We previously reported a signal transducing adaptor molecule, STAM, which is associated with Janus kinase (Jak)2 and Jak3 and is involved in signal transduction mediated by IL-2 and GM-CSF (26
). We have also recently cloned a cDNA clone encoding a novel molecule, named AMSH, which binds to STAM (our unpublished results). To address the functional significance of AMSH, we attempted to identify molecules associated with AMSH using the yeast two-hybrid assay system. One full-length cDNA clone was isolated from a human PHA-PBL cDNA library. The cDNA clone encodes a molecule homologous to Grb2, named Grf40 (for Grb2 family member of 40 kD). The nucleotide sequence of the Grf40 gene has been deposited with GenBank, and is available from EMBL/GenBank/DDBJ under accession no. AF042380
. The deduced amino acid sequence of Grf40 consists of 330 amino acid residues. The schematic structure of Grf40 was compared with Grb2 (7
) and Grap, another Grb2 family member (27
). The NH2
- and COOH-terminal SH3 domains and an intermediate SH2 domain of Grf40 are highly homologous to those of Grb2 and Grap, while a unique insert region (amino acid position Arg156
) containing proline/ glutamine-rich sequences was seen in Grf40 but not Grb2 and Grap (Fig. A). These results indicate that Grf40 is a new member of the Grb2 family.
Figure 1 Schematic structure and expression of Grf40. (A) The schematic structure of Grf40 was compared with Grb2 and Grap. The percentages of amino acid identity of the SH3 and SH2 domains of Grf40 with Grb2 and Grap were indicated in terms of their SH3 and (more ...)
Various human cell lines were examined for expression of Grf40 by immunoblotting with anti-Grf40 Ab. Two T cell lines, MOLT-4 and Jurkat, were strongly positive for expression of the 40-kD Grf40, and two B cell lines, Daudi and Raji, were weakly positive, but the other cell lines, including a B cell line (Ramos), myeloid cell lines (THP-1, TF-1, and Eol-3), and the nonhematopoietic cell lines (HeLa and WI-26) were all negative for this expression (Fig. B). In contrast to Grf40, appreciable expression of Grb2 was seen in all the cell lines (Fig. B). Northern blot analyses on various human cell lines and tissues showed two Grf40-specific transcripts at 3.5 and 1.5 kb in Jurkat, MOLT-4, three myeloid cell lines (KU812, K562, and M-TAT) and PHA-PBL (data not shown), and in human immunotissues such as thymus, spleen, small intestine, and PBL, whereas marginal levels of the transcripts were detected in other tissues, including prostate, testis, ovary, colon, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Fig. C). These results suggest that Grf40, unlike Grb2, is predominantly expressed in immunotissues and hematopoietic cells, particularly T cells.
Since Grb2 has been shown to bind to SLP-76 and LAT, which are 76- and 36/38-kD tyrosine-phosphorylated proteins essential for TCR-mediated signaling, respectively (10
), we asked ourselves whether or not Grf40 is also associated with SLP-76 and LAT. We detected 76- and 36/38-kD tyrosine-phosphorylated proteins that coimmunoprecipitated with Grf40 in Jurkat cells after stimulation by TCR cross-linking with OKT3 (Fig. A). We then confirmed that the 76- and 36/38-kD tyrosine-phosphorylated proteins were SLP-76 and LAT, respectively, by stimulating Jurkat cells with OKT3. Their lysates were immunoprecipitated with anti-Grf40 Ab, and the immunoprecipitates were then immunoblotted with anti-LAT, anti–SLP-76, or anti-Grf40 Ab. Grf40 precipitated SLP-76 irrespective of TCR stimulation, but precipitated LAT only after TCR stimulation (Fig. B). These results indicate the association of Grf40 with SLP-76 and LAT in Jurkat cells. To determine the association site of Grf40 for SLP-76, we carried out further coimmunoprecipitation assays between the various deletion mutants of Grf40 and SLP-76. COS7 cells were transiently transfected with Myc-tagged wild-type Grf40 and four Grf40 mutants deleted of the NH2
-terminal SH3 domain (Grf40-dSH3N), the COOH-terminal SH3 domain (Grf40-dSH3C), both the NH2
