The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling.

We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-γ1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle.

paired tyrosine residues within ITAM motifs of the and CD3 cytoplasmic tails. The ZAP-70 tyrosine kinase is recruited to the activated complex by binding phosphorylated ITAM motifs via its tandem SH2 domains. Sequential or combinatorial activation of the ZAP-70 and Src family kinases leads to phosphorylation of numerous additional proteins, including Shc, p36/38, Lnk, SLP-76, phospholipase C-␥1 (PLC-␥1), 1 and the protooncoproteins c-Cbl and Vav. Many of these substrates contain SH2 domains. Several additional SH2 domain proteins that are not tyrosine-phosphorylated, such as Grb2 and phosphatidylinositol (PI) 3-kinase, are also recruited to the activated receptor. SH2 domain-phosphoprotein interactions, therefore, provide a mechanism for the assembly of protein complexes that participate in propagating antigen receptor signals to downstream cellular targets. Similar events follow antigen receptor activation in B lymphocytes.
Many critical proteins involved in lymphocyte signal transduction are ubiquitously expressed, including enzymes in phosphoinositide pathways such as PLC-␥1 and PI 3-kinase and Ras effectors like Grb2 and Sos. In contrast, the expression of signaling proteins such as Lck, ZAP-70, SHP1, Vav, SLP-76, Lnk, and the T cell receptor/CD3 components is much more restricted. Recent advances in cloning and gene sequencing technology, particularly random genome sequencing efforts, allow the identification of additional cell type-specific proteins with potential roles in cell signaling. Based on sequence homology, a cDNA clone encoding a new adaptor protein was identified 2 in the Human Genome Sciences expressed sequence tag (EST) data base (7). This protein has an SH3-SH2-SH3 domain architecture and 58% sequence identity to Grb2. Northern analyses of mRNA derived from multiple human tissues show restricted expression in spleen, thymus, and peripheral blood leukocytes. Using a specific antibody, we detected the protein in isolated peripheral blood lymphocytes and in all established T and B cell lines tested. These findings suggest potential functions for this protein in T cell signaling. Independently, an identical protein, designated Grap (Grb2-like accessory protein), was identified in hematopoietic cells and found to have potential functions downstream from erythropoietin and stem cell receptors (8).
A large number of studies have established an important role for Grb2 in linking tyrosine kinase receptors to downstream effector pathways. For example, the SH2 domain of Grb2 binds activated growth factor receptors either directly or indirectly via Shc, an adapter protein with single SH2 and PTB domains (9). The specificity for these interactions is determined by the selectivity of the Grb2 SH2 domain for phosphorylated YXN motifs (10,11). In many cells the SH3 domains of Grb2 are constitutively associated with proline-rich motifs in mSos, a guanine nucleotide exchange factor that activates Ras (12)(13)(14)(15). The SH3 domains of Grb2 may also bind such signaling molecules as Vav (16), dynamin (17,18), c-Cbl (19 -21), and GTPaseactivating protein-associated p62 (22). By this mechanism Grb2 mediates the recruitment of mSos and additional potential effector enzymes to activated receptor signaling complexes. In T cells, Grb2 recruitment also occurs via its SH2 domain, by binding T cell receptor ITAM motifs directly or indirectly through Shc or p36/38. Grb2 also binds mSos in T cells, although association is low under basal conditions and up-regulated significantly upon T cell receptor activation (23,24). The ability of Grb2 to form complexes with additional potential effectors and the roles of these complexes in signaling have not been adequately studied in T cells.
Given the predominant expression of Grap in lymphoid tissues, we have analyzed its role in T cell signaling. Grap associates prominently in activated Jurkat cells with p36/38, a major tyrosine kinase substrate in T cells. In contrast, low levels of Grap associate with Shc. These interactions are through its SH2 domain. Grap also interacts directly via its SH3 domains with the Ras activator, mammalian Sos, the GTPase dynamin, and the mitosis-related tyrosine kinase substrate, Sam68. Associations with p36/38, Shc, Sos, and dynamin are up-regulated upon T cell activation, whereas Sam68 is bound at similarly high levels in stimulated and unstimulated cells. PLC-␥1 and Fyn are also found in activated Grap signaling complexes, although these interactions may be indirect, through p36/38 and Sam68, respectively. Based on the critical functions associated with each of these effector molecules, Grap is likely to play a crucial role in lymphocyte activation.

