In this study, we have demonstrated Grb10 association with activated PDGFRβ in response to PDGF-BB stimulation (Fig. A). The Grb10 SH2 domain was found to be sufficient for this interaction (Fig. A) and, when used as a competitor, to essentially block the interaction with cellular Grb10 (Fig. A). Of the major tyrosine autophosphorylation sites of PDGFRβ, only one, Y771 when mutated, resulted in the lack of association with the Grb10 SH2 domain (Fig. A). Synthetic phosphopeptides representing Y771 interfered with PDGFRβ binding of the Grb10 SH2 domain (Fig. B) and indicated that the motif around phosphotyrosine 771 actually binds to Grb10. PDGFRβ mutation Y771F similarly interfered with the association of complete cellular Grb10 (Fig. C). This binding site is shared by Ras GAP, which has been shown to be involved in the chemotactic response to PDGF (51
). Since transgenic mice lacking GAP demonstrate normal PDGF-BB-mediated mitogenesis, GAP does not appear to participate in that response unless it plays an unidentified putative role by down-modulating Grb10 action, possibly by competing for the same PDGFRβ binding motif (51
). It is conceivable that Grb10 may modulate GAP action or otherwise participate in the chemotactic signal, but this issue is outside the scope of this study.
Grb10 association with the IGF-IR had been demonstrated earlier (8
) and was confirmed in this study (Fig. B); however, a preference of Grb10 for the IR had been observed in a direct comparison (20
). This was investigated by others (15
), who reported that IR associates with the SH2 and BPS domains of Grb10, resulting in high-affinity binding, which explains the significant but partial interference by the SH2 domain (Fig. C), whereas IGF-IR primarily associates with the BPS domain, which explains the minimal interference observed by the SH2 domain (Fig. B). Of a number of point mutations at major IGF-IR autophosphorylation sites, only mutation Y1316F at the carboxyl terminus interfered with the association of the Grb10 SH2 domain (Fig. A). This finding is supported by our earlier studies which implicated the homologous carboxyl-terminal autophosphorylation motif Y1322 of IR in Grb10 SH2 domain binding, based on analysis of receptor mutants and the binding of GST-Grb10 SH2 domain fusion proteins to immobilized IR phosphopeptides (14
). Phosphopeptides representing the major IGF-IR autophosphorylation sites were not available for our studies; however, new experiments with IR phosphopeptides confirmed that only a peptide representing Y1322 (homologous to IGF-IR Y1316 [50
]) specifically interfered with IR binding to the Grb10 SH2 domain and showed that this sequence actually interacts with Grb10 (Fig. B). However, the SH2 domain association likely represents only a minor aspect of the interaction between Grb10 and IGF-IR, which appears to be largely carried out by the Grb10 BPS domain (15
). The consensus receptor phosphotyrosine sequence motif which is recognized by the Grb10 SH2 domain has not yet been established to our knowledge, and our comparison of the implicated PDGFRβ, IR, and IGF-IR sites of association has not been able to define it (not shown).