- and COOH-terminal SH3 domains (Grf40-dSH3NC), or the SH2 domain (Grf40-dSH2). The transfected COS7 cells were immunoprecipitated with anti–SLP-76 Ab or anti-Myc mAb, and then immunoblotted with anti-Myc mAb or anti–SLP-76 Ab. Wild-type Grf40 and the Grf40-dSH2 and Grf40-dSH3N mutants were coimmunoprecipitated with SLP-76, but the Grf40-dSH3C and Grf40-dSH3NC mutants were not tested (Fig. C). Conversely, SLP-76 was coimmunoprecipitated with Grf40-dSH2, Grf40-dSH3N, and wild-type Grf40, but not with Grf40-dSH3C and Grf40-dSH3NC mutants (data not shown). These results indicate that the COOH-terminal SH3 domain of Grf40 is an association site for SLP-76. We next determined the association site of SLP-76 for Grf40 by using various SLP-76 mutants. Flag-tagged wild-type and four mutants of SLP-76 were introduced into COS7 cells together with Myc-tagged Grf40, and then immunoprecipitated and immunoblotted with anti-Flag and anti-Myc Abs. Myc-tagged Grf40 was coimmunoprecipitated with the SLP-76 mutants consisting of and containing the amino acid position Glu217
, but not with the SLP-76 mutant consisting of the amino acid position Pro241
. These results indicate that the Grf40 binding site is located in the amino acid position Glu217
of SLP-76 (Fig. D). This Grf40 binding site of SLP-76 almost overlaps the amino acid position Asn224
, which has been shown to be the Grb2 binding site (15
). On the other hand, the binding site of Grb2 for LAT has been shown to be the SH2 domain of Grb2, which is thought to bind to the phosphorylated tyrosine residue (18
). Together with this notion, we showed that LAT is tyrosine phosphorylated and subsequently coimmunoprecipitated with Grf40 after TCR stimulation, suggesting that the SH2 domain of Grf40 is possibly the binding site for LAT.
Figure 2 Coimmunoprecipitation of SLP-76 and LAT with Grf40 in Jurkat cells. Jurkat cells were stimulated with (+) or without (−) OKT3 for 3 min, and their lysates were immunoprecipitated (IP) with anti-Grf40 or preimmune (control) serum. (more ...)
Since the COOH-terminal SH3 domain of Grb2 has been shown to be the binding site for SLP-76 (10
), we examined the competitive binding ability between Grf40 and Grb2 to SLP-76. COS7 cells were transiently transfected with 2.5-μg plasmids of Myc-tagged Grf40 and Myc-tagged Grb2 in association with different doses (0–1.0 μg) of Flag-tagged SLP-76 plasmid. Their lysates were immunoprecipitated with anti-Flag mAb and then immunoblotted with anti-Myc polyclonal Ab. Coimmunoprecipitation of Myc-tagged Grf40 with SLP-76 gradually decreased upon reducing the SLP-76 plasmid dose to 0.05 μg, whereas the Myc-tagged Grb2 coimmunoprecipitation with SLP-76 was detectable only at a 1.0-μg dose of SLP-76 plasmid (Fig. A). Expression levels of the plasmids introduced were quantified by immunoblotting, confirming that there was no significant difference in the amounts between Myc-tagged Grf40 and Myc-tagged Grb2 (Fig. A). These results suggest the possibility that Grf40 associated much stronger with SLP-76 than did Grb2. To confirm this further, COS7 cells were transiently transfected with low doses of SLP-76 plasmid (0.2 μg) and Myc-Grf40 plasmid (2.5 μg) together with various doses (0–10 μg) of Myc-tagged Grb2 plasmid. Their lysates were immunoprecipitated with anti-Flag mAb and then immunoblotted with anti-Myc polyclonal Ab. Even when up to 10-μg plasmid doses of Myc-tagged Grb2 were cotransfected, coimmunoprecipitation of Myc-tagged Grf40 with SLP-76 was still unchanged (Fig. B). Furthermore, although the increased expression of Myc-tagged Grb2 was dependent on its plasmid dose, which was considerably higher than that of Grf40 at a 10-μg plasmid dose of Myc-tagged Grb2, Myc-tagged Grb2 was not coimmunoprecipitated with SLP-76 (Fig. B). These results indicate that Grf40 competes with Grb2 in its binding to SLP-76, and the binding affinity of Grf40 to SLP-76 is apparently higher than that of Grb2. Since the SLP-76 mutant deleted of the Grb2 binding site, which overlaps the Grf40 binding site, failed to increase IL-2 promoter activity upon TCR stimulation (15
), it is possible that not only Grb2 but also Grf40 plays a critical role in the SLP-76–dependent increase in IL-2 promoter activity.
