin Lackmann §, W. Bret Church , Georgina M. Sanderson Dagger , Robert L. Sutherland Dagger and Roger J. Daly Dagger par

From the Dagger  Cancer Research Program and the  Neurobiology Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia and the § Growth Regulation Laboratory, Ludwig Institute for Cancer Research, P. O. Royal Melbourne Hospital, Victoria 3050, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The Src homology 2 (SH2) domain-containing protein Grb7 and the erbB2 receptor tyrosine kinase are overexpressed in a subset of human breast cancers. They also co-immunoprecipitate from cell lysates and associate directly in vitro. Whereas the Grb7 SH2 domain binds strongly to erbB2, the SH2 domain of Grb14, a protein closely related to Grb7, does not. We have investigated the preferred binding site of Grb7 within the erbB2 intracellular domain and the SH2 domain residues that determine the high affinity of Grb7 compared with Grb14 for this site. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 of erbB2 is the major binding site for the Grb7 SH2 domain, indicating an overlap in binding specificity between the Grb7 and Grb2 SH2 domains. Substituting individual amino acids in the Grb14 SH2 domain with the corresponding residues from Grb7 demonstrated that a Gln to Leu change at the beta D6 position imparted high affinity erbB2 interaction, paralleled by a marked increase in affinity for the Tyr-1139 phosphopeptide. The reverse switch at the beta D6 position abrogated Grb7 binding to erbB2. This residue therefore represents an important determinant of SH2 domain specificity within the Grb7 family.


INTRODUCTION

Src homology 2 (SH2)1 domains are conserved noncatalytic regions of approximately 100 amino acids found in a variety of cytoplasmic signaling proteins. They bind to specific phosphotyrosine-containing sequences within autophosphorylated RTKs and intracellular phosphoproteins and, along with SH3 and pleckstrin homology domains, mediate inter- and intramolecular interactions involved in signal transduction from activated RTKs (1, 2).

Grb7 is an SH2 domain-containing signaling protein encoded by a gene commonly co-amplified with the ERBB2 gene in human breast cancer cell lines and primary breast cancers (3). Overexpression of the erbB2 receptor tyrosine kinase occurs in approximately 20% of breast cancers, where increased expression correlates with poor patient prognosis (4-6). Grb7 also associates strongly with erbB2 via its SH2 domain in co-immunoprecipitation experiments (3), although the Grb7 binding site on erbB2 has not been determined. The simultaneous overexpression of Grb7 with erbB2 in breast cancer cells therefore suggests greatly amplified signaling through these proteins. Although the precise function of Grb7 is not known, it probably serves as an "adapter" protein, linking tyrosine phosphorylated proteins to downstream effectors, since it lacks a known catalytic region but possesses multiple domains capable of mediating intermolecular interactions.

Grb7 is a member of an emerging family of signaling proteins that also includes Grb10 (7) and Grb14 (8). Another related protein, Grb-IR, is highly conserved with mouse Grb10 and may represent an alternatively spliced version of the human homologue (9). The family members share a common overall structure consisting of an N-terminal region harboring a conserved proline-rich motif (8), a central region exhibiting homology to the Caenorhabditis elegans protein Mig10 that also contains a pleckstrin homology domain, and a C-terminal SH2 domain. The SH2 domains of Grb7 and Grb14 share 67% amino acid identity, yet despite this high conservation, fusion proteins containing these SH2 domains display differences in binding preference for RTKs in whole cell extracts. In particular, the Grb7 SH2 domain interacts strongly with erbB2 in vitro, whereas the affinity of the Grb14 SH2 domain for erbB2 is very weak (8). This therefore represents a novel system in which to study the determinants of SH2 specificity that enable two highly conserved SH2 domains to retain distinct RTK binding preferences.

The specificity of high affinity SH2 binding is conferred both by amino acids in the SH2 domain and by residues flanking the phosphotyrosine in the target sequence. In the target sequence, the amino acids immediately C terminal to the phosphotyrosine are most important in controlling specificity, especially the first three to six residues, as shown by structural studies (10, 11) and experiments using phosphopeptide libraries randomized at +1 to +3 (12, 13). Within the SH2 domain, selectivity is determined by variation of specific residues that interact with these positions of the phosphopeptide, whereas the overall structure is well conserved. All SH2 domain structures solved to date consist of a large central beta  sheet with an associated smaller beta  sheet flanked by two alpha  helices (10, 11, 14-16). One residue important for specificity is beta D5, which interacts with both the +1 and +3 positions (10). Group I SH2 domains are distinguished from other types by possessing a residue with a bulky hydrophobic side chain at the beta D5 position (Tyr or Phe) that acts as a divider between two pockets that bind the phosphotyrosine and +3 positions. In Group III SH2 domains this is absent, creating a groove between the binding pockets that can extend out beyond +3 to the +6 position (11, 15). Introduction of a tyrosine at beta D5 into a Group III SH2 domain is sufficient to shift its phosphopeptide specificity to resemble that of a Group I SH2 domain (17).