MATERIALS AND METHODS
Northern Analyses-A DNA fragment encompassing the entire open reading frame of Grb2 (kindly provided in a pGEX vector by M. Moran, University of Toronto) and the full-length cDNA of Grap (supplied by D. Dunnington, SmithKline Beecham Pharmaceuticals) were excised from the appropriate vectors, labeled with [ 32 P]dATP to greater than 2 ϫ 10 9 cpm/g by the random hexamer method, and hybridized to Northern blots of multiple human tissue mRNAs as recommended by the manufacturer (Clontech). The membranes were washed at high stringency, and hybridized mRNA was detected using a PhosphorImager (Molecular Dynamics).
Bacterial Expression Vectors-BamHI and XhoI restriction sites were introduced by polymerase chain reaction into the Grap cDNA immediately upstream and downstream of the start and stop codons, respectively. The restricted DNA fragment was subcloned into the corresponding sites of the pGEX-4T-1 (Pharmacia Biotech Inc.) plasmid for expression as a fusion protein of glutathione S-transferase (GST) and full-length Grap. Polymerase chain reactions with mutant primers were used to introduce mutations into the Grb2 and Grap coding sequences to provide proteins with deficient binding, either to the SH2 domains (R86K within the FLRVES motif) or both SH3 domains (P49L/ P208L of Grap and P49L/P206L of Grb2). A fragment of mSos1 cDNA encoding residues 1135-1322 was excised with endonucleases BglII and NsiI and subcloned into the BamHI and HindIII sites of the plasmid pET30c (Novagen) for expression of histidine-tagged protein. The DNA sequence of each plasmid was verified.
Transient Expression of Grap in JMC-T Cells-An XhoI restriction site was introduced by polymerase chain reaction into the Grap cDNA immediately upstream of the start codon. Simultaneously, nucleotides encoding a Myc epitope tag (EQKLISEDL) were introduced between the site encoding Grap residue 217 and the stop codon, and a BamHI restriction site was introduced downstream from the stop codon. The XhoI/BamHI fragment was subcloned into the pSR␣neo vector (30) to generate pGrap-Myc. The pGrap-Myc (50 g/1.2 ϫ 10 7 cells) was introduced into Jurkat-derived JMC-T cells expressing the SV40 large T antigen (31) by electroporation. Cells were cultured 72 h before collection, anti-CD3 activation, and lysis.
Protein Precipitation with Antibodies, Fusion Proteins, and Immobilized Peptides-In typical pull-down experiments, lysates from 10 7 Jurkat cells were incubated with 4 -10 g of GST fusion protein bound to glutathione-agarose or immobilized peptide for 1.0 h at 4°C. For immunoprecipitations, lysates from 10 8 Jurkat cells were incubated with 50 g of anti-phosphotyrosine antibody immobilized on protein A-Sepharose. Alternatively, lysates from 10 7 transfected JMC-T cells were incubated with 6 g of immobilized anti-Myc antibody. All precip-itates were washed four times with lysis buffer E. Proteins were eluted with Laemmli sample buffer and separated by SDS-PAGE.
Immunoblots-Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membranes (Immobilon; Millipore Corp.) by electroblotting. Membranes were blocked with TBS-T buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 2% gelatin for Ͼ12 h at 22°C, reacted with optimal concentrations of specific antibodies in TBS-T containing 2.0% bovine serum albumin for 1.0 h at 22°C, and washed five times with TBS-T. Proteins were identified following incubation with horseradish peroxidase-conjugated goat antirabbit or goat anti-mouse Ig (Sigma) or streptavidin (KPL) by enhanced chemiluminescence, according to the manufacturer's protocol (DuPont).

Tissue Distribution of Grap mRNA and Protein Expression-
The entire cDNA for Grap and the coding sequence for Grb2 were radiolabeled and used to probe multiple-tissue Northern blots. A 2.1-kb transcript was detected for human Grap, compared with a 3.5 kb transcript for human Grb2 (Fig. 1). Highest levels of Grap mRNA were found in spleen and thymus, with an intermediate amount in peripheral blood leukocytes. Much less of the transcript was detected in alternative tissues, suggesting that Grap expression is predominant in lymphoid tissues. Apparent expression of Grap at low levels may reflect the presence of immune cells within these tissues. For comparison, equivalent amounts of a 3.5-kb human Grb2 transcript were found in all tissues ( Fig. 1), as previously reported (32).