Figure 3 Competitive binding ability of Grf40 and Grb2 to SLP-76. COS7 cells were transiently transfected with the indicated doses of pFlagSLP (SLP-76), 2.5 μg of pMycGrf40 (Grf40), and 2.5 μg of pMycGrb2 (Grb2) (A), or 0.2 μg of pFlagSLP, (more ...)
To address the functional significance of Grf40 in TCR-mediated signaling, we performed luciferase assays with reporter genes containing the IL-2 promoter and the NF-AT binding domain. Overexpression of wild-type Grf40 did not lead to either basal or TCR-mediated activation of the IL-2 promoter and NF-AT (data not shown). Since Grf40 interacts with SLP-76 in Jurkat cells, and overexpression of SLP-76 is known to augment TCR-mediated stimulation of the IL-2 promoter and NF-AT activity (15
), we used Jurkat cells transiently transfected with SLP-76 to examine the effect of Grf40 in TCR-mediated signaling. Transfections of wild-type Grf40 and Grf40-dSH3N mutant into Jurkat cells overexpressing SLP-76 led to significant increases in IL-2 promoter activity upon stimulation with OKT3 plus PMA, whereas transfection of Grf40-dSH2 mutant induced a marked inhibition of IL-2 promoter activity compared with transfection of an empty vector (Fig. A). These results indicate that Grf40-dSH2 mutant has a dominant-negative effect in TCR stimulation, suggesting that the SH2 domain of Grf40 interacts with an essential molecule for TCR-mediated signaling, which is possibly LAT (18
). Similar results were obtained in NF-AT luciferase assays with Jurkat cells stimulated with OKT3 (Fig. B). Furthermore, the Grf40 mutants (Grf40-dSH3C and Grf40-dSH3NC) deleted of the COOH-terminal SH3 domain, which is the binding site for SLP-76, also lost their ability to increase IL-2 promoter activity (Fig. A), suggesting that a Grf40–SLP-76 complex formation is required for SLP-76–dependent TCR stimulation. These results indicate that Grf40 is involved in signaling the stimulation of the IL-2 promoter and NF-AT activities mediated by OKT3 and PMA.
Figure 4 Effects of Grf40 mutants on TCR-mediated activation of IL-2 promoter and NF-AT activities. Jurkat cells were transfected with 10 μg of IL-2-Luc (A) or 10 μg of NF-AT-Luc (B), along with 10 μg of SLP-76 and 10 μg of an empty (more ...)