In this study we investigated the preferred binding site of the Grb7 SH2 domain in erbB2 and identified amino acids in the SH2 domain important for determining the high affinity binding of Grb7 to this site compared with Grb14. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 in erbB2 is specifically bound by the Grb7 SH2 domain. Interestingly, this also corresponds to the major Grb2 binding site (18). Six SH2 domain residues implicated in determination of SH2 domain binding specificity were replaced in Grb14 with the corresponding amino acid from Grb7. Two substitutions increased binding of the Grb14 SH2 domain to erbB2, and the reciprocal change at one of these positions (beta D6) abrogated Grb7 binding to erbB2. The beta D6 amino acid therefore plays an important role in determining the affinities of the Grb7 and Grb14 SH2 domains for Tyr-1139 of erbB2, and possible mechanisms for its action are discussed.


MATERIALS AND METHODS

Cell Culture and Lysis

HER14, HER1-2, and HEK 293 cells were maintained and subjected to growth factor stimulation as described previously (8, 19, 20). SK-BR-3 human breast cancer cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained according to Janes et al. (21). Lysates were prepared as described previously (21) and normalized for protein content using a Bradford-based protein assay (Bio-Rad).

Transfection Procedures

HEK 293 cells were plated at a density of 1.6 × 106/dish on 10-cm-diameter tissue-culture dishes. Prior to transfection the medium was changed to Dulbecco's modified Eagle's medium (CSL Biosciences, Parkville, Victoria, Australia) containing 5% fetal calf serum (CSL Biosciences). The cells were then transfected for 6 h using the calcium phosphate co-precipitation method (22), treated with 15% glycerol in 5% fetal calf serum/Dulbecco's modified Eagle's medium for 1 min at 37 °C, and then returned to maintenance medium for 24 h. The precipitates contained 5 µg of expression vectors encoding HER1-2 (23) or HER1-2 Y1139F, made up to 20 µg with vector DNA, whereas control cells received 20 µg of vector DNA alone. Prior to EGF treatment the cells were starved overnight in medium containing 0.5% fetal calf serum. Receptor expression and stimulation was confirmed by Western blotting cell lysates with anti-erbB2 (Novocastra, Newcastle, United Kingdom) and anti-phosphotyrosine (PY20, Transduction Laboratories, Lexington, KY) antibodies. Visualization of bound antibodies was by ECL (Amersham Corp.).

Generation of GST Fusion Proteins

A DNA fragment corresponding to the SH2 domain of Grb14 (amino acids 426-540) was amplified from GRB14 cDNA (8) using flanking primers containing BamHI (forward primer) and EcoRI (reverse primer) restriction sites to enable subcloning into the pGEX2T expression vector (Amrad-Pharmacia Biotech Inc., Melbourne, Victoria, Australia). DNA encoding the human Grb7 SH2 domain (amino acids 415-532) and the mouse Grb7 SH2 domain (amino acids 418-535) was amplified by reverse transcription-polymerase chain reaction from SK-BR-3 human breast cancer cell RNA and mouse liver RNA, respectively, using primers containing appropriate restriction sites and cloned into pGEX2T. The mouse and human Grb7 SH2 domains were used interchangeably with identical results. Recombinant pGEX plasmids were transformed into Escherichia coli DH5alpha cells and verified by DNA sequencing. Fusion proteins were expressed and purified from isopropyl-beta -D-thiogalactopyranoside-induced bacterial cultures as described previously (24), and purity was confirmed by SDS-PAGE and silver staining.

Fusion Protein Binding Experiments

Binding experiments were typically performed by mixing 2.5-5 µg of GST-SH2 fusion protein bound to glutathione-Sepharose beads (Sigma) with 150-300 µl of lysate (approximately 5 mg/ml total protein) for 2 h at 4 °C. The beads were then washed three times with cell-lysis buffer (21) and boiled for 3 min in SDS-PAGE sample buffer. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and Western blotted with an anti-erbB2 monoclonal antibody. In experiments using different GST fusion proteins, equal loading was verified by SDS-PAGE followed by Coomassie Blue staining.

