From the Department of Pharmacology, The University of Texas Health Science Center, San Antonio, Texas 78284-7764
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ABSTRACT |
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hGrb10 is a newly identified Src homology 2 (SH2)
and pleckstrin homology (PH) domain-containing protein that binds to
autophosphorylated receptor tyrosine kinases, including the insulin and
insulin-like growth factor receptors. To identify potential downstream
proteins that interact with hGrb10, we screened a yeast two-hybrid
cDNA library using the full-length hGrb10 as bait. A fragment of
hGrb10, which included the IPS (insert between the PH and SH2 domain) and the SH2 domains, was found to bind with high affinity to the full-length protein. The interaction between the IPS/SH2 domain and the
full-length hGrb10 was further confirmed by in vitro
glutathione S-transferase fusion protein binding studies.
Gel filtration assays showed that hGrb10 underwent tetramerization in
mammalian cells. The interaction involved at least two functional
domains, the IPS/SH2 region and the PH domain, both of which interacted
with the NH2-terminal amino acid sequence of hGrb10
(hGrb10
C, residues 4-414). Competition studies showed that
hGrb10
C inhibited the binding of hGrb10 to the
tyrosine-phosphorylated insulin receptor, suggesting that this region
may play a regulatory role in hGrb10/insulin receptor interaction. We
present a model for hGrb10 tetramerization and its potential role in
receptor tyrosine kinase signal transduction.
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INTRODUCTION |
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Growth factors and hormones initiate and regulate cell growth, differentiation, and metabolism by binding to receptors on the cell membrane. The binding of ligands to their receptors results in receptor tyrosine phosphorylation and kinase activation. Through an intracellular molecule relay, signals are amplified and transmitted from receptors to downstream targets. Under physiological conditions, these signaling processes are accurately regulated through mechanisms such as phosphorylation/dephosphorylation, coordinate localization of enzymes/substrates, and assembly of signaling molecule complexes by scaffolding, anchoring, and adaptor proteins (1).
Many of the adaptor proteins involved in receptor tyrosine kinase signaling contain specific function modules such as the Src homology 2 (SH2)1 domain and the PH domain. One such example is Grb10 (2). Grb10 was first identified by screening a bacterial expression library with the autophosphorylated carboxyl terminus of the epidermal growth factor receptor as a probe (3). This newly identified adaptor protein has been shown to bind directly to several autophosphorylated receptor tyrosine kinases, including the IR (4-8), IGF-1R (5, 6, 9), ELK (10), and Ret (11, 12). Grb10 contains several functional domains including an SH2 domain at the extreme COOH terminus, a PH domain in the central region, and a proline-rich sequence near the NH2 terminus, suggesting that it is capable of interacting with multiple signaling proteins. The SH2 domain of the protein has been shown to be essential for the protein to interact with the autophosphorylated IR and IGF-1R (4-6, 13), whereas the proline-rich sequence of hGrb10 has been shown to bind SH3 containing sequence (6). The function of the PH domain of hGrb10 is currently unknown but may also play an important role in signaling.
Several isoforms of Grb10 have recently been identified from human
species, including Grb-IR/hGrb10 (4), hGrb10
(5, 6), and
hGrb10
(14). These isoforms differ in their PH domains and extreme
NH2-terminal sequences, probably because of alternative splicing events. By using the yeast two-hybrid system and site-directed mutagenesis, we have recently shown that, unlike other SH2
domain-containing proteins such as Shc and the 85-kDa subunit of
phosphatidylinositol 3-kinase, hGrb10 binds to the autophosphorylated
tyrosine residues in the kinase domain of the IR (13). We also found
that hGrb10 isoforms are expressed differentially in cells, suggesting
that these isoforms may play different roles in receptor tyrosine
kinase signal transduction (14). hGrb10 undergoes insulin-stimulated phosphorylation (14) and membrane translocation (6, 14), suggesting
that the protein is a potential signaling molecule downstream of the
IR. However, little is known about the functional role of Grb10 in
signal transduction pathways. We previously found that overexpression
of Grb-IR/hGrb10
, which has a 46-amino acid deletion in the PH
domain, in Chinese hamster ovary cells overexpressing the insulin
receptor (CHO/IR) inhibited insulin-stimulated phosphatidylinositol 3-kinase activity (4). On the other hand, microinjection of the SH2
domain of the protein in fibroblasts was found to inhibit insulin and
IGF-1-mediated mitogenesis, suggesting that the endogenous protein may
have a positive role in signaling (5). More recently, Baserga and
colleagues (15) have shown that overexpression of mGrb10 in mouse
embryo fibroblast cells inhibits IGF-1-mediated cell proliferation by
causing a delay in the S and G2 phases of the cell cycle.