Polyclonal antisera were generated against the C-terminal 19 residues of Grap to detect the expressed protein. The corresponding region provides useful anti-Grb2 antibodies (e.g. Santa Cruz), and the protein sequences diverge within this region. Antibodies were concentrated and nonreacting species were eliminated by immobilized Grap peptide affinity purification. GST-Grap was readily recognized by the anti-Grap antibodies in immunoblots. GST-Grb2 was recognized by commercial anti-Grb2 antibodies (Santa Cruz). There was no detectable cross-reaction (i.e. affinity-purified anti-Grap antibodies did not recognize Grb2, and vice versa) (Fig. 2). However, the Grap antibodies were not useful for immunoprecipitation, and gave high backgrounds when attempts were made to immunoblot SDS-PAGE-separated cell lysates. Therefore, endogenous Grap and Grb2 proteins were quantitatively extracted from cell lysates with immobilized phosphopeptide beads. The peptide sequence was derived from the region surrounding Tyr 177 of the breakpoint cluster region of BCR-ABL (Lys-Pro-Phe-Tyr(P)-Val-Asn-Val-Glu-Phe), which binds Grb2 and Grap with equally high affinity (Refs. 11 and 33 and this study; see below).
Proteins from lysates of several cell lines were precipitated using an excess of immobilized BCR-ABL peptide beads, separated by SDS-PAGE, and detected by immunoblotting. Cells of lymphocytic origin contained Grap, including normal peripheral T cells (phytohemagglutinin blasts) and cultured T (Jurkat) and B (Ramos, Raji, and Daudi) cell lineages (Fig. 2). In contrast, all cells tested, including erythroleukemia line K562 and Va2 fibroblasts, contained Grb2. In conjunction with the Northern analyses, these results suggest a restricted role for Grap in B and T lymphocyte signaling, compared with a more general role for Grb2 in the signaling pathways of many cells. Because the phosphopeptide beads extracted both Grap and Grb2 quantitatively, relative amounts of the proteins could be estimated by comparing blotting intensities with various fusion protein dilutions. Grap is present in B and T lymphocytes at about 5 ng/mg total protein. Grb2 is present in the same cells at approximately 10-fold higher concentration (Fig. 2).
p36/38 Is a Phosphoprotein Partner of Grap-Jurkat cells were activated by anti-CD3 cross-linking to determine whether Grap is involved in T cell signaling. Phosphoproteins were immunoprecipitated with anti-phosphotyrosine antibodies and Grap was detected by immunoblotting with the anti-Grap antibody. Receptor activation led to a substantial increase in the amount of Grap that co-precipitated with phosphoproteins ( Fig.  3), indicating that at endogenous levels of all cellular constituents, Grap functions in a signaling pathway downstream from the T cell receptor.
To begin to learn which cellular proteins interact with Grap, Jurkat cells were activated, and proteins were precipitated with GST-Grap and GST-Grb2 fusion proteins. Two prominent, tyrosine-phosphorylated, 36-and 38-kDa proteins co-precipitated with GST-Grap in lysates of stimulated but not unstimulated cells (Fig. 4). No other phosphoproteins were readily detected. In contrast, GST-Grb2 interacted with numerous tyrosine-phosphorylated proteins in Jurkat cell lysates, including 36-and 38-kDa proteins (Fig. 4). Previous studies suggest that in lymphocytes, Grb2 associates directly or indirectly with Shc (p52), PLC-␥1, p36/38, c-Cbl (p120), SLP-76 (p76), and ZAP-70 (p70). Several of these phosphoproteins account for additional bands present in GST-Grb2 precipitates. Whereas Grap and Grb2 precipitate equivalent amounts of p36/38, Grap does not associate substantially with the alternative Grb2-bound phosphoproteins. Based on the time course of phosphorylation (not shown) and relative mobilities by SDS-PAGE, the Grap-associated p36/38 appears to be the same protein(s) known to interact with Grb2, PLC-␥1, and PI 3-kinase p85 in activated T cells (31, 34 -36).