Peptide Competition Assays

Synthetic phosphopeptides were synthesized by Chiron Mimotopes (Clayton, Victoria, Australia). These were purified by reverse phase high pressure liquid chromatography to >= 95% and their identity confirmed by ion spray mass spectrometry. Peptide competition of GST fusion protein binding to the erbB2 intracellular domain was performed as follows. 5 µg of fusion protein on glutathione-Sepharose beads was preincubated for 30 min in 300 µl of lysis buffer with each individual peptide or with no peptide (control), and this was then added to 300 µl of HER1-2 cell lysate and mixed for 2 h at 4 °C (final peptide concentration of 50 µM). The beads were then washed, and bound proteins were analyzed as described for the fusion protein binding assays. Densitometric analysis of autoradiographs was performed using the IP Lab Gel analysis program (Signal Analytics Corp., Vienna, VA).

Site-directed Mutagenesis

Site-directed mutagenesis of recombinant pGEX plasmids encoding the Grb14 and Grb7 SH2 domains and the HER1-2 expression plasmid was performed using a method that incorporates both the mutagenic primer and a selection primer, allowing selection of mutated plasmids due to alteration of a restriction site in the vector sequence (Transformer System, Clontech, Palo Alto, CA). Sequences of mutagenic and selection primers will be made available on request. All mutated SH2 domains were fully sequenced to verify correct incorporation.

BIAcore Affinity Measurements

The general operation principles of the BIAcore biosensor (Pharmacia) have been described previously (25). Parallel channels of CM-5 sensor chips were derivatized (45 µl at 2 µl/min) with phosphorylated or nonphosphorylated peptide solutions (2 mg/ml in 50 mM HEPES, pH 7.5, 150 mM NaCl) to yield a 250-350 resonance units response increase. The integrity and accessibility of the phosphopeptide was confirmed routinely by monitoring the response to an anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). Interaction of soluble GST fusion proteins (0.01-10 µM) with immobilized peptide was measured at 5 µl/min in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 0.005% Tween 20 with kinetic constants derived from raw data of the BIAcore sensograms using kinetic models included in the BIAevaluation software (version 2.1, 1995, Pharmacia). Equilibrium constants were derived from steady state binding responses by Scatchard analysis, according to Req/C = KaRmax - KaReq (where Req is the equilibrium response, C is the analyte concentration, Rmax is the saturation response, and Ka is the association constant), as outlined in the BIAcore user manual.

In addition, the interactions of the fusion proteins with Tyr-1139 phosphopeptide were studied in solution by reacting constant fusion protein concentrations with increasing concentrations of soluble peptide (0.016-20 µM). Free analyte concentrations, estimated from the BIAcore responses of known samples (BIAevaluation software, version 2.1) were used to calculate the concentration of SH2 domain-bound receptor peptide and yielded the equilibrium constant Kd by Scatchard analysis as described previously (25).

Molecular Modeling

Structures of the Grb7 and Grb14 SH2 domains were modeled on the Src SH2 domain structure by substituting sequences of the SH2 domain and the complexed peptide. We assumed that the overall fold was strictly conserved in terms of secondary structure elements and did not attempt to move the backbone in these regions but rather considered compatible rearrangements of side chains (according to Homology Users Guide version 95.0, Molecular Simulations Inc., San Diego, CA). Sequence alignment of Grb7 and Grb14 with Src showed differences in the lengths of the CD and EF loops, which in the case of the EF loop led to clashes with the peptide, so it was assumed that structural homology was conserved in this area and that the insertion in Grb7 and Grb14 is later in the EF loop than is shown in the alignment. Side chains identical between Src and Grb7 or Grb14 were copied from the Src structure, whereas all nonidentical side chains were adjusted using a side chain rotamer library (26), an automated procedure that attempts to minimize the energy of groups of side chains by selecting rotamers in a systematic fashion. Figures were generated using the programs MOLSCRIPT (27) and Insight II (Molecular Simulations Inc.).


RESULTS

Phosphorylated Tyr-1139 of the erbB2 Receptor Represents the Major Binding Site for the Grb7 SH2 Domain

The specific site (or sites) within the erbB2 receptor recognized by Grb7 has not been clearly determined. The Grb7 binding site in Shc is likely to be the YVNV motif at Tyr-317, since Grb2, which recognizes this motif, can compete with Grb7 for binding to Shc (3). A Grb2 binding site comprising a similar motif (YVNQ) is also present at Tyr-1139 of erbB2 (18) and is therefore a likely candidate as a Grb7 binding site. However, Stein et al. (3) found that Grb2 was unable to compete for binding of Grb7 to erbB2, suggesting that either Grb7 binds to this site in erbB2 at a much higher affinity than Grb2 or that Grb7 recognizes a different site.

