(Received for publication, September 20, 1995; and in revised form, February 9, 1996)
From the
Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P. P., and Dixit, V. M.(1995) J. Biol. Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell.
The ret protooncogene encodes a transmembrane receptor
protein-tyrosine kinase (RPTK). ()The extracellular domain
of Ret is unrelated to any other subfamily of RPTKs and contains
multiple cadherin-like repeats and a cysteine-rich region(1) .
Ret plays a critical role in renal development as well as that of
endocrine organs derived from the neural crest including the adrenal
medulla and the thyroid gland. Mutations in the ret protooncogene have been shown to result in a variety of disorders
including Hirschsprung's disease, multiple endocrine neoplasias
2A and 2B, and familial medullary thyroid
carcinoma(2, 3, 4, 5, 6, 7, 8) .
More recently, ret has been shown to be a dominant acting
oncogene capable of germline transmission. Knockout studies have
demonstrated that mice lacking ret have renal agenesis or
severe dysgenesis and lack enteric neurons(9) . Despite the
fact that the ligand for Ret has yet to be identified, several
signaling molecules have been identified that interact with Ret
including PLC
, rasGAP, Shc, Grb2, and
paxillin(10, 11, 12, 13) .
In an effort to identify additional signaling intermediates capable of transducing signals originating from Ret, we had previously used the cytoplasmic domain of Ret as bait in a yeast two-hybrid screen. This resulted in the identification of Grb10 as a molecule that specifically bound the activated form of Ret(14) . Grb10 is a recently described SH2 domain containing adapter protein that belongs to an emerging family of adapter proteins. This family includes the previously described protein Grb7 and is characterized by a carboxyl-terminal SH2 domain, a central domain, and a proline-rich region(15, 16, 17) . The central domain (also termed GM domain), is similar to the Caenorhabditis elegans gene mig-10 that is crucial for the migration of a subset of neuronal cells and contains a pleckstrin homology domain. The central domains of Grb7 and Grb10 are 54% identical to each other(16) . Since Grb10 interacted with the Ret receptor protein-tyrosine kinase in an activation dependent manner, we asked if Grb7 might also associate with Ret in a similar fashion. This reasoning was based on the fact that since the SH2 domains of Grb7 and Grb10 are 64% identical to each other, it was possible that they engaged similar receptor protein-tyrosine kinases. Grb7 has previously been shown to associate with HER2/neu, a receptor closely related to the epidermal growth factor receptor (EGFR)(18) . Activation of the HER2 receptor led to association and tyrosine phosphorylation of Grb7 in cells overexpressing the chimeric EGFR/HER2 receptor(18) . In this study we demonstrate that Grb7 directly associates with Ret via its SH2 domain, and unlike Grb10, it undergoes tyrosine phosphorylated in response to Ret activation.
For in vitro competition assays, 293 cells were transfected with EGFR/Ret, the EGFR/ret receptor immunoprecipitated using anti-EGFR antibody (Upstate Biotechnology, Inc.), and the immunoprecipitates phosphorylated in vitro in the presence of 45 µM ATP. The samples were then divided into equal parts, and preincubated with the indicated amounts of soluble His-tagged SH2 domains for 30 min at room temperature. The samples were then incubated with 5 µg of soluble GST fusion of SH2 domains for 1 h at room temperature followed by washing and SDS-PAGE. The gels were transferred onto nitrocellulose, and immunoblotted with an anti-GST monoclonal antibody (Santa Cruz Biotechnology, Inc., CA) to detect the bound SH2 domain GST fusion protein.
For in vivo binding studies using SKBR-3 cells, these cells were transiently transfected with EGFR/Ret using the calcium phosphate method. 48 h after transfection, cells were starved in 1% BSA overnight and left untreated or treated with EGF followed by lysis as described above. The lysates were immunoprecipitated with anti-Grb7 (N-20) (Santa Cruz Biotechnology, Inc.) followed by Western blot analysis with anti-phosphotyrosine as described below.
Given the importance of Ret in mammalian development and
oncogenesis, characterization of molecules that participate in its
signaling is of paramount importance. Since the ligand for Ret is
unidentified, a strategy was utilized to exploit the finding that the
cytoplasmic domain of Ret mimics the activated form of the receptor as
it possesses constitutive tyrosine kinase activity that results in
autophosphorylation. The phosphorylated tyrosines in turn, should be
capable of engaging downstream signaling molecules. Using the Ret
cytoplasmic domain fused to the GAL4 DNA binding domain as bait in the
yeast two-hybrid screen, we had previously identified Grb10, an
SH2-containing adapter protein, as a downstream target of
Ret(14) . Grb10 belongs to an emerging family of SH2 containing
adapter proteins with Grb7 as the prototypical
member(15, 16, 17) . Both of these proteins
contain a single SH2 domain at the COOH terminus, a central region
containing a pleckstrin homology domain, and a proline-rich region at
the NH terminus.