In independent studies of several research teams, the exact sites of interaction with Grb10 are controversial for both IGF-IR and IR. Using a yeast two-hybrid interaction approach, an 800-bp fragment of Grb10 was independently found to associate with the IGF-IR carboxyl terminus, but at a site reported to lie between aa 1229 and 1245 (30
). In contrast to that study, the activation loop had been implicated in the association with IR and IGF-IR by using a yeast two-hybrid approach combined with receptor mutants (9
). Based on IR mutants and phosphopeptides, the kinase activation loop and the juxtamembrane region have been implicated in the interaction with Grb10 in another study (13
). However, the juxtamembrane region and the carboxyl terminus were not found to be essential in a further study based on yeast two-hybrid mapping and IR mutant analysis of Grb10 association (38
). Part of the differences may be explained by the observation that in addition to the SH2 domain, other Grb10 sequences have been implicated in the interaction with IR and IGF-IR (13
). A new domain, termed BPS (or IPS) to reflect its location between the PH and SH2 domains at aa 358 to 434, has been implicated in Grb10 binding to the insulin, IGF-I, and EGF receptors (11
). Whereas our studies are exclusively based on the association of Grb10 SH2 domain fusion peptides, other studies have used complete Grb10 or larger fragments of Grb10 and included the interactions of the BPS domain. A recent study has compared various IR mutants for their interaction with the Grb10 SH2, the BPS, and a combination of both domains (15
). Only mutation Y1150/1151F in the activation loop was found to essentially abolish the association of IR with Grb10, surprisingly with any isolated domain either SH2 or BPS, or both in combination. This finding suggests either that both Grb10 domains bind to the same IR site, which is not supported by the model presented in this study (15
), or that the mutation in the activation loop abolishes other putative IR binding sites of Grb10, which would impair the specificity of the analysis. Since phosphorylation of the activation loop is one of the first steps in IR activation and a requirement for subsequent steps such as phosphorylation of carboxyl-terminal sites (53
) and since carboxyl-terminal mutations have not been tested, the reported finding does not address the specific binding of Grb10 to other receptor sites.
Other factors which may help to explain some of the differences observed by different research teams are the distinct Grb10 sequence variants which have been studied and may be explained by differential splicing; more than one Grb10 protein band has typically been identified in many experiments, which may also be due in part to variable translation starts and/or modification (9
). In addition to the original mouse Grb10 (39
), a related human protein termed Grb-IR or hGrb10α had been identified as an IR partner with a truncated PH domain (21
). Several human sequence variants at the amino terminus termed Grb10/IR-SV1 or hGrb10β, hGrb10γ, hGrb10δ (10
) and an additional variant in the mouse which lacks a sequence upstream of the amino-terminal end of the GM region (20
) have been reported (42
). Most available polyclonal antibodies are expected to cross-react with several of the known variants; however, only specific forms will be expressed in specific cells and tissues. The set of three major protein bands which are frequently identified in fibroblasts unlikely represent three distinct splice variants since mouse Grb10 (39
), when overexpressed, results in overall similar patterns of intensified protein bands which must all result from the same cDNA (Fig. A). Many cell types including NIH 3T3 fibroblasts may express only few Grb10 variants which may be represented by several distinct protein bands due to the use of alternative translation start signals and differences in posttranslational modification (20
To learn more about the role of Grb10 as a mitogenic mediator, we investigated its interaction with other receptor tyrosine kinases. In addition to the observed association with PDGF, insulin, and IGF-I receptors, we found the SH2 domain to associate with activated HGFR (Met) and FGFR but not with EGFR and NGF receptors (TrkA) (Fig. ). Since our experiments were based on GST-Grb10 SH2 domain fusion proteins, it is possible that association with TrkA involves additional Grb10 sequences such as the BPS domain. This probably does not apply to the putative association with the EGFR (15
). When combined with the reported Grb10 association with other receptor tyrosine kinases such as Ret (12
) and the Eph-related receptor tyrosine kinase ELK (48
), our data suggest a role for Grb10 downstream of most receptor tyrosine kinase subfamilies (52
) except for the EGF and NGF subfamilies. This inference is based on at least one member of each subfamily (52
) which was tested for Grb10 association (Fig. ) and is compatible with a putative role of Grb10 as a general mitogenic mediator, in addition to other putative functions of Grb10 downstream of the ELK receptor in axonal guidance, neuronal bundling, or angiogenesis (48
The role of Grb10 in EGF action is still controversial. Grb10 was originally cloned with an activated carboxyl-terminal EGFR fragment as a probe but was found to interact only weakly with the activated receptor (13
). However, serine phosphorylation of Grb10 has been reported in response to EGF stimulation (39
). Interaction with the EGFR has not been observed in our study (Fig. ) despite the fact that the Grb10 SH2 domain had been implicated in this association (15
), and changes in Grb10 function were not found to affect EGF-stimulated DNA synthesis (Fig. and B).