The inhibitory effect of Grb10 has also been suggested from recent
genomic imprinting studies. The gene coding for mouse Grb10 has been
identified as a maternally expressed gene located on proximal
chromosome 11 (16). Genetic studies have shown that maternal
duplication of chromosome 11 proximal to the translocation breakpoint
cause prenatal growth retardation (17). In humans, Grb10 is located on
chromosome 7p11.2-12 (14, 18). Maternal disomy of human chromosome 7 has been shown to cause Silver-Russell syndrome whose symptoms include
pre- and postnatal growth retardation and other dysmorphologies
(19).
To further understand the functional role of Grb10, we attempted to identify molecules that interacted with the protein using the yeast two-hybrid system. Here we present evidence that hGrb10 undergoes tetramerization in cells. In addition, we have found that the interaction involves at least two functional domains: the IPS/SH2 domain and the PH domain. Furthermore, we have shown that the NH2-terminal sequence of hGrb10 inhibited the binding of the protein to the autophosphorylated IR, suggesting that the oligomerization may play a role in the regulation of insulin signaling. We present a model for the mechanism of the tetramerization and its potential role in receptor signal transduction.
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EXPERIMENTAL PROCEDURES |
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Materials--
The yeast two-hybrid system were from
CLONTECH. Bacterial protein expression vectors
pGEX-4T-1 and pET21a were from Amersham Pharmacia Biotech and Novagen
(Madison, WI), respectively. A polyclonal antibody to the COOH terminus
of hGrb10 and Chinese hamster ovary cells expressing the IR and hGrb10
isoforms (CHO/IR/hGrb10 and CHO/IR/hGrb10
) were described
previously (4, 14).
Construction of Plasmids--
The cDNAs encoding the
full-length (residues 4-594) and various truncated mutants of
hGrb10 were generated by polymerase chain reaction using hGrb10
cDNA (14) as a template (Fig. 3A). Convenient
restriction endonuclease sites were introduced to allow the in-frame
insertion of the cDNAs into the yeast two-hybrid plasmids pGBT9 and
pGAD424, the GST fusion protein expression plasmid pGEX-4T-1, and the
bacterial expression vector pET21a. All recombinant plasmid constructs
were verified by restriction mapping and/or DNA sequencing (detailed
cloning strategies available upon request).
Yeast Two-hybrid Studies--
A yeast two-hybrid cDNA
library derived from HeLa cells was screened with plasmid
pGBT9-hGrb10 as bait. Positive clones were identified by selection
of transformants on minimum medium without Leu, Trp, and His and by
-galactosidase filter assays. For interaction studies, SFY526 cells
were cotransfected with various truncated forms of hGrb10
fused in
frame with either the DNA binding domain or the activation domain of
GAL4 protein. Interactions were monitored by
-galactosidase filter
or liquid assays described previously (13).
Expression of GST Fusion Proteins--
DH5 cells containing
plasmids encoding for various recombinant GST/hGrb10 fusion proteins
were grown in LB medium at 37 OC overnight. This culture
was diluted 1:10 and grown at 30 °C for 80 min. Expression of the
fusion protein was induced by the addition of
isopropyl-
-D-thiogalactoside to a final concentration of
1 mM. After 3.5-h induction, cells were harvested by
centrifugation at 5,000 × g for 10 min, washed with 10 mM Tris-HCl, pH 8.0, and suspended in bacterial lysis
buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM
KCl, 1 mM dithiothreitol, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.1% (v/v) Triton
X-100, and 1 mg/ml lysozyme and lysed by sonication. Cell lysates were
clarified by centrifugation at 12,000 × g for 15 min.
The GST/hGrb10 fusion proteins were purified by affinity chromatography
with glutathione-agarose beads (Sigma).