Grb7 was initially identified by
screening a gt11 expression library with a phosphorylated
P-labeled carboxyl-terminal segment of EGFR(17) .
Despite this, it has not been shown to bind to EGFR in vivo.
It does, however, associate with HER2/neu, a receptor closely
related to the EGF receptor (18) . As a result of this binding,
Grb7 is tyrosine-phosphorylated which may be sufficient to initiate
further downstream signaling. Since the SH2 domain of Grb7 is 64%
identical to Grb10, we asked whether Grb7 could associate with Ret in a
manner similar to Grb10. To directly ascertain if the SH2 domain of
Grb7 could bind the cytoplasmic domain of Ret, human embryonic kidney
293 cells were transfected with an expression vector encoding
hemagglutinin-tagged Ret cytoplasmic domain (HA-Ret). The lysates were
incubated with Grb7 SH2-GST, NH
- and COOH-terminal SH2
domains of PLC
, or GST alone. As shown in Fig. 1, the SH2
domain of Grb7 bound the Ret cytoplasmic domain as did the
COOH-terminal SH2 domain of PLC
. The COOH-terminal SH2 domain of
PLC
has been previously shown to bind Ret, and served as an
important positive control. This interaction was specific since the
NH
-terminal SH2 domain of PLC
or GST by itself did not
bind.
Figure 1: SH2 domain of Grb7 specifically associates with Ret in vitro. 293 cells were transfected with an expression vector containing the cytoplasmic domain of Ret that was HA epitope-tagged (HA-Ret). Cells were then lysed and the lysates incubated with the indicated GST fusion proteins. Bound material was dissociated by boiling in sample buffer, subjected to SDS electrophoresis, transferred onto nitrocellulose, and immunoblotted with anti-HA antibody.
To confirm that the binding was due to the ability of the Ret kinase to autophosphorylate itself, a kinase-defective mutant of Ret (K758M) was generated by replacing lysine in the ATP binding pocket of the kinase domain with methionine(20) . 293 cells were transfected either with wild type or kinase-defective Ret receptor, and the receptors were immunoprecipitated, phosphorylated in vitro, and incubated with a soluble GST fusion protein of the SH2 domain of Grb7. Although Grb7 efficiently bound the wild type receptor, it showed no detectable binding to the kinase defective mutant of Ret (data not shown).
Since the ligand for Ret is not known, it is difficult to activate the native receptor in vivo and determine if the tyrosine-phosphorylated cytoplasmic domain has engaged Grb7. To circumvent this problem, we have used an EGFR/Ret chimera in which the extracellular domain of EGFR is fused to transmembrane and cytoplasmic regions derived from Ret. When such a chimeric receptor is tested, the tyrosine kinase activity of Ret is increased substantially upon addition of EGF. 293 cells were either transfected with Grb7 alone, EGFR/Ret and Grb7, or EGFR/Ret and an unrelated gene product CD40bp-Flag to serve as a control. Cells were then either left untreated or treated with EGF, and anti-Grb7 or anti-Flag immunoprecipitates were subjected to immunoblotting with anti-phosphotyrosine antibody to detect coprecipitating EGFR/Ret. As shown in Fig. 2A, Grb7 associated with EGFR/Ret in an activation-dependent manner, and as expected, the negative control, CD40bp, did not show any detectable binding. The bottom panel shows equal expression of EGFR/Ret in the various samples. Fig. 2B shows that Grb7 underwent an increase in tyrosine phosphorylation upon addition of EGF. This is analogous to the situation with cells expressing the EGFR/HER2 receptor chimera where stimulation of HER2 by EGF leads to increased tyrosine phosphorylation of Grb7. This suggests that Grb7, besides binding receptor kinases, also serves as a substrate being phosphorylated on tyrosine residues. In order to eliminate the possibility that the observed association of Grb7 with Ret was due to overexpression of Grb7, the SKBR-3 cell line which expresses endogenous Grb7 was used for analysis. This cell line was transiently transfected with the EGFR/Ret plasmid, and left untreated or treated with EGF. Anti-Grb7 immunoprecipitates were then immunoblotted with an anti-phosphotyrosine antibody to detect co-precipitated EGFR/Ret. As shown in Fig. 2C, the endogenous Grb7 again associated with the activated chimeric Ret receptor but not with the unactivated receptor. There was equal expression of EGFR/Ret in the unactivated and activated samples (data not shown).
