(Received for publication, June 22, 1995; and in revised form, July 21, 1995)
From the
Ret is a receptor protein tyrosine kinase that has been implicated in the development of the enteric nervous, endocrine, and renal systems. Mutations associated with multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B) have been shown to activate the intrinsic kinase and transforming ability of ret (Santoro, M., Carlomagno, F., Romano, A., Bottaro, D. P., Dathan, N. A., Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. H., and Paolo DiFiore, P.(1995) Science 267, 381-383). Using the cytoplasmic domain of Ret as bait in a yeast two-hybrid screen of a mouse embryonic library, it was discovered that the src homology 2 (SH2) domain containing protein Grb10 bound Ret. Grb10 belongs to an emerging family of SH2 containing adapter proteins, the prototypical member being Grb7. Using glutathione S-transferase fusion proteins, it was demonstrated that the SH2 domain of Grb10 specifically interacted with Ret. Additionally, using an EGFR/Ret chimera, it was shown that Grb10 bound Ret in an activation dependent manner in vivo. This is the first description of a receptor protein tyrosine kinase that utilizes Grb10 as a signaling intermediate.
Cells respond to a variety of extracellular stimuli, including
nutritional deprivation, osmotic stress, growth factors, and hormones.
Many of these responses are mediated by transmembrane receptors and
activation of these receptors can trigger cellular proliferation,
differentiation, survival, or alterations in
metabolism(1, 2) . Alteration in protein
phosphorylation is the major conduit for the flow of information from a
cell's exterior to the interior. Receptor protein tyrosine
kinases (RPTKs) ()are important mediators of such signals.
The ret protooncogene encodes a transmembrane tyrosine kinase
receptor with a cadherin and cyteine-rich extracellular domain and a
tyrosine kinase containing intracellular domain(2) . ret was originally identified as a transforming gene detected by
transfection of NIH 3T3 cells with DNA from a human T cell
lymphoma(3) . Subsequently, it was discovered that the
activated ret transforming gene was generated during a
recombination of its carboxyl-terminal kinase domain with unrelated 5`
sequences(4) . ret is found to be rearranged and
constitutively activated in a large proportion of thyroid papillary
carcinomas(5, 6, 7) . Dominant transforming
mutations of the ret protooncogene in the germ line have been
shown to result in multiple endocrine neoplasia types 2A and 2B (MEN 2A
and 2B) (8, 9, 10, 11) . Studies
based on ret expression have suggested that ret may
encode the receptor for a factor that is involved in the migration,
proliferation, or survival of a variety of neuronal
lineages(12) . The importance of ret during
development is underscored by the finding that mice deficient in the
expression of the ret protooncogene display renal agenesis and
lack enteric neurons throughout the digestive tract(13) .
Since the ligand for Ret has not been identified, studies of the signaling pathways initiated by the ret receptor kinase have been difficult to perform. To overcome this problem, we have used an EGFR/Ret chimera in which the extracellular ligand binding domain of the EGF receptor is fused in frame to the cytoplasmic domain of the Ret RPTK(14) . Such a receptor chimera is potently activated by EGF. Additionally, we have employed the yeast two hybrid system to identify downstream signaling molecules engaged by the cytoplasmic domain of Ret. When the Ret cytoplasmic domain was used as bait in the two hybrid system to screen for interacting molecules, a partial cDNA encoding the SH2 domain of Grb10 was isolated that specifically bound Ret. Grb10 is a recently discovered molecule that contains an SH2 domain, a central domain, and a proline-rich region(15) . The central domain of Grb10 is similar to the putative Caenorhabditis elegans gene F10E9.6 and contains a pleckstrin homology domain(16, 17) . It has been suggested that F10E9.6 represents mig-10, a gene that is crucial for the embryonic migration of a subset of C. elegans neuronal cells(18) . The exact function of pleckstrin homology domain is not known, but it has been proposed to be involved in protein-protein or protein-lipid interactions(17, 19, 20) . The SH2 domain of Grb10 is 64% identical to the SH2 domain of a previously described gene designated Grb7(15, 21) . SH2 domains have been shown to mediate binding to phosphotyrosines embedded in an appropriate sequence context(22, 23, 24) . Thus Grb10 and Grb7 define a new family of SH2 domain containing adapter proteins that may serve to link activated receptor tyrosine kinases to downstream signaling molecules.
Grb7 has previously been shown to bind to
activated HER2/neu, a receptor with close similarity to the
EGF receptor, and was found to be coamplified and overexpressed along
with HER2/neu in certain forms of breast cancer(21) .
Grb10 was cloned by screening a gt11 expression library with a
phosphorylated
P-labeled carboxyl-terminal segment of the
EGF receptor (15) . Despite being cloned in this manner, Grb10
has not been shown to bind the EGFR or any other RPTK, in
vivo. However, we now show that the SH2 domain of Grb10
specifically associates with the cytoplasmic domain of Ret.
Furthermore, using an EGFR/Ret receptor chimera, it was determined that
Grb10 bound Ret in an activation-dependent fashion in vivo.
This is the first identification of a receptor that is engaged by
Grb10.
DNA sequence analysis was carried out on both strands using the Sequenase kit (U. S. Biochemical Corp.) and custom synthetic oligonucleotide primers. Homology searching against GenPept, PIR, and SwissProt data bases was performed using the on-line BLAST network service.
293T cells were cotransfected with EGFR/Ret and either Grb10 or A20 expression vectors, starved in 1% BSA for 24 h, and either not treated or treated with 100 ng/ml of EGF for 5 min and lysed in lysis buffer. Cleared cell lysates were incubated with 5 µg of anti-Grb10 (number 309) (15) or anti-A20 (32) for 2 h, followed by incubation with protein A/G, washed, and bound material eluted by boiling in 1% SDS. The eluates were subjected to Western blot analysis as described below.