To test the putative mitogenic role of Grb10 directly, we used four independent experimental strategies to interfere with cellular Grb10 function or increase cellular Grb10 levels. Initially, we microinjected GST-Grb10 SH2 domain fusion protein to test its impact on PDGF-BB- and insulin-mediated mitogenesis in NIH 3T3 fibroblasts. The high fusion protein concentration in the injected cells is expected to exert a dominant-negative effect on cellular Grb10 function (37
). The observed reduction in DNA synthesis suggests a positive, stimulatory role of cellular Grb10 in that pathway (Fig. A). Microinjection of GST-Grb10/IR-SV1 or hGrb10β had been shown earlier to interfere with IGF-I- and insulin-mediated, but not with EGF-mediated, DNA synthesis in Rat-1 fibroblasts (38
). The observed reduction in DNA synthesis (Fig. A) was specific for the Grb10 SH2 domain since a control Crk SH2 domain did not exert a significant effect.
In an alternative, dominant-negative strategy, the Grb10 SH2 domain was introduced into NIH 3T3 fibroblasts as a cell-permeable fusion peptide with a 16-aa fragment of the Drosophila
antennapedia homeodomain protein, which mediates the effective transfer of fusion peptides across the cell membrane (41
). A tyrosine-phosphorylated, cell-permeable Grb2 binding peptide based on EGFR sequences was shown to specifically block the mitogenic response to PDGF and EGF but not to FGF, demonstrating that selective pathways are inhibited by this approach (54
). Activation of the mitogen-activated protein kinase cascade was inhibited in response to up to 10-ng/ml but not to higher 50-ng/ml) EGF concentrations, suggesting that the peptide does not exert an excessive inhibitory function (54
). In our experiments, we observed a substantial decrease in PDGF-BB-, IGF-I-, and insulin-mediated DNA synthesis, as measured by the incorporation of [3
H]thymidine into DNA, which corresponded to the dose of the cell-permeable Grb10 SH2 domain fusion peptide (Fig. B). These results directly confirmed those shown in Fig. A from microinjection studies using an alternative approach.
In addition, these studies indicate a role of the Grb10 SH2 domain in IGF-I-mediated mitogenesis (Fig. B) but not through binding to the IGF-IR (Fig. B), since this is largely carried out by the Grb10 BPS domain (15
). The SH2 domain is consequently implicated in the association with another unknown signaling mediator, probably at an activated phosphotyrosine which may be represented by another (receptor) tyrosine kinase or by an alternative signaling mediator. Such a mechanism could assemble different receptors into a joint signaling complex and regulate cross talk between distinct receptor pathways. Grb10 may represent a shortcut in the Raf-mediated signaling cascade by directly interacting with Raf1 and MEK1 via its SH2 domain and linking growth factor receptors to these mediators (35
We have to consider possible cross talk of the Grb10 SH2 domain and Pro-rich fusion peptides with mechanisms involving the related mediators Grb7 and Grb14. However, functional selectivity has been reported for SH2 domains in the Grb7 family (17
) and for Pro-rich SH3 domain ligands (46
), which was also observed in control experiments with other Pro-rich regions (not shown). The responses shown in Fig. to point to a positive stimulatory role of Grb10 in the mitogenic actions of PDGF-BB, IGF-I, and insulin but have not been observed for Grb7 or Grb14 (5
), suggesting that these mediators do not affect our results.