Five in vivo tyrosine autophosphorylation sites have been identified in the C terminus of erbB2, at positions 1139, 1196, 1221/1222, and 1248 (28, 29), and an additional site of in vitro phosphorylation maps to Tyr-1023 (29) (Fig. 1A). We therefore used synthetic phosphopeptides spanning these tyrosine residues to compete for binding of a GST-Grb7 SH2 fusion protein to the autophosphorylated erbB2 intracellular domain in cell lysates. Lysates of HER1-2 cells were used that express a chimera (HER1-2) of the extracellular domain of the EGF receptor fused to the intracellular domain of erbB2, enabling study of erbB2 interactions in a ligand (EGF)-inducible manner (20). Lysates from EGF-treated cells were incubated with a Grb7 SH2 fusion protein coupled to Sepharose beads, and associated receptor was detected by SDS-PAGE and Western blotting. When the Grb7 SH2 fusion protein was preincubated with each peptide individually, only the Tyr-1139 peptide inhibited binding (Fig. 1B). This strongly suggests that the preferred binding site of the Grb7 SH2 domain is the YVNQ motif at Tyr-1139 of erbB2.


Fig. 1. Mapping of the Grb7 binding site on erbB2 by phosphopeptide competition analysis. A, schematic representation of the erbB2 receptor showing identified autophosphorylation sites and synthetic phosphopeptides used to compete fusion protein binding to these sites. B, phosphopeptide competition analysis of GST-Grb7 SH2 fusion protein binding to the HER1-2 chimeric receptor. GST-Grb7 SH2 fusion protein immobilized on Sepharose beads was incubated with lysis buffer alone (control) or containing individual synthetic phosphopeptides (100 µM) corresponding to erbB2 autophosphorylation sites. Following dilution (1:1) with lysates from EGF-stimulated HER1-2 cells and further incubation, the beads were washed and bound receptor was detected by Western blot analysis.
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To verify this result, the Tyr-1139 residue of the HER1-2 chimera was mutated to Phe to create HER1-2 Y1139F, and the receptor was then tested for its association with the Grb7 SH2 domain. Plasmid constructs encoding wild type HER1-2 and HER1-2 Y1139F were transiently transfected into HEK 293 cells and lysates prepared from control and EGF-stimulated cells. Western blotting of these lysates with either anti-erbB2 or anti-phosphotyrosine antibodies confirmed equivalent expression and tyrosine phosphorylation of the wild type and mutated receptors (Fig. 2A). Incubation of the lysates with GST-Grb7 SH2 fusion protein immobilized on Sepharose beads followed by detection of bound receptor with anti-erbB2 antibodies revealed that introduction of the Y1139F mutation reduced binding by approximately 95% compared with the wild type receptor (Fig. 2B), confirming that Tyr-1139 is the major Grb7 binding site in erbB2.


Fig. 2. Confirmation of Tyr-1139 as the major Grb7 binding site on erbB2 by site-directed mutagenesis. A, expression and activation of the HER1-2 and HER1-2 Y1139F receptors in HEK 293 cells. Following transient transfection with vector alone (lanes 1 and 2) or expression constructs encoding HER1-2 (lanes 3 and 4) or HER1-2 Y1139F (lanes 5 and 6), the cells were serum starved and then either left untreated or stimulated with EGF. Normalized cell lysates were then subjected to Western blot analysis with the indicated antibodies. alpha -PTyr, anti-phosphotyrosine. B, binding of a GST-Grb7 SH2 fusion protein to HER1-2 and HER1-2 Y1139F receptors. The cell lysates analyzed in A were incubated with either GST alone (upper panel) or a GST-Grb7 SH2 fusion protein (lower panel) immobilized on Sepharose beads. Following washing, bound receptor was detected by Western blotting with an anti-erbB2 antibody.
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Substitution of Specific Grb14 SH2 Domain Residues with the Corresponding Amino Acids from Grb7 Increases erbB2 Association

Having demonstrated binding of Grb7 to Tyr-1139 of erbB2, we attempted to identify SH2 domain residues determining the high affinity interaction of Grb7, but not Grb14, with this site. Fig. 3 shows an alignment of the SH2 domains from Grb7, Grb14, and Src in the context of the secondary structure of the Src SH2 domain as deduced by Waksman et al. (10). Secondary structure elements are labeled using the notation of Eck et al. (30). All three SH2 domains fall into Group I since they contain Phe or Tyr at beta D5. The six amino acids that were replaced in the Grb14 SH2 fusion protein by the corresponding amino acid in Grb7 are indicated. It should be noted that the EF loop in Grb7 and Grb14 is extended compared with that in Src, and the EF1 position refers to that in the latter SH2 domain. The choice of the targeted residues was based on 1) being nonidentical between Grb14 and Grb7, and 2) their likely interaction with the +1 to +3 positions of target sequences, as suggested by crystal structures of other Group I SH2 domains complexed with a high affinity peptide (10, 30). Thus, beta D5 interacts with +1, beta D'1 interacts with +2, and beta D5, beta E4, and EF1 interact with +3. beta D6 forms part of the phosphotyrosine binding site and also contributes to a hydrogen-bonded network involving water molecules that interact with the +1 and +2 positions. alpha B9 forms part of the +3 binding pocket.