p36/38 Binds Grap in Cells-Grap was expressed in Jurkat cells in order to learn more about its role in T cell receptor signaling. An anti-Myc antibody epitope was fused to the C terminus of Grap for immunoidentification. The Myc-tagged Grap was expressed transiently in an SV40 large T antigenexpressing Jurkat cell line, JMC-T (31), and after 72 h, cells were stimulated with anti-CD3 antibodies. Grap-Myc protein was immunoprecipitated from lysates using anti-Myc antibodies. Lysates from unstimulated and stimulated cells contained equivalent amounts of the protein, as detected by immunoblotting with anti-Grap antibodies (Fig. 5A). Associated proteins Membranes containing mRNA from multiple human tissues were probed as described under "Results." The 2.1-kb Grap transcript is expressed predominantly in spleen and thymus, with less in peripheral blood leukocytes. The 3.5-kb Grb2 is present in all tissues.

FIG. 2. Grap protein is present in T and B lymphocytes.
Proteins were precipitated from the indicated cell lysates (800 g of total protein from 4 -8 ϫ 10 6 cells) using immobilized BCR-pY177 peptide beads. Proteins were separated by SDS-PAGE and detected by immunoblotting with affinity-purified anti-Grap (top) or anti-Grb2 (bottom) antibodies. Known amounts of recombinant protein standards were analyzed under identical conditions for quantitative comparison (right).
were detected in anti-phosphotyrosine immunoblots (Fig. 5B). Only p36/38 co-immunoprecipitated with Grap-Myc in stimulated but not unstimulated cells. Although it is not abundant in cell lysates, p36/38 is concentrated in immunoprecipitates. We conclude that the Grap-p36/38 association is of high affinity and that p36/38 associates preferentially with Grap in activated T lymphocytes.
Grap Binds p36/38 with Its SH2 Domain-p36/38 could bind Grap through its SH2 domain or SH3 domains. Blocking mutations in GST-Grap fusion proteins were used to discriminate between these possibilities. R86K mutations within the critical FLVRES motifs were introduced into GST-Grb2 and GST-Grap to ablate SH2 domain binding. Interactions with the N-terminal SH3 domains of GST-Grb2 and GST-Grap were blocked with P49L mutations, while the C-terminal SH3 domains of GST-Grb2 and GST-Grap were altered with P206L and P208L mutations, respectively. Previous studies confirmed that these mutations block binding to the SH2 and SH3 domains of Grb2 (20). Mutation in the Grap SH2 domain blocked the interaction with p36/38 (Fig. 4). In contrast, mutation of both SH3 domains had no effect. Therefore, the Grap SH2 domain mediates its interaction with p36/38, similar to previous findings with Grb2 (31,35,36).
Peptide Binding Specificity of the Grap SH2 Domain-Grb2 interacts with numerous phosphoproteins in activated T cells, while Grap binds primarily to p36/38 (Fig. 4). Phosphopeptide binding specificities of Grb2 and Grap were compared in order to test whether a restricted SH2 domain binding specificity might account for the limited number of proteins interacting with Grap. Immobilized GST-Grb2 and GST-Grap were used to precipitate p36/38 in the presence of varying concentrations of a Shc-derived phosphopeptide (Fig. 6, A and B). The Shc peptide corresponds to the sequence surrounding Tyr 317 (Pro-Ser-Tyr(P)-Val-Asn-Val-Gln-Asn-Leu), a major Shc phosphorylation site and recognition motif for Grb2. This peptide blocks interactions with both proteins at equally low concentrations (ID 50 ϳ1 M). Findings are similar with isolated peptide binding assays. ID 50 values for Grb2 and Grap binding to the Shc-pY317 peptide are 4.4 Ϯ 3.2 M and 1.6 Ϯ 0.4 M, respectively (Fig. 6, C and D). The oncoprotein BCR-ABL binds Grb2 at Tyr 177 within a similar motif (33). A BCR pY177 peptide (Lys-Pro-Phe-Tyr(P)-Val-Asn-Val-Glu-Phe) binds Grb2 and Grap SH2 domains with equivalent high affinity (ID 50 ϭ 2.1 Ϯ 0.4 M and 0.8 Ϯ 0.3 M, respectively). Asparagine at the Tyr(P) ϩ 2 position is critical for binding to both proteins, since substitution with alanine substantially reduced affinity (ID 50 Ͼ300 in both cases; Fig. 6, C and D), and tyrosine phosphorylation is necessary as well (data not shown). These results demonstrate that Grap and Grb2 SH2 domains have similar peptide binding specificities. The p36/38 bands could correspond to Lnk, a recently cloned, lymphoid tissue-restricted SH2 domain phosphoprotein with a predicted molecular mass of 34 kDa (37). In vitro, Lnk isolated from lymphocytes co-precipitates with Grb2, PLC-␥1, and PI 3-kinase p85. It was reported that a single peptide (Cys-His-Leu-Arg-Ala-Ile-Asp-Asn-Gln-Tyr(P)-Thr-Pro-Leu-Ser-Gln-Leu) derived from Lnk blocks this interaction by binding SH2 domains of all three proteins. This would be unusual in that the peptide lacks appropriate SH2 domain binding motifs. We show that a related peptide (Asp-Asn-Gln-Tyr(P)-Thr-Pro-Leu--Ser-Gln-Leu) does not bind Grb2 or Grap directly (Fig. 6, A  and B). Since the published protein sequence of Lnk has no consensus motif for Grb2 binding, the reported association between Lnk and Grb2 may be mediated indirectly through another protein.