Expression and Purification of the Full-length and the IPS/SH2
Truncated Mutant of hGrb10 in Bacterial Cells--
BL21(DE3)
bacterial cells expressing the full-length hGrb10
with a His tag at
its COOH terminus and a truncated form of hGrb10
with a deletion in
the IPS/SH2 region (hGrb10
(IPS/SH2), Fig. 3A) were
grown in LB medium. The expression and cell lysis procedures were
similar to those used to purify the GST fusion protein described above.
The His-tagged hGrb10
was affinity-purified with Ni-NTA-agarose beads according to the protocol of the manufacturer (Qiagen,
Chatsworth, CA). To purify hGrb10
(IPS/SH2), solid
(NH4)2SO4 was added to cell lysates
to a final concentration of 10% (v/v). The suspension was centrifuged
at 15,000 × g for 20 min. Additional solid
(NH4)2SO4 was added to the
supernatant to a final concentration of 15% (v/v). The suspension was
centrifuged for 20 min at 15,000 × g, and the pellet
containing the recombinant protein was retained and resuspended in a
minimal volume of buffer containing 20 mM Hepes, pH 7.0, 50 mM KCl, 1 mM
-mercaptoethanol, and 0.1%
(v/v) Triton X-100. The suspension was loaded onto a gel filtration
Sephacryl S-300 column, and elution of the recombinant protein was
performed at a flow rate of 0.2 ml/min in WGA buffer (50 mM
Hepes, pH 7.6, 0.15 NaCl, and 0.1% (v/v) Triton X-100). The fractions
containing the protein were pooled and the purity of the protein was
determined by Coomassie Blue staining.
In Vitro Binding Studies--
Lysates (0.3 ml) from
CHO/IR/hGrb10 or CHO/IR/hGrb10
cells (in 100-mm plates) were
mixed with 15 µg of freshly made GST or GST/hGrb10 fusion proteins
coupled to glutathione-agarose. After incubation at 4 °C overnight,
the beads were washed extensively with cold WGA buffer. hGrb10 isoforms
associated with the GST/hGrb10 fusion proteins were separated by
SDS-PAGE, transferred to a nitrocellulose membrane, and detected by
immunoblot with anti-hGrb10 antibody.
Competition Studies--
To purify the IR, cell lysate from
CHO/IR cells were incubated with WGA beads for 4 h at
4 OC. After extensive washing with WGA buffer, the IR
attached to the beads was in vitro phosphorylated by
incubation at room temperature for 1 h with kinase buffer
containing 50 mM Hepes, pH 7.6, 150 mM NaCl,
0.1% Triton X-100, 5 mM MgCl2, 5 mM MnCl2, 1 mM ATP, and 1 µM insulin and then eluted with 0.3 M
N-acetylglucoseamine. The partially purified,
tyrosine-phosphorylated IR (10 µg) was added to tubes containing the
bacteria expressed His-tagged hGrb10 (15 µg) coupled to Ni-NTA
agarose beads and different concentrations of purified hGrb10
C.
After incubation at 4 OC overnight, the beads were washed
extensively with WGA buffer, and the IR associated with hGrb10
was
separated by SDS-PAGE, transferred to a nitrocellulose membrane, and
detected by a polyclonal antibody to the
-subunit of the receptor
(Santa Cruz Biotechnology, Santa Cruz, CA).
Size Exclusion Chromatography--
A Sephacryl S-300 column
(16 × 60 cm) was equilibrated with buffer containing 50 mM Hepes, pH 7.0, and 0.15 M NaCl and
calibrated with standards: thyroglobulin, 669 kDa; ferritin, 440, kDa;
catalase, 232 kDa; aldolase, 158 kDa; and ovalbumin, 44 kDa. Lysates
from CHO/IR/hGrb10 or CHO/IR/hGrb10
cells were clarified by
centrifugation at 12,000 × g for 15 min at 4 °C.
The clarified supernatant (0.5 ml) was applied to the column and eluted
with the same buffer at a flow rate of 0.2 ml/min. Fractions (1 ml)
were collected and proteins were precipitated by the addition of 100 µl of 0.15% sodium deoxycholate and 100 µl of 72% trichloric
acid, separated by SDS-PAGE, and the position of hGrb10
or hGrb10
in the elution profile was determined by Western blot using an
anti-hGrb10 antibody.