Fig. 3. Amino acid sequence alignments of the SH2 domains of Grb14, Grb7, and Src based on Margolis et al. (38). Secondary structure elements according to Waksman et al. (10) are also shown. Asterisks denote residues in the Grb14 SH2 domain subjected to site-directed mutagenesis. Shading indicates residues that are identical in two or more of the SH2 domains. The positions of the residues in the amino acid sequence are indicated.
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Amino acid changes were incorporated into the GST-Grb14 SH2 fusion protein by site-directed mutagenesis, and the resulting fusion proteins were coupled to Sepharose beads and tested for their ability to bind the intracellular domain of erbB2 in control or EGF-treated HER1-2 cell lysates. Association of receptor with the beads was detected by Western blotting. As described previously (8), binding of the wild type Grb14 SH2 domain was not detected. However, two mutations increased Grb14 SH2 domain affinity for the EGF receptor/erbB2 chimera (Fig. 4A). A Gln to Leu change at beta D6 caused a marked increase in binding, whereas a Phe to Tyr change at beta D5 resulted in a more modest increase. To evaluate the combined effect of the beta D5 and beta D6 substitutions, both mutations were introduced into the Grb14 SH2 fusion protein. This resulted in a further increase in affinity above that achieved by the single mutations (Fig. 4B).


Fig. 4. Effect of substitution of specific Grb14 residues with the corresponding Grb7 amino acids on binding to HER1-2. A, binding of wild type Grb14 and Grb7 SH2 domains and mutant Grb14 SH2 domains incorporating Grb7 residues to the HER1-2 receptor. Lysates of control (-) or EGF-stimulated (+) HER1-2 cells were incubated with Sepharose beads bound to the indicated GST fusion proteins or GST alone. Bound receptor was detected as in previous figures. B, binding of Grb14 SH2 domains mutated at beta D5 and beta D6, separately and in combination, to the HER1-2 receptor. The experimental protocol was as described in A.
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Introduction of Specific Grb14 Residues into Grb7 Abrogates Grb7 SH2 Domain Binding to erbB2

To further investigate the significance of the beta D5 and beta D6 residues in controlling Grb7 SH2 domain specificity toward erbB2, the reverse experiment was performed by introducing corresponding Grb14 residues, either separately or in combination, into the Grb7 SH2 fusion protein. The resulting mutant Grb7 SH2 domains were then analyzed for their ability to bind the erbB2 intracellular domain in HER1-2 cell extracts. The Leu to Gln change at beta D6 consistently abrogated binding, whether introduced on its own or simultaneously with the beta D5 change (Fig. 5). However, binding of the mutant Grb7 SH2 domain incorporating the beta D5 change alone was not reduced compared with the wild type Grb7 SH2. Similarly, switching the alpha B9 residues did not affect binding of the Grb7 SH2 domain (Fig. 5). These results highlight the importance of beta D6 in Grb7 binding to erbB2.


Fig. 5. Effect of substitution of specific Grb7 residues with the corresponding Grb14 amino acids on binding to the HER1-2 receptor. Lysates of EGF-stimulated HER1-2 cells were incubated with Sepharose beads bound to the indicated fusion proteins or GST alone, and bound receptor was detected as in previous figures.
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Grb14 SH2 Mutants Incorporating beta D6 from Grb7 Recognize the Same Site in erbB2 as the Grb7 SH2 Domain

To confirm that the interaction of the beta D6 and beta D5/beta D6 mutant Grb14 SH2 domains with erbB2 was mediated via the same binding site as Grb7, phosphopeptide competition analysis was performed as described previously. Only the phosphopeptide corresponding to Tyr-1139 of erbB2 significantly inhibited binding to the HER1-2 chimera, reducing the interaction of the beta D6 mutant to 11.5% and that of the beta D5/beta D6 mutant to 9% of control values (Fig. 6). These mutants therefore display Grb7-like SH2 domain specificity in that they bind Tyr-1139 of erbB2.


