(Received for publication, March 5, 1996; and in revised form, March 22, 1996)
From the
A human cytoplasmic signaling protein has been cloned that possesses the same structural arrangement of SH3-SH2-SH3 domains as Grb2. This protein is designated Grap for Grb2-related adaptor protein. The single 2.3-kilobase (kb) grap transcript was expressed predominantly in thymus and spleen, while the ubiquitously expressed grb2 gene produced two mRNA species of 3.8 and 1.5 kb. Grap and Grb2 consist of 217 amino acids and share 59% amino acid sequence identity, with highest homology in the N-terminal SH3 domain. The GrapSH2 domain interacts with ligand-activated receptors for stem cell factor (c-kit) and erythropoietin (EpoR). Grap also forms a stable complex with the Bcr-Abl oncoprotein via its SH2 domain in K562 cells. Furthermore, Grap is associated with a Ras guanine nucleotide exchange factor mSos1, primarily through its N-terminal SH3 domain. These results show that a family of Grb2-like proteins exist and couple signals from receptor and cytoplasmic tyrosine kinases to the Ras signaling pathway.
Grb2, the mammalian homologue of Caenorhabditis elegans Sem-5 and Drosophila Drk, is composed of a single Src
homology 2 (SH2) ()domain flanked by two SH3
domains(1, 2, 3, 4, 5) .
Genetic analyses of sem-5 in C. elegans and of drk in Drosophila and biochemical dissection of Grb2
in mammalian cells revealed a highly conserved pathway governing the
stimulation of Ras proteins by receptor protein-tyrosine kinases
(PTKs)(4, 5, 6, 7, 8, 9, 10, 11) .
Sem-5 functions in a signaling pathway that involves the
Let-23/receptor tyrosine kinase and Let-60/Ras(1) . Similarly, Drosophila Drk was found to be essential for signaling by
Sevenless receptor PTK to Son of Sevenless (Sos), an activator of Ras
guanine nucleotide exchange, in the development of R7 photoreceptors in Drosophila eye(4, 5) . Mammalian Grb2
physically interacts with a number of autophosphorylated receptor PTKs
through its SH2 domain, while its SH3 domains associate with the
proline-rich motifs in the C-terminal tail of
mSos1(6, 7, 8, 9, 10, 11) .
These interactions apparently mediate the growth factor stimulation of
Ras proteins by promoting the conversion of inactive GDP-bound Ras to
the active GTP-bound form(8, 9) . Human grb2 and Drosophila drk genes specifically rescue the defects
of sem-5 mutations in C. elegans, indicating a high
degree of conservation of this signaling pathway throughout evolution (12) . In addition to direct interaction with receptor PTKs,
Grb2 binds through its SH2 domain to several cytoplasmic proteins,
including Shc, Syp, and Bcr-Abl, in a tyrosine
phosphorylation-dependent
manner(13, 14, 15) .
In this paper, we identify a novel human signaling protein Grap that has the Grb2-like architecture (SH3-SH2-SH3 domains) and is highly expressed in thymus and spleen. Our experiments suggest that Grap and Grb2 are members of the same adaptor protein family with overlapping yet distinct expression patterns and possibly functions.
Figure 1: Deduced amino acid sequence of Grap. The deduced amino acid sequence of Grap is shown in an alignment with Grb2. Identical and conserved amino acid residues are printed in bold capital letters, and the other amino acid residues are shown in small letters. The SH2 domain is indicated with a solid line, and the two SH3 domains are pointed out with a dashed line.
Figure 2: Grap is predominantly expressed in thymus and spleen. Northern blotting was performed on two human multiple tissue Northern blots as described in the text. Lanes 1-16, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocyte.
By immunizing rabbits with a purified GST-fusion protein containing the GrapSH2 domain, we have successfully produced specific antiserum against Grap that works very well in immunoprecipitation and immunoblotting. The antibody specifically recognized a 27-kDa polypeptide in human cells, as compared to 23-kDa Grb2 (Fig. 3). It should be noted that most commercially available anti-Grb2 antibodies cross-react with Grap (Fig. 3). Conversely, a little cross-reaction of our anti-Grap antiserum with Grb2 was observed only after a long exposure of the immunoblot filters. The slower migration of Grap than Grb2 distinguishes the two proteins despite their identical predicted size. Highest amounts of Grap protein were in mouse thymus and spleen; lower levels were detected in lung, liver, kidney, heart, and brain (data not shown). These results suggest that Grap is predominantly expressed in hematopoietic and lymphocytic cells, and, indeed, Grap was easily detected in MO7e, MO7eR, TF1, and K562 cells by immunoblot analysis (Fig. 3).
Figure 3: Immunoblot analyses of Grap and Grb2. Equal amounts of K562 (lane 1), MO7e (lane 2), MO7eR (lane 3), and TF1 (lane 4) cell lysates (40 µg/lane) were loaded on SDS-PAGE and subjected to immunoblotting with anti-Grap and anti-Grb2 antibodies, respectively.