Additional Grap Partners in T Cells: Shc, mSos, Sam68, and Dynamin-Since Grap and Grb2 SH2 domains have similar binding specificities, one would expect that Grap might coprecipitate with Shc, a major Grb2 SH2 domain partner in many cells. Shc is present in JMC-T cells, although the -fold increase in its phosphorylation is quite low (Fig. 5). In an additional experiment, Grap-Myc was immunoprecipitated from transfected JMC-T cells, and associated Shc was identified by immunoblotting (Fig. 7). T cell receptor activation does induce an association between Shc and Grap, although less than 2% of cellular Shc associates with Grap-Myc in these experiments. This is similar to the situation for Grb2 in Jurkat cells, where Shc association appears to be transient and of low stoichiometry (31,35,36).
Additional studies aimed at identifying potential SH3 domain partners of Grap. Two phosphoproteins interact with the SH3 domains of Grb2 in lymphocytes: the SH2 domain protein, SLP-76 (38,39), and the protooncoprotein c-Cbl (19 -21). SLP-76 and c-Cbl are usually detected in anti-phosphotyrosine immunoblots due to abundant phosphorylation. Although appropriate 76-and 120-kDa proteins were precipitated from activated Jurkat cell lysates by GST-Grb2, only p36/38 coprecipitated with GST-Grap (Fig. 4). These results suggest that SLP-76 and c-Cbl may not be major partners for Grap. Additional SH3 domain-associated proteins may not be detected as phosphoproteins. While mSos is the best characterized of the Grb2 SH3 domain-binding proteins, the p85 subunit of PI 3-kinase (40 -42), dynamin (17,18), Vav (16), c-Abl (43), and Sam68 (22,44) also have proline-based motifs that may bind Grb2 SH3 domains. Grap-Myc was immunoprecipitated from transfected JMC-T cells, and the presence of certain candidates was probed by specific immunoblotting. mSos co-immunoprecipitates with Grap-Myc (Fig. 8). Furthermore, the amount of co-precipitated mSos increases with T cell receptor activation, analogous to the increase in Grb2/Sos association upon T cell activation (23,24). Sam68 co-immunoprecipitates with Grap as well. However, Grap and Sam68 exhibit constitutive association, since there is little change in the amounts of co-precipitated proteins upon T cell activation. Dynamin also co-immunoprecipitates with Grap. As seen with Sos, the association between Grap and dynamin appears to be regulated by T cell activation. Previously, we showed that PI 3-kinase p85 co-precipitates with Grb2 and p36/38 in activated Jurkat cells (31). We have not detected p85 or c-Cbl in Grap-Myc immunoprecipitates (Fig. 8).