Fig. 6. Phosphopeptide competition analysis of binding of mutant Grb14 SH2 domains to the HER1-2 receptor. Competition of binding of Grb14 SH2 domains mutated at the beta D6 position (upper panel) and the beta D5 and beta D6 positions (lower panel) to the HER1-2 receptor was performed as described in Fig. 1B.
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Determination of the Relative Affinities of Wild Type Grb7 and Grb14 and Mutant Grb14 SH2 Domains for Tyr-1139 of erbB2

The interaction of these SH2 domains with the erbB2 Tyr-1139 site was further investigated using real time biosensor (BIAcore) analysis of the GST-SH2 fusion proteins binding to sensor chip-immobilized Tyr-1139 phosphopeptide. Since GST dimerization can lead to an overestimation of binding affinities by this method (31), these experiments enabled investigation of binding in the absence of interference from other autophosphorylation sites but only estimated the relative, not absolute, affinities of the SH2 domains for the phosphopeptide. Parallel channels of a sensor chip coupled to either the Tyr-1139 phosphopeptide or the nonphosphorylated version were exposed to increasing concentrations of soluble fusion proteins. Table I shows apparent affinity constants estimated from equilibrium responses by Scatchard analysis of BIAcore progress data (see "Materials and Methods"). No binding of the SH2 domain fusion proteins to the nonphosphorylated peptide was detected, and the interaction of the GST-Grb14 SH2 fusion protein with phosphorylated Tyr-1139 was too weak for kinetic analysis. Equilibrium responses of the beta D5 mutant binding to the phosphopeptide demonstrated an apparent Kd of 438 nM, whereas a higher affinity was derived for the beta D6 mutant (Kd = 224 nM). The apparent Kd for interaction of the beta D5/beta D6 mutant was slightly lower again (203 nM), approaching that of wild type Grb7 (126 nM). These results therefore parallel those obtained upon binding of the fusion proteins to the intracellular region of erbB2 (Fig. 4) and provide additional evidence for the interactions being mediated via the Tyr-1139 autophosphorylation site.

Table I.

Apparent equilibrium dissociation constants from BIAcore analysis of GST fusion protein binding to the Tyr-1139 phosphopeptide


GST fusion protein Dissociation constanta r2b

nM
GST-Grb14 SH2 n.d.c -
GST-Grb14 beta D5 SH2 438 0.995
GST-Grb14 beta D6 SH2 224 0.986
GST-Grb14 beta D5/beta D6 SH2 203 0.998
GST-Grb7 SH2 126 0.989

a Determined by Scatchard analysis of steady state binding response.
b r2, correlation of experimental data with the line of best fit.
c n.d., not determined, interaction too weak to obtain kinetic data from BIAcore analysis.

Molecular Modeling of the Impact of beta D6 Substitutions on Grb7/Grb14 SH2 Domain-Phosphopeptide Interactions

From all the available structures of Group I SH2 domains in complex with a high affinity phosphopeptide, the SH2 domains of Grb7 and Grb14 share the highest overall homology with that of Src, with 33 and 31% amino acid identity, respectively. For this reason the structure of the Src SH2 domain, determined by x-ray crystallography (10), was used to model the structure of the Grb7 and Grb14 SH2 domains. The Src sequence was substituted by the sequences of Grb7 and Grb14, and the sequence of the complexed phosphopeptide was replaced by the Tyr-1139 peptide sequence. It was assumed that the backbone conformations of the peptide and of the SH2 domain secondary structure elements were the same as in the Src structure, and identical side chains were copied and compatible rearrangements of nonidentical side chains were considered (see "Materials and Methods"). In this way the relative influences of the beta D6 residues from Grb7 and Grb14 on SH2 interaction with the Tyr-1139 peptide could be modeled.

The overall structure deduced for the Grb7 SH2 domain is shown in Fig. 7A with the position of the bound Tyr-1139 peptide. Comparison of the Grb7 and Grb14 SH2 domains revealed differences in the predicted orientation of the beta D6 side chain with respect to the peptide, whereas conformations of adjacent side chains were identical. In this model the Leu of Grb7 is directed away from the Asn at +2 of the peptide, superimposable with the Lys at beta D6 of Src, whereas the Gln in Grb14 is predicted to extend toward the Asn side chain (Fig. 7B). Without further refinement of the model, the distance between the side chain amide heavy atoms is 3.95 Å. This is suggestive of the opportunity for hydrogen bonding between these side chains, so this distance may in fact be smaller. The conformation of the Asn BC3 side chain is also predicted to change in Grb14 to a position sufficiently close (3.24 Å between heavy atoms) for hydrogen bonding with Gln beta D6 (Fig. 7B). Hydrogen bonding of Gln beta D6 with Asn at +2 of the peptide may add rigidity to the structure of the complex that could be detrimental to efficient binding. In addition, hydrogen bonding and/or the orientation of the beta D6 Gln may disrupt other interactions required for high affinity phosphopeptide binding.