Based on the putative role of Grb10 as a signaling adapter, we tested an additional domain, the Grb10 amino-terminal Pro-rich region, as a synthetic cell-permeable fusion peptide with a 16-aa fragment of the Drosophila
antennapedia homeodomain protein. With this approach, the IGF-I and insulin-stimulated mitogenic response was almost eliminated with increasing doses of peptide in a dose-responsive fashion, whereas the EGF and PDGF-BB responses were not significantly affected (Fig. ). Since the high concentration of the peptide is expected to represent a dominant-negative form, this result is consistent with a stimulatory role of the Grb10 Pro-rich region in IGF-I and insulin action. This is compatible with a role of Grb10 as a mitogenic signaling adapter which may involve its SH2 or BPS and Pro-rich domains to form a signaling complex between an activated receptor tyrosine kinase and an SH3 domain mediator such as Abl (13
). In contrast to the Grb10 SH2 domain, the Pro-rich region appears not to participate in PDGF-BB action (Fig. ). A role as a putative signaling adapter in this pathway may consequently involve an alternative Grb10 domain, such as the PH region, which remains to be identified. The functional differences observed for these two growth factor pathways at the level of the involved Grb10 domain structure may reflect a specific role of Grb10 in either pathway which remains to be elucidated.
A stimulatory role for Grb10 in PDGF-BB-, IGF-I-, and insulin- but not EGF-mediated mitogenesis was directly shown by cDNA expression from an ecdysone-regulated Grb10 expression plasmid. Stimulation of DNA synthesis was ecdysone dose responsive, with the highest level observed for PDGF-BB (sixfold over background levels [Fig. B]). The Grb10-mediated mitogenesis correlated with increased Grb10 expression levels which had been reproducibly observed on immunoblots (Fig. A). A wide range of ecdysone-adjustable expression levels has been found in cultured cells or in whole experimental mice, which in combination with the reported lack of basal expression surpassed tetracycline-based expression systems in a direct comparison (36
). Few if any side effects were described for ecdysone, including lack of toxicity at functional doses in vivo, even in whole mice (36
The effect of Grb10 on mitogenesis is controversial. Interference of the Grb10 SH2 domain with insulin-stimulated DNA synthesis upon microinjection into fibroblasts suggested a positive role for Grb10 (38
), which is also supported by the implicated stimulatory role of Grb10 in Bcr-Abl-mediated oncogenicity (2
) and by its implicated protective function in apoptosis (35
). On the other hand, the possible implication of Grb10 as a candidate for the Silver-Russel syndrome gene would point to a negative role in mitogenesis (29
). In addition, an inhibitory effect on IGF-I-mediated cell growth was reported for increased expression levels of mouse Grb10α (39
) in fibroblasts which were transformed by IGF-IR overexpression (31
). These cells express very high IGF-IR levels (500,000 per cell) and grow independently of growth factors except for IGF-I. Increased levels of stably expressed Grb10 have been reported to interfere with the transformed phenotype as well as with cell growth by delaying the S and G2
phases of the cell cycle (31
). Control experiments in the same cell line have not been performed; however, overexpression of Grb10 in different, normal, IGF-IR-disrupted fibroblasts did not interfere with cell growth. A Myc-tagged cDNA of Grb10 was expressed in these experiments, and it remains possible that the tag affects Grb10 function so as to result in a dominant-negative form. Alternatively, the transformed phenotype of this experimental system may have an impact on the observed function of Grb10 when compared to normal fibroblasts.
We addressed this question in our own study by monitoring the effect of increased Grb10 levels or of interference with cellular Grb10 on the cell doubling time in normal fibroblasts. We observed that Grb10 overexpression consistently increased the cell proliferation rate, whereas cell-permeable SH2 fusion peptides resulted in a dose-dependent substantial decrease (Fig. ). The observed opposite effects of Grb10 overexpression and of the dominant-negative SH2 domain peptides are complementary, confirming and expanding the observed changes in DNA synthesis shown in Fig. to . Consequently, all findings presented in this study support a positive, stimulatory role of Grb10 as a cellular partner of the PDGFβ, IGF-I, and insulin receptors, based on several independent dominant-negative as well as positive experimental strategies. Such a role as a general mitogenic signaling adapter is suggested in many growth factor signaling pathways by the observed association between Grb10 and other receptor tyrosine kinases (Fig. ). It is possible that Grb10 acts positively or negatively in mitogenesis dependent on the specific cellular context. Such a variable role has been described for other signaling mediators, including Myc, one important example, and its role in apoptosis (16