Sos, Sam68, and Dynamin Bind Grap via Its SH3 Domains-Since mSos is not tyrosine-phosphorylated and is known to bind the SH3 domains of Grb2, it is likely that it also binds the SH3 domains of Grap. Indeed, the C-terminal tail of Sos that contains four proline-rich motifs for SH3 domain recognition (BSos, Sos1 residues 1135-1322) binds Grap directly and with high affinity (Fig. 9B). Constitutive association between Grap-Myc and Sam68 suggests an SH3 domain-mediated interaction, as well (Fig. 9A). This association is decreased dramatically at low Sos tail concentrations (30 nM), indicating that Sam68 binds Grap through its SH3 domains. The association between Grap and dynamin is inhibited by the Sos tail as well, suggesting that this interaction is also mediated by Grap SH3 domain binding to proline-rich motifs in dynamin. Like Sos, dynamin is known to bind the SH3 domains of Grb2 (17,45). However, there is a suggestion in the literature that Sam68 might bind Grb2 via its SH2 domain (22) (this paper describes binding between Grb2 and GTPase-activating protein-associated p62, but, because the identity of these proteins has been confused, this is probably the protein referred to here as Sam68 (44)). To investigate further the mechanism of Grap and Grb2 binding with Sam68, mutated forms of the proteins were used in pull down experiments (Fig. 10). The Grb2 or Grap SH2 domain mutations do not prevent precipitation of Sam68 from Jurkat cell lysates, so neither protein binds Sam68 through its SH2 domain. In contrast, SH3 domain mutations resulted in much less precipitated Sam68. We conclude that Grap and Grb2 proteins bind Sam68 primarily via their SH3 domains and not their SH2 domains.
Additional Proteins in Grap Signaling Complexes: PLC-␥1 and Fyn-Grb2 and PLC-␥1 appear to interact with one another in activated Jurkat cells, either directly or indirectly (22,31,35). We have confirmed these findings using immobilized GST-Grb2 for protein precipitation and anti-PLC-␥1 antibodies for immunodetection (Fig. 11A). PLC-␥1 also precipitates with GST-Grap under these conditions, although less PLC-␥1 protein was precipitated by an equivalent amount of fusion protein. Additional studies with mutated proteins demonstrate that the Grap SH2 domain, and not its SH3 domains, is necessary for the effect. Weiss and colleagues (35) have suggested that Grb2 and PLC-␥1 both bind p36/38 rather than one another and that this is the basis for co-precipitation of Grb2 and PLC-␥1 (35). Our results with Grap and PLC-␥1 are consistent with a similar mechanism, particularly since an abundance of p36/38 precipitates with Grap, the Grap SH2 domain mediates both interactions, and PLC-␥1 has no Grb2/Grap SH2 domain binding site.
Sam68 interacts with and is phosphorylated by c-Src and Fyn (22,46,47). Having now identified Sam68 in T cells and shown that it associates with Grap and Grb2, it seemed possible that Fyn or perhaps Lck might be present in Grap signaling complexes. This was tested by immunoprecipitating Grap-Myc and immunoblotting for Fyn and Lck. An abundance of Fyn co-precipitates with Grap, and the amount increases with T cell activation (Fig. 11B). Association with Lck is less obvious in our experiments, but a small increase may occur with activation. Therefore, in T cells, Fyn (and possibly Lck) is recruited to activated Grap signaling complexes. Although we have not eliminated alternative possibilities, it is most likely that Grap and Fyn both bind proline-rich sites of Sam68 via their SH3 domains. Notably, in addition to a region that binds RNA, Sam68 has several potential sites for SH3 domain binding. Taken together, these data indicate that Grap might play a central role in the formation of signaling complexes in activated T cells. Grap binds p36/38 and Sam68 via its SH2 and SH3 domains, respectively, while p36/38 binds PLC-␥1 and Sam68 binds Fyn. Therefore, the potential exists for the formation of pentameric signaling complexes surrounding Grap. Further studies are needed to test these possibilities.

DISCUSSION
Grap is a newly identified adaptor protein composed entirely of two SH3 domains that flank a single SH2 domain. Our clone has an identical sequence to the one recently published (8). Grb2 is the only other known protein with this domain architecture (32,48). Several splice variants of Grb2 have been reported. All are encoded by the same gene, including one with an altered SH2 domain and apoptotic properties (49) and others with modified C-terminal SH3 domains (50). Grap arises from a distinct genetic locus. Overall, Grap and Grb2 share 58% protein sequence identity. This is distributed more or less evenly throughout the three domains.