Figure 4: Protein-protein interactions mediated by the GrapSH2 and -SH3 domains. A and B, GrapSH2 binds to activated c-kit and EpoR. Serum-starved MO7e cells were stimulated with 100 ng/ml stem cell factor for 5 min, and MO7eR cells were treated with 80 units/ml Epo for 5 min, respectively. Control and factor-stimulated cell lysates containing 1 mg of total protein were mixed with 5 µg of purified GST-fusion proteins of GrapSH2 or Grb2SH2 immobilized on glutathione-Sepharose beads. The precipitates were immunoblotted with monoclonal anti-PY antibodies (4G10 + PY20). The positions of c-kit and EpoR were indicated by an arrow. After the anti-PY blotting, the filters were reprobed with anti-c-kit and anti-EpoR, and the indicated phosphoproteins were confirmed as phosphorylated c-kit and EpoR. C, binding of GrapSH2 to Bcr-Abl. K562 cell lysates containing 1 mg of total proteins were immunoprecipitated by 2 µl of anti-Grap antiserum or 1 µg of anti-Grb2 antibodies, or by 5 µg of purified GST-fusion proteins of GrapSH2 or Grb2SH2. The precipitates were immunoblotted with anti-c-Abl. The K562 cell lysate (40 µg) was also loaded on one lane. D, association of Grap with mSos1 via its SH3N domain. The TF1 cell lysates (1 mg of protein) were precipitated with antibodies against Grap or Grb2, or purified GST-fusion proteins containing the full-length Grap, or the SH2, SH3N, and SH3C domains of Grap. The precipitated proteins were immunoblotted with anti-mSos1. On the left lane was loaded 40 µg of TF1 cell lysate.
Bcr-Abl fusion protein is an up-regulated PTK implicated in the pathogenesis of chronic myelogenous leukemia and acute lymphoblastic leukemia. It has been demonstrated that Bcr-Abl forms a physical complex with Syp and Grb2(15, 21, 22) , and binding to Grb2 is required for activation of Ras proteins and the transforming activity of Bcr-Abl (15) . To detect the interaction between Grap and Bcr-Abl, Grap was immunoprecipitated from a K562 cell lysate and the precipitates were blotted with anti-c-Abl. As shown in Fig. 4C, Grap co-precipitated from K562 cells with Bcr-Abl and purified GST-GrapSH2 bound Bcr-Abl in vitro. These results indicate that Grap, like Grb2, forms a stable complex through its SH2 domain with the Bcr-Abl oncoprotein in leukemia cells.
In summary, we have described a human cytoplasmic signaling protein that shares structural and functional similarity with Grb2. Both proteins contain a central SH2 domain sandwiched by two SH3 domains. The SH2 domains directly recognize phosphotyrosine-containing sites on activated receptor PTKs and cytoplasmic proteins. Its binding specificity is often dictated by the three amino acids C-terminal to phosphotyrosine (the +1- to +3-positions). A Trp residue in the EF1 position of the +3 binding pocket in Grb2SH2 was critical in defining specificity(24) . In this regard, it is of great interest that the GrapSH2 has a Trp at the same position (Fig. 1) in addition to its high homology with Grb2SH2. Results from Fig. 4, demonstrate that the SH2 domains of Grap and Grb2 bind to tyrosine-phosphorylated c-kit, EpoR, and Bcr-Abl. We also observed that the two SH2 domains bind to a similar group of phosphoproteins in v-src transformed cells in vitro (data not shown). Further experiments are needed to determine whether the two SH2 domains recognize and compete for the same phosphotyrosine motifs in these activated receptors in vivo and select identical phosphopeptide sequences in vitro(25) .
Grap appears to form a physical complex
with mSos1 through its SH3N domain (Fig. 4D). This
agrees with previous observations that the SH3N domain is primarily
involved in association of Drk and Grb2 to Sos and mSos1,
respectively(7, 23) . Notably, higher similarities
between these proteins are often seen in the SH3N region, and the SH3C
domains appear less conserved. It seems reasonable that the SH3C domain
of Grap may couple to other signaling pathways by binding yet-to-be
identified proteins. For example, Grap may couple to other guanine
nucleotide exchange factors for Ras, Rac, or Rho. The SH3 domains of
Grb2 are also reported to associate with c-Cbl(26) , a p75
protein(27) , the p85 subunit of the phosphatidylinositol
3-kinase (28) , -dystroglycan(29) , and
Vav(30) , although the physiological significance of these
interactions has not been documented. It will be worthwhile to examine
whether the GrapSH3 domains form complexes with these proteins.
Finally, generation of Grb2 and Grap knockout mice will give important
clues to their overlapping yet distinct functions in vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U52518 [GenBank](BankIt41164).