Fig. 7. A, schematic diagram of the modeled Grb7 SH2 domain in complex with the Tyr-1139 peptide from erbB2 viewed from the peptide-binding surface and showing secondary structural elements and notation used. alpha  Helices appear as ribbons, and beta  strands appear as arrows. The peptide is represented in ball-and-stick fashion, encompassing the phosphotyrosine (P-Tyr), +1 (Val), +2 (Asn), and +3 (Gln) positions. The modeled structure was derived from the coordinates of the Src SH2 domain structure (10), as described under "Materials and Methods." B, comparison of the predicted conformation of key residues in the SH2 domains of Grb7 (yellow) and Grb14 (cyan) with respect to the Tyr-1139 peptide. Peptide atoms are colored as follows: carbon, green; nitrogen, blue; oxygen, red; phosphorus, pink. The residues from the Grb7 and Grb14 SH2 domains are overlaid to show differences in predicted side chain conformations. Dotted lines indicate potential hydrogen bonds.
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DISCUSSION

The receptor tyrosine kinase erbB2 and the signaling protein Grb7 are overexpressed in a subset of human breast cancer cell lines and primary breast cancers due to co-amplification of their respective genes. They are also functionally linked as they co-immunoprecipitate from cell lysates and interact directly in vitro (3). This study investigated the interaction between these two proteins by first determining the binding site of Grb7 and then identifying Grb7 SH2 domain residues conferring specificity toward this site.

Five putative in vivo autophosphorylation sites and an additional site of in vitro autophosphorylation have been identified in the erbB2 C terminus (28, 29). Since point mutation of the five tyrosines phosphorylated in vivo reduced receptor tyrosine phosphorylation by 95% (28), the existence of major autophosphorylation sites other than the six pictured in Fig. 1A is unlikely. Of these six sites, peptide competition revealed Tyr-1139 as the preferred binding site of the Grb7 SH2 domain (Fig. 1B). This was supported by binding studies involving the HER1-2 Y1139F receptor, in which mutation of this site reduced association of the Grb7 SH2 domain by 95% compared with that observed with the wild type receptor (Fig. 2B).

Interestingly, Tyr-1139 also represents the major Grb2 binding site on erbB2 (18), and there is evidence for interaction of Grb7 with Grb2 binding sites on other proteins. Both bind Tyr-580 of the tyrosine phosphatase SH-PTP2 (32), and Grb7 probably also recognizes the Grb2 binding site at Tyr-317 of Shc (3). The potential interaction with Grb2 binding sites on other signaling molecules such as FAK (33), IRS-1 (34), and RPTPalpha (35) awaits further investigation. The binding of different SH2 proteins to a single phosphotyrosine site is not unprecedented. Nck and the p85 subunit of phosphatidylinositol 3'-kinase both bind and compete for Tyr-751 in the platelet-derived growth factor beta  receptor (36), and the Met receptor has a multiple SH2 protein docking site at Tyr-1349 (37). Since the downstream effectors of the Grb7 family have yet to be identified, the consequences of these interactions are not clear. However, since Grb7 exhibits a relatively tissue-specific expression profile (38) and the GRB7 gene resides on the same breast cancer amplicon as ERBB2 (3), it is clear that competition between Grb2 and Grb7 provides a potential mechanism for modulation of the Ras signaling pathway in specific tissues and/or cancer cells. In the latter context, amplification and overexpression of the ERBB2 gene in a series of breast cancer cell lines that exhibit concomitant Grb7 overexpression correlates with increased erbB2-Grb2 interaction and mitogen-activated protein kinase stimulation, indicating that the Ras pathway is not markedly down-regulated by Grb7 competition for Grb2 binding sites (3, 21). However, since either proliferation or differentiation can be specified by the kinetics of mitogen activated protein kinase activation (39), the consequences of erbB2 signaling via the Ras pathway may be different in the presence or absence of Grb7. This is currently under investigation.

The Grb7 and Grb14 SH2 domains are classified into Group I due to the presence of Phe or Tyr at the beta D5 position. Most members of this group exhibit a preference for the target amino acid sequence pTyr-hydrophilic-hydrophilic-Ile/Pro, where the selectivity at the +2 position is lower or similar to that at +1 and +3 (12, 13). However, Grb2 has an atypical Group I SH2 domain that selects pY-Q/Y/V-N-Y/Q/F, with the strongest preference at the +2 position (13). As expected, this consensus exhibits homology to the Grb7 binding sites on Shc (YVNV) (3), SHPTP2 (YENV) (32), and erbB2 (YVNQ), with the conservation of Asn at +2 particularly apparent. The poor selectivity of the Grb2 SH2 domain toward the +3 position is thought to be partly due to the bulky Trp residue at the EF1 position closing up the +3 binding pocket (12, 40). Two features of the Grb2 and Grb7 SH2 domains may contribute to their similar phosphopeptide selectivity. First, the insertion in the Grb7 SH2 EF loop region relative to Src (Fig. 3) may restrict the +3 binding pocket in a manner similar to Trp EF1 in the Grb2 SH2. Second, selectivity of the Src SH2 domain at the +2 position is influenced by the beta D'1 residue, which in Src is Arg but in Grb2 and Grb7 is Leu, which may favor selectivity for Asn at +2. The low affinity of the Grb14 SH2 domain, which has Ile at beta D'1, for such phosphopeptide sequences may be due to structural changes induced by the nonconservative change at the beta D6 position relative to Grb7, as discussed below.