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RESULTS |
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Identification of hGrb10 Dimerization by the Yeast Two-hybrid
System--
To identify hGrb10-associated proteins, we screened a
yeast two-hybrid library derived from HeLa cell cDNAs using the
full-length hGrb10 cDNA as bait. From 4 million colonies
screened, we identified over 50 positives that grew on synthetic medium
without Leu, Trp, and His and remained positive during the
-galactosidase filter assays.
hGrb10 Dimerized in Vitro--
To test whether the dimerization
was through a direct interaction between two hGrb10 molecules or
mediated by an auxiliary protein(s) in yeast cells, we first
investigated whether the IPS/SH2 domain interacted with hGrb10 in
vitro by GST fusion protein pull-down studies. As shown in Fig.
1, both Grb-IR/hGrb10 (lanes
1 and 2) or hGrb10
(lanes 3 and
4) were precipitated by the GST-IPS/SH2 fusion proteins.
Under similar condition, no hGrb10 isoforms were pulled down by the
control GST (data not shown). The GST-IPS/SH2 fusion protein
precipitated a lesser amount of Grb-IR/hGrb10
than hGrb10
(Fig.
1, lanes 1-4), probably because of a lesser expression of
this isoform in cells (Fig. 1, lanes 9-12). However, no
significant difference was observed between hGrb10 isoforms precipitated from cells treated with or without insulin (Fig. 1,
lanes 1-4).
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hGrb10 Underwent Tetramerization in Mammalian Cells--
To see
whether the dimerization also occurred in mammalian cells,
Grb-IR/hGrb10 and hGrb10
expressed in either CHO/IR/hGrb10
or
CHO/IR/hGrb10
cells were subjected to size exclusion chromatography analysis. As shown in Fig. 2A,
hGrb10
was eluted as two peaks of approximately 320 and 80 kDa,
respectively, suggesting that hGrb10
exists as both a tetramer and a
monomer in mammalian cells. Similar elution profile was observed for
hGrb10
(Fig. 2B), suggesting that this isoform can also
form a tetramer in mammalian cells. Although no apparent peaks were
observed in the 120-240 kDa range, we could not exclude the
possibility that some dimeric and trimeric Grb10 might also be present
in cells. It is possible that the signal of these dimers and trimers
may be masked by the smearing peaks of the more abundant tetramers and
monomers.
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The IPS/SH2 Domain of hGrb10 Interacts with the NH2
Terminus of the Protein--
Having found that hGrb10 tetramerized in
cells and that the IPS/SH2 domain was involved in the interaction, we
attempted to identify the sequence in hGrb10 that interacts with this
domain. We constructed various yeast two-hybrid plasmids in which
different regions of hGrb10 were fused in frame with either the GAL4
DNA binding domain (in plasmid pGBT9) or with the GAL4 transcription activation domain (in plasmid pGAD424, Fig.
3A). We tested the interaction
between these fusion proteins by the yeast two-hybrid system. As shown
in Fig. 3B, The IPS/SH2 region interacted with both the
wild-type and a mutant form of hGrb10 in which the critical arginine
residue in the SH2 domain was changed to glycine (R520G). This mutant
of hGrb10 did not bind the IR in the yeast two-hybrid system (13).
These data suggest that the structural requirement for hGrb10
dimerization and for its interaction with the IR are different. The
IPS/SH2 protein also bound to a truncated form of hGrb10 in which the
IPS/SH2 region was deleted (hGrb10
(IPS/SH2)). In addition, no
-galactosidase activity was detected between two IPS/SH2 domains
using the yeast two-hybrid system (Fig. 3B). These results
suggest that the dimerization is not through a direct interaction
between two IPS/SH2 regions. To delimit the boundary of the IPS/SH2
interaction sequence, additional truncated versions were generated.
However, no interaction was detected between these mutants and the
IPS/SH2 domain in the yeast two-hybrid system (Fig. 3B),
suggesting that a specifically folded structure of the N terminus,
which could be disrupted by the truncation, may be required for the
IPS/SH2 region to bind. The interaction seems also to require an intact
IPS/SH2 domain as neither the SH2 nor the IPS region alone bound with a
significant affinity to the full-length form of hGrb10
(data not
shown).