Figure 2: Grb7 binds activated Ret RPTK in vivo and is tyrosine-phosphorylated upon activation of Ret. A and B, 293 cells were cotransfected with expression plasmids containing EGFR/Ret and either Grb7 or Cd40bp-Flag, or with Grb7 alone as indicated. After starvation in 1% BSA for 24 h, cells were untreated or treated with EGF (100 ng/ml). Anti-Grb7 or anti-Flag immunoprecipitates were run on an SDS-PAGE gel and subjected to immunoblotting with an anti-phosphotyrosine antibody (anti-pTyr) (top panels, A and B). The bottom panel of A shows parallel samples that were immunoprecipitated and immunoblotted with anti-EGFR antibody to demonstrate equal expression of EGFR/Ret. The bottom panel of B shows the blot in the top panel of B stripped and reprobed with anti-Grb7 antibody. C, SKBR-3 cells which express endogenous Grb7 were either untransfected or transfected with the EGFR/Ret expression vector as described under ``Materials and Methods.'' Untreated or EGF-treated cells were lysed, and the lysates were immunoprecipitated with anti-Grb7 antibody followed by anti-phosphotyrosine antibody to detect coprecipitating EGFR/Ret.
The SH2 domain of Grb7 could either bind the autophosphorylated Ret receptor directly, or this binding could be mediated by adapter proteins such as Shc which are phosphorylated by Ret. In order to distinguish between these two possibilities, a direct binding assay using a soluble GST fusion of the SH2 domain of Grb7 was carried out. EGFR/Ret was immunoprecipitated from transfected cells that were either untreated or treated with EGF and the immunoprecipitates were resolved by SDS-PAGE followed by transfer onto a nitrocellulose membrane. This membrane was then incubated with soluble SH2 GST Grb7 followed by conventional immunoblotting with an anti-GST monoclonal antibody to detect the GST fusion bound to immunoprecipitated EGFR/Ret receptor. As shown in Fig. 3A, SH2 GST Grb7 directly bound the activated chimeric receptor on the membrane. This confirms that Grb7 does not need an intermediate molecule to bind to the activated Ret receptor.
Figure 3:
Grb7 interacts directly with Ret, and
Grb10 competes with Grb7 for binding to Ret. A,
immunoprecipitated EGFR/Ret from 293 cells treated as indicated was
resolved by SDS-PAGE and transferred onto nitrocellulose. The membrane
was incubated with 5 µg/ml soluble SH2 Grb7 GST followed by
immunoblotting with anti-GST antibody. The arrow indicates the
position of EGFR/Ret where bound SH2 Grb7 can be detected. B, in vitro competition assay. In vitro phosphorylated
EGFR/Ret receptor was incubated with soluble SH2 Grb7 GST with or
without a preincubation with the indicated excess of soluble His-tagged
SH2 Grb10 or COOH-terminal SH2 PLC. The blot was immunoblotted
with an anti-GST antibody to detect bound Grb7. C, same as
panel B except that soluble SH2 Grb10 GST was used instead of
SH2 Grb7 GST, and His-tagged SH2 Grb7 was used for
competition.
In order to compare the binding abilities of Grb7 and Grb10, in
vitro experiments utilizing soluble forms of His-tagged and
GST-tagged SH2 Grb7 and Grb10 were carried out. EGFR/Ret receptors were
immunoprecipitated and phosphorylated in vitro, and then
incubated with soluble GST fusions of the SH2 domains. In parallel
competition experiments, the samples were preincubated with increasing
concentrations of His-tagged fusion protein of the corresponding SH2
domain. As shown in Fig. 3B, a 50-fold molar excess of
competing Grb10 SH2 domain was able to completely abrogate the binding
of Grb7 to Ret suggesting that they bind to the same site. However, a
10-fold excess of His-tagged Grb10 was not as effective as a 2-fold
excess of His-tagged Grb7 which totally abolished binding to Ret
implying a higher affinity of the Grb7 SH2 domain for Ret. Importantly,
a 20-fold excess of His-tagged COOH-terminal SH2 PLC had no effect
on binding of Grb7 suggesting that the observed inhibition was not due
to nonspecific binding. Additionally, when the experiment was repeated
in reverse, a 10-fold molar excess of Grb7 SH2 domain was sufficient to
completely abolish binding of Grb10 to Ret (Fig. 3C).
In summary, we have shown that Grb7 associates with the Ret receptor
tyrosine kinase through its SH2 domain and that activation of Ret
results in tyrosine phosphorylation of Grb7. Given the importance of
Ret during development and in carcinogenesis, signaling molecules
downstream of Ret are likely to be important as they may mediate some
of these actions of Ret. Clearly, determining the binding sites of Grb7
and Grb10 will be important to further dissection of the signaling
pathways initiated by Ret. However, it should be noted that it has been
exceedingly difficult to map the Grb7 binding sites on the HER2/neu
receptor(18) . Nevertheless, studies are ongoing in our
laboratories to delineate the site(s) on the Ret receptor
protein-tyrosine kinase responsible for binding to these adapter
molecules. Further, it should be noted that though Grb7 is tyrosine
phosphorylated in response to Ret activation, we have failed to see a
similar phosphorylation in the case of Grb10. ()However, the
significance of this phosphorylation is not clear at the present time.
One explanation may be that this difference in tyrosine phosphorylation
may serve to recruit distinct downstream effectors. Identification of
these downstream effectors will be important in dissecting Ret
signaling pathways.