Residues within the Grb7 SH2 domain that determine the specificity of its interaction with erbB2 were identified by substitution analysis using the closely related Grb14 SH2 domain, which exhibits a low affinity for erbB2. A Phe to Tyr change at beta D5 in the Grb14 SH2 domain caused a modest increase in binding to the intracellular domain of erbB2 (Fig. 4A), whereas the reverse change in the Grb7 SH2 domain had no discernible effect on binding (Fig. 5). At the adjacent beta D6 position a Gln to Leu change caused a marked increase in Grb14 SH2 binding to erbB2, and the reverse change in Grb7 abrogated its ability to bind the receptor. The erbB2 affinity of the beta D6 mutant was further augmented by simultaneously substituting the beta D5 residue (Fig. 4B). Substitution at the other four candidate positions, which included beta D'1, had minor or undetectable effects on erbB2 binding. Importantly, phosphopeptide competition analysis demonstrated that the increases in affinity toward erbB2 exhibited by the Grb14 beta D6 and beta D5/beta D6 mutants were directed toward the same autophosphorylation site as that preferentially bound by Grb7, Tyr-1139. Moreover, the apparent equilibrium affinity constants of these mutants for interaction with the Tyr-1139 phosphopeptide were determined by BIAcore analysis and paralleled their binding to erbB2. Minimal binding was observed with the Grb14 SH2, whereas the apparent equilibrium constants for the beta D6 and beta D5/beta D6 mutants approached that of wild type Grb7. Taken together, our studies of SH2 domain interactions with both the erbB2 intracellular domain and the Tyr-1139 peptide pinpoint the beta D6 Leu as an important determinant of Grb7 SH2 specificity.

We have modeled the interaction of the Grb14 and Grb7 SH2 domains with the Tyr-1139 phosphopeptide, using the structure determined for the Src SH2 domain complexed with a high affinity peptide, in an attempt to understand the impact of the different beta D6 residues. The conformation of the peptide was assumed to be the same as in the Src structure, lying perpendicular to the central beta  sheet, between the BG and EF loops (Fig. 7A). Indeed, this peptide position is very similar in the SH2 domain structures of Lck, PLC-gamma 1, and Syp, although in Shc the peptide is positioned lower, between the BG and DE loops (16). Recalculation of side chain configurations using a rotamer library revealed a difference in the predicted position of the beta D6 side chain of Grb14 compared with Grb7, whereas adjacent residues, including beta D5, were superimposable (Fig. 7B). The side chain of Asn BC3 in the phosphate-binding BC loop also changed conformation in the Grb14 model. Therefore, since Gln at beta D6 abrogates Grb7 binding to erbB2, the orientation of this beta D6 residue might introduce rigidity and/or interference and hence be detrimental to binding. However, in the recently described crystal structure of the Grb2 SH2 domain complexed with a high affinity peptide (41), the latter adopts a novel conformation, incorporating a beta  turn, and the +2 Asn hydrogen bonds to the backbone carbonyl and amide groups of the SH2 domain beta D6 residue. This is intriguing in light of the overlap in SH2 specificity between Grb2 and Grb7 and our data highlighting the beta D6 residue as an important selectivity determinant of the Grb7 SH2 domain, and will be investigated further once the Grb2 structural coordinates become available.

In summary, the nature of the beta D6 residue is a critical determinant of the high affinity interaction of the Grb7 SH2 domain with Tyr-1139 of the erbB2 intracellular domain. Switching this single amino acid between the Grb14 and Grb7 SH2 domains exchanges their respective affinities for erbB2, analogous to the effect of swapping the EF1 residue between the Src and Grb2 SH2 domains (40). These observations therefore provide a clear example of how heterogeneity at a single residue allows two highly conserved SH2 domains to retain distinct preferences for RTK binding.


FOOTNOTES

*   This work was supported by research grants from the National Health and Medical Research Council of Australia and the New South Wales State Cancer Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be sent: Tel.: 61-2-92958100; Fax: 61-2-92958321; E-mail: r.daly{at}garvan.unsw.edu.au.
1   The abbreviations used are: SH, Src homology; EGF, epidermal growth factor; GRB and Grb, growth factor receptor bound, GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; RTK, receptor tyrosine kinase.

ACKNOWLEDGEMENTS

We thank Dr. J. Schlessinger for the HER1-2 cell line and plasmid encoding the Grb2 fusion protein and Dr. Axel Ullrich for the HER1-2 expression vector.


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