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The Involvement of the PH Domain in hGrb10 Dimerization--
The
domain structure of hGrb10 consists of several functional regions: a
COOH-terminal SH2 domain, a central PH domain, and an
NH2-terminal proline-rich sequence (Fig. 3A).
Although our data showed that the IPS/SH2 domain was sufficient to bind
the full-length protein, we also found that the full-length hGrb10
underwent dimerization with a higher
-galactosidase activity in the
yeast two-hybrid system (data not shown). This observation suggested
that additional region(s) may also be involved in the interaction. To
test this hypothesis, we subcloned cDNAs encoding various truncated
form of hGrb10 (Fig. 3A) into both the "bait" (pGBT9)
and the "prey" (pGAD424) plasmids and tested the interaction between these yeast two-hybrid fusion proteins. Our studies showed that, in addition to the IPS/SH2 region, the PH domain (residues 290-414) was sufficient to interact the full-length hGrb10 (Fig. 3C). This observation was confirmed by in vitro
binding studies, which showed that the GST-hGrb10(PH) fusion protein
interacted with both hGrb10 isoforms expressed in CHO/IR/hGrb10 cells
(Fig. 1, lanes 5-8). No
-galactosidase activities were
observed when other fragments were tested against the full-length Grb10
in the yeast two-hybrid system (data not shown). To map the binding
region for the PH domain, we tested the interaction between the PH
domain and various truncated forms of hGrb10 in the yeast two-hybrid system. We found that the minimum sequence that retained binding to the
PH domain was between residues 4-290 (
(PH/IPS/SH2); Fig. 3,
A and C). These data suggest that although both
the IPS/SH2 and the PH domain interacts with the
NH2-terminal region of the protein, the detailed structural
requirement for the binding of the IPS/SH2 and the PH domains may be
different.
The NH2-terminal Region Negatively Regulates the
Binding Affinity of hGrb10 to the IR--
We previously showed that
the SH2 domain of hGrb10 was essential for hGrb10 binding to the
autophosphorylated IR in vitro and in cells (13). The
finding that the IPS/SH2 region of hGrb10 interacts with the
NH2-terminal region of the protein raised an interesting
question of whether this interaction affects the binding of the SH2
domain to the IR. To address this question, we expressed a truncated
form of hGrb10 with a deletion in the IPS/SH2 region (hGrb10
(IPS/SH2), Fig. 3A) in bacteria and purified it
to near homogeneity (Fig. 4A).
We tested the effect of the recombinant protein on the interaction
between hGrb10
and the IR in vitro. As expected,
significant autophosphorylated IR was pulled down by the His-tagged
hGrb10
protein bound to Ni-NTA-agarose beads (Fig. 4B,
lane 2), but not by the Ni-NTA beads control (Fig.
4B, lane 1). The interaction between the IR and
hGrb10
, however, was competitively inhibited by increased
concentrations of hGrb10
(IPS/SH2) (Fig. 4B,
lanes 3-7). The inhibition was specific as unrelated proteins such as bovine serum albumin at similar concentrations had no
effect on the binding (data not shown). In addition, the hGrb10
(IPS/SH2) recombinant protein did not bind to the IR
in vitro (data not shown), suggesting that the inhibition
was because of a competitively binding of the polypeptide to a site on
hGrb10
which affected the binding of the protein to the
tyrosine-phosphorylated IR.
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DISCUSSION |
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We report here the oligomerization of an adaptor protein, hGrb10.
To our knowledge, this is the first indication that an SH2 and PH
domain-containing adaptor protein undergoes tetramerization in cells,
and the oligomerization is mediated by at least two functional domains,
the IPS/SH2 and the PH domain. These findings are quite interesting as
oligomerization of adaptor proteins in cells may have important
physiological relevance. One possible function for hGrb10
tetramerization may be to provide a reservoir of latent hGrb10
molecules and prevent the protein from nonspecific binding to cellular
phosphotyrosine-containing proteins. We have shown previously that the
SH2 domain of hGrb10 binds the autophosphorylated tyrosine residues in
the kinase domain of the IR (13). Recently, Dr. Gustafson and
colleagues (20) have found that the IPS region alone is also capable of
interacting with the IR and IGF-1R in a kinase dependent manner. The
finding that the IPS/SH2 domain is one of the determinants involved in
the hGrb10 tetramerization suggests that the tetramerization may
regulate the binding of the protein to the autophosphorylated IR.