The binding specificities of the Grb2 and Grap SH2 and SH3 domains are related. Both proteins bind p36/38 and Shc using their SH2 domains. The peptide binding specificities of Grap and Grb2 SH2 domains are correspondingly similar (both require asparagine at the Tyr(P) ϩ 2 position) and are determined in part by a tryptophan in the EF loops of the SH2 domains of both proteins (51). Since the sequence of the Grb2 EF loop is WVV, while that of Grap is WEE, it is possible, for example, to unmask differences in binding specificity using peptides with acidic residues at positions C-terminal to asparagine. 3 We do not know whether this finding has physiological relevance. Peptide binding specificities for Grap SH3 domains were not determined. However, residues lining the peptide binding pockets of Grb2 SH3 domains are known (52)(53)(54). Many corresponding residues of Grap are identical, suggesting similar binding specificities. Consistent with this suggestion, we have observed a similar subset of proteins binding the SH3 domains of Grap and Grb2.
Northern analyses show restricted expression of Grap in spleen, thymus, and circulating leukocytes, implying a function for Grap in lymphocytes. Its SH3-SH2-SH3 domain composition suggested that this function is in signal transduction. We show that the Grap protein is present in lymphocytes and acts downstream from antigen receptors (Fig. 12). In Jurkat cells, Grap associates avidly with p36/38 and to a lesser degree with Shc. These interactions require T cell receptor activation, phosphorylation of p36/38 or Shc, and a functional Grap SH2 domain. Grap also associates with mSos, the guanine nucleotide exchange factor for Ras, with Sam68, an RNA-binding protein potentially involved in cell division, and with dynamin, a guanosine triphosphatase involved in protein trafficking. Sos, Sam68, and dynamin all bind Grap via its SH3 domains. The binding of Sos and dynamin are up-regulated by T cell activation, whereas Sam68 association is constitutive. These findings allow tentative placement of Grap into the complex network of interactive proteins that propagate signals downstream from activated T cell receptors. T cell receptor-associated tyrosine kinases, including Lck, Fyn, and ZAP-70, phosphorylate several cellular proteins upon receptor activation. Presumably one or more of these kinases phosphorylates p36/38, and this leads to Grap association. Since p36/38 (35,36) and the kinases are associated with membrane components, phosphorylation must occur at or near the plasma membrane. It is likely that the SH3 domain-binding proteins are integrated individually into p36/38-Grap-mSos, p36/38-Grap-Sam68, and p36/38-Grap-dynamin complexes. While PLC-␥1 and Fyn are also present in Grap signaling complexes, we do not think that these are direct interactions. PLC-␥1 has been reported to bind p36/38 (35) and Sam68 (22) in T cells, and Fyn binds Sam68 directly (22,47). Therefore, PLC-␥1 might be present in p36/38-Grap complexes with mSos, Sam68, and dynamin. Fyn may be found in p36/38-Grap-Sam68 complexes. Additional studies are needed to determine which of these ternary, quaternary, or higher order complexes actually form in activated lymphocytes.
Biological roles of Grap signaling complexes can be considered based on the biochemical functions of the SH3 domainbound effector proteins and potential links to downstream cellular effects. IL-2 production provides a paradigm for studying immediate effects of T cell receptor activation on the nucleus (1,3). Changes in IL-2 gene expression are influenced by intracellular calcium and the activation state of Ras. Significant headway has been made mapping molecular components of the Ca 2ϩ and Ras pathways, which converge at the level of IL-2 gene transcription through synergistic effects on components of the nuclear factor of activated T cells (NF-AT) (55,56). Activated Ras associates with Raf family serine/threonine kinases, leading sequentially to the phosphorylation and activation of MEK and the mitogen-activated protein kinases, ERK1 and ERK2. The mitogen-activated protein kinases can translocate to the nucleus and regulate nuclear components of NF-AT (57,58). Since Grap-Sos likely functions much like Grb2-Sos as a guanyl nucleotide exchange factor for Ras, Grap may be an important link between the activated T cell receptor and the Ras/Raf/ MEK/ERK cascade.