Since Grb2 has also been considered to be involved in the modulation of TCR-mediated signal transduction (9
), we compared the functional significance of Grf40 and Grb2 in TCR-mediated stimulation of IL-2 promoter activity. Jurkat cells overexpressing SLP-76 were transfected with the wild-types and SH2 deletion mutants of Grf40 and Grb2, in association with an IL-2 promoter–driven luciferase construct. They were stimulated with OKT3 plus PMA, and assayed for luciferase activity. An appreciable enhancement of IL-2 promoter activity was seen with wild-type Grf40, but scarcely with wild-type Grb2, whereas Grf40-dSH2 mutant showed a marked dominant-negative effect in the IL-2 luciferase assay compared with Grb2-dSH2 mutant (Fig. A). The plasmid dose dependency in the IL-2 luciferase assay was compared between the Grf40-dSH2 and Grb2-dSH2 mutants. The suppressive effects on the luciferase activities were significantly stronger in Grf40-dSH2 mutant than Grb2-dSH2 mutant at various plasmid doses (Fig. B). These results indicate that Grf40 is involved in TCR-mediated signaling more effectively than Grb2. This conclusion is in accordance with the observation of greater binding affinity of Grf40 to SLP-76 compared with Grb2.
Figure 5 Comparison of blocking effects between Grf40-dSH2 and Grb2-dSH2 on TCR-mediated stimulation of IL-2 promoter activity. (A) Jurkat cells were transfected with 10 μg of IL-2-Luc along with 10 μg of SLP-76 and 10 μg of an empty vector (more ...)
This study showed a critical involvement of Grf40 in the SLP-76–dependent signaling mediated by the TCR. One might consider the possibility that Grf40 mutants exert their effects in TCR-mediated signaling by altering expression levels of SLP-76. However, this possibility is negligible because the expression of pCX-SLP76 was confirmed to be unaffected by transient expression of Grf40, Grb2, and their mutants in COS7 cells (data not shown). Hence, the interaction of Grf40 with SLP-76 and LAT is thought to be critical for TCR-mediated signaling.
-terminal SH3 domain of Grb2 is known to be a binding site for Sos, a Ras guanine nucleotide exchange factor, which has been considered to be involved in Ras activation upon TCR stimulation (9
). We confirmed the association of Grb2 with Sos in Jurkat cells; however, a complex formation between Grf40 and Sos was undetectable in these cells (data not shown). Therefore, we suspect that Grf40 does not direct the Ras activation signaling mediated by the TCR. However, Grf40 contributes to TCR-mediated activation of the IL-2 promoter and NF-AT more effectively than Grb2, suggesting the critical involvement of Grf40 in TCR-mediated signaling. In this context, it is of interest that SLP-76 associated with Grf40 also binds to Vav, a Rac/Rho guanine nucleotide exchange factor, and that the interaction between SLP-76 and Vav has been shown to participate in IL-2 gene activation upon TCR stimulation (17
). These observations provide a model pathway in which activated ZAP-70 tyrosine kinase after TCR ligation phosphorylates LAT (18
), which then binds to the preformed Grf40–SLP-76 complex and recruits it to ZAP-70 (29
), which further phosphorylates SLP-76 to be associated with Vav, leading to the downstream signaling of the TCR.
Northern blot and immunoblot analyses revealed that Grf40 is expressed predominantly in immunotissues and hematopoietic cells, particularly T cells, in contrast to Grb2. Such restricted distribution of Grf40 may reflect the more efficient involvement of Grf40 in TCR-mediated signaling compared with Grb2. Although Grap has also been shown to be specific for hematopoietic and lymphocytic cells (28
), the functional significance of Grap is still unknown.
The genome sequence of GRB2L has been registered in GenBank/EMBL/DDBJ (under accession no. Z82206
), which contains the entire sequence of Grf40. Since GRB2L has been mapped to human chromosome 22q12, Grf40 is thought to have the same chromosomal location. Furthermore, cDNA clones identical to Grf40 were reported as Grap2 (30
), and human Gads (31
) and their mouse homologues, named Mona (32
) and mouse Gads (33
), were also reported after the submission of this paper. Although the report regarding human Gads showed similar results as our study, they did not show any comparative study between Gads and Grb2. We here show evidence suggesting that Grf40 plays a more critical role in the TCR-mediated signaling than Grb2.