The cytoplasmic domain (amino acids 660-1115) of the murine
Ret RPTK(25) , which when expressed in 293T cells displayed
constitutive tyrosine kinase activity (data not shown), was fused in
frame to the GAL4 DNA-binding domain in the yeast vector pAS1CYH2. This
was used as bait to detect interacting proteins encoded by library
cDNAs fused to the VP16 activation domain. The rationale for this
strategy was that the truncated cytoplasmic domain of Ret would
autophosphorylate itself as a result of constitutive kinase activity
and bind downstream signaling molecules in the yeast two-hybrid screen.
A total of approximately 10 transformants were screened by
expression in a yeast strain harboring lacZ and HIS3 genes under control of the GAL4 upstream activating sequence.
Transformants were plated on histidine-deficient media to select for
interacting clones as described previously(28) .
One
interacting clone that grew on histidine-deficient media and was also
strongly positive in the -galactosidase filter assay was
characterized further. As shown in Table 1, this clone interacted
specifically with the Ret cytoplasmic domain, but not with cytoplasmic
domains from the unrelated Eck RPTK, Fas, or CD40. Upon sequencing,
this clone was found to encode an SH2 domain (amino acids
466-622) of a recently described protein, Grb10(15) . To
obtain independent confirmation of the interaction, cell lysates from
cells transfected with an expression construct encoding
hemagglutinin-tagged Ret cytoplasmic domain (HA-Ret) were incubated
with GST fusions of SH2 domains from Grb10, Grb2, or PLC
. As shown
in Fig. 1, the SH2 domain of Grb10 bound the Ret cytoplasmic
domain as did the SH2 domain of Grb2 and the carboxyl-terminal SH2
domain of PLC
. This binding is presumably mediated by tyrosines
that have been autophosphorylated as a result of constitutive kinase
activity of the HA-Ret fusion protein. Grb2 and PLC
have been
shown previously to bind to the Ret RPTK in vitro and in
vivo, respectively, and therefore served as important positive
controls(14, 33) . This interaction was specific,
since the NH
-terminal SH2 domain of PLC
or GST by
itself did not bind. To determine whether the binding of the Grb10 SH2
domain was specific for Ret, a similar set of experiments was conducted
using lysates from cells transfected with the cytoplasmic domain of an
unrelated RPTK, Eck. The Eck cytoplasmic domain did not bind Grb10, but
as reported earlier it did bind the COOH-terminal SH2 domain of the p85
subunit of phosphatidylinositol kinase(28) .
Figure 1: SH2 domain of Grb10 specifically binds Ret in vitro. 293T cells were transfected with an expression vector containing either the cytoplasmic domain of Ret that was HA epitope-tagged (HA-Ret) or a similar expression vector containing HA-Eck. Cells were then metabolically labeled, and lysates were incubated with GST fusion proteins as indicated in the figure. Bound material was dissociated by boiling in 1% SDS, diluted, and reimmunoprecipitated with anti-HA antibody.
Since the ligand for the Ret RPTK has not been identified, the EGFR/Ret chimeric construct was used for in vivo binding studies. In this receptor chimera, the cytoplasmic domain is derived from the Ret RPTK, and its tyrosine kinase activity is increased substantially upon addition of EGF(14) . This increase in catalytic activity of Ret (autoactivation) can be easily assessed by measuring the degree of autophosphorylation. Cells were cotransfected with expression vectors containing EGFR/Ret and Grb10 or an unrelated cytoplasmic protein A20, treated or not treated with EGF, and Grb10 or A20 immunoprecipitates subjected to immunoblotting with an anti-phosphotyrosine antibody to detect coprecipitating EGFR/Ret. As shown in Fig. 2, Grb10 bound EGFR/Ret chimera in an activation-dependent manner and as expected, the negative control, A20, did not show any detectable binding. In spite of efficient binding of SH2 Grb2-GST to the cytoplasmic domain of Ret (Fig. 1), no binding of Grb2 to the EGFR/Ret construct was observed in 293T cells (data not shown). The EGFR/Ret construct used in these experiments lacks the carboxyl-terminal 42 amino acids that are missing in some isoforms of ret, whereas the HA-Ret construct encodes the entire cytoplasmic domain found in the longest isoforms of Ret, including these 42 amino acids. The discrepancy in the binding of Grb2 can be explained by the fact that there are two consensus binding sites for Grb2 (pYXNX) in these last 42 amino acids. Since, Grb10 is still capable of binding to the EGFR/Ret chimera, we conclude that the predominant binding site(s) for Grb2 on Ret are distinct from those for Grb10 and are likely located in the carboxyl-terminal 42 amino acids of the long isoform of ret.
Figure 2: Grb10 binds activated Ret RPTK in vivo. 293T cells were cotransfected with expression plasmids containing EGFR/Ret and either Grb10 or A20. After starving in 1% BSA for 24 h, cells were untreated or treated with EGF (100 ng/ml). Anti-Grb10 or anti-A20 immunoprecipitates were run on an SDS-PAGE gel and subjected to immunoblotting with an anti-phosphotyrosine antibody (anti-pTyr, top panel). The bottom panel is an immunoblot of respective cell lysates with anti-Ret antibody.
Taken together, these data indicate that Grb10 binds to the activated Ret RPTK. In keeping with the observation that SH2 domains of other adapter proteins such as Grb2 and Grb7 bind phosphotyrosines on activated RPTKs, the binding of Grb10 to activated Ret is likely mediated by its SH2 domain. This binding is specific, since Grb10 did not bind an unrelated receptor protein tyrosine kinase Eck. This report establishes Grb10 as a component of the signal transduction machinery engaged by the potentially oncogenic Ret RPTK.