Calcium effects on NF-AT induction are likely through calcineurin, a two-subunit serine/threonine phosphatase whose activity is potently inhibited by cyclosporin-cyclophilin and FK506-FKBP complexes (55,56). The catalytic subunit binds calmodulin, and the regulatory subunit binds Ca 2ϩ . Cal-3 G. Wolf and S. E. Shoelson, unpublished observations. Grap-dynamin complexes may be involved in T cell receptor trafficking. Dynamin is a GTPase that contains a pleckstrin homology domain and a proline-rich region that binds SH3 domain proteins (17). These latter two regions may participate in the subcellular targeting of dynamin. As is true for many signaling proteins, much of what is known about dynamin function was originally inferred from studies in lower organisms. Dynamin is homologous with the Drosophila shibire gene product (60) and yeast Vps1p and Dnm1p proteins. Flies carrying the temperature-sensitive shibire allele exhibit a defect in protein endocytosis (61), and Vps1p and Dnm1p are involved in protein trafficking to the yeast vacuole (62,63). In mammalian cells that overexpress GTP binding-defective mutants of dynamin, receptors sort to clathrin-coated pits, but the pits fail to constrict and invaginate (64,65). In vitro dynamin assembles into stacked rings resembling the electron-dense collars that accumulate at nerve terminals of shibire flies (66,67). GTP hydrolysis correlates with a change in ring structure that may be associated with vesicle fusion. Grb2-dynamin complexes present in numerous cell types may play a role in receptor internalization (17,18,45). Since T cell activation leads to T cell receptor endocytosis and down-regulation (68,69), Grapdynamin and Grb2-dynamin complexes may have important functions in these events.
Grap and Grb2 also interact in T cells with Sam68, a protein with homology to the heterogeneous nuclear ribonucleoproteins (44,70). Sam68 is phosphorylated by Src (or Src-like kinases) during G 1 of the cellular interphase (22,46,47). It appears that the Src SH3 domain has a role in this, by binding one the putative proline-rich binding motifs in Sam68. Selective phosphorylation by Src kinase during G 1 implies a role in cell division, although cellular functions of Sam68 and biochemical effects of phosphorylation are unknown. Nevertheless, Sam68 binds RNA like a heterogeneous nuclear ribonucleoprotein and contains a KH domain common to this protein class. By extrap-olation, its function might be in the editing, stabilization, or metabolism of RNA. Since Sam68 may be a nuclear protein, Src and Sam68 may gain access to one another during breakdown of the nuclear envelope at the beginning of mitosis (71). In lymphocytes, Fyn and possibly Lck may take over functions of Src, to interact with and phosphorylate Sam68 during lymphocyte proliferation. Therefore, Grap-Sam68 and Grb2-Sam68 complexes may have roles in the entry of T lymphocytes into the cell cycle as a response to antigen receptor triggering.
Given its prominent expression in the thymus, Grap may function in lymphocyte differentiation and development. In this regard, Grap may have potential roles downstream from activated cytokine receptors that are distinct from its functions in T cell receptor signaling. Although lymphocytes contain numerous cytokine receptors, we have not tested this possibility. However, since Grap reportedly binds the stem cell factor receptor (c-Kit) isolated from MO7e cells and the erythropoietin receptor from the same cells that overexpress the receptor (8), a related function in lymphocytes is possible. These are obvious areas for future investigation.
It is important to consider why lymphocytes should have an auxiliary pathway, containing an extra substrate (p36/38) and linker protein (Grap), compared with most alternative cells, and why both pathways are activated by the T cell receptor. Many concepts in tyrosine kinase signal transduction, including the Grb2 paradigm, derive from studies with growth factor receptors. Growth factors and related hormones circulate freely or as dimers in solution and bind their receptor with high affinity and specificity. Typically one of these ligands (or ligand pairs) binds one receptor or receptor dimer. In this fashion, each ligand evokes a signaling response. Once many receptors have been occupied, a threshold is reached and the cell responds. Mechanisms for T cell activation are different. T cell receptors bind peptide/major histocompatibility complexes on the surface of antigen-presenting cells. These interactions are of low affinity and exhibit rapid dissociation, which enables each peptide/major histocompatibility complex to serially engage many T cell receptors (68). Since each ligand activates numerous receptors, a very few peptide/major histocompatibility complexes can trigger the lymphocyte response. Although certain functions of Grap and Grb2 must differ (e.g. Figs. 3 and 4), several effectors associate with both, including Sos, dynamin, Sam68, and (indirectly) PLC-␥1. We speculate that the additive effects of Grap and Grb2 and the numerous associated effectors may help to amplify the unique T cell receptor signal inside the cell.