Consistent with this hypothesis, we have found that the NH2
terminus of hGrb10 (hGrb10(IPS/SH2)), which interacted with the
IPS/SH2 domain of the protein (Fig. 3B), competitively
inhibited the binding of hGrb10 to the IR (Fig. 4B). These
data suggest that the tetramerized hGrb10 may be unable to interact
with phosphotyrosine-containing peptides because of the blockage of its
IPS/SH2 domain by the NH2-terminal sequence. The
stimulation of cells by insulin or other growth factors may induce a
conformational change of hGrb10 so that the SH2 domain of the protein
is accessible for binding to phosphotyrosine-containing receptors.
Tetramerization may thus provide a mechanism to regulate the
interaction between the adaptor protein and the receptor in cells.
Another possible role of hGrb10 oligomerization in cells may be to serve as a docking complex to recruit multiple signaling molecules. As Grb10 monomer contains several functional domains such as an SH2 domain, a PH domain, and a proline-rich sequence, all of which are able to interact with other cellular proteins, oligomerization of Grb10 should enhance the binding capacity of the protein to these signaling molecules. Although we were unable to determine whether hGrb10 binds to the IR or IGF-1R as a monomer or oligomer because of technical difficulties, this hypothesis is quite interesting, especially with the findings that hGrb10 is capable of binding simultaneously to the IR and other signaling proteins in cells (5, 6, 21). Therefore, oligomerization of the adaptor protein may provide a novel mechanism to colocalize multiple signaling molecules to regulate cell signaling processes.
In this study, we have also found that the PH domain is sufficient to
interact with the full-length hGrb10 (Fig. 1, lanes 5-8,
and Fig. 3C). PH domains have been shown to be involved in both protein-lipids or protein-protein interactions (22). Examples of
the latter include -adrenergic receptor kinase, IRS-1, and the Btk
tyrosine kinase, whose PH domains bind to the
-subunits of
trimeric G proteins (23), the IR (24), and protein kinase C (25),
respectively. The finding that the PH domain is involved in hGrb10
dimerization in vitro and in the yeast two-hybrid system provides new evidence that PH domains are functional motifs and can
function independently to mediate regulated protein-protein interactions in signaling. However, it should be pointed out that the
IPS/SH2 domain of hGrb10 is sufficient to interact with the full-length
protein and that Grb10
, the isoform which has a 46-amino acid
deletion, including part of the PH domain, was still be able to undergo
tetramerization in cells (Fig. 2B). These data suggest that
the PH domain may not be essential for hGrb10 tetramerization. One
possible role for the involvement of the PH domain may be to provide
additional specificity for the interaction. Further studies will be
needed to test this hypothesis.
A simple model, which accounts for the role of hGrb10 tetramerization, is depicted in Fig. 5. In this model, hGrb10 tetramerization in cells involves two functional motifs: the IPS/SH2 domain and the PH domain, both interacting with the NH2-terminal region of its partner. The tetramerization may bury the functional domains of hGrb10 and prevent them from nonspecifically interacting with the tyrosine-phosphorylated receptors or other cellular signaling molecules. The balance between monomeric and oligomeric hGrb10 may thus provide a specificity for the adaptor to transduce or regulate receptor tyrosine kinase signaling processes. This balance may be regulated by mechanisms such as phosphorylation, which could result from stimulation by insulin or other growth factors (14). The modification may cause a conformational change of the protein and lead to the access of the SH2 domain by the autophosphorylated receptor. The binding affinity of the SH2/phosphotyrosine containing sequence interaction may be much greater than that of the interaction between the SH2 domain and the non-tyrosine-phosphorylated hGrb10 NH2-terminal sequence so that the hGrb10-receptor complex is stabilized. The binding of hGrb10 to the IR may result in a further conformational change for hGrb10, which may allow other functional domains such as the PH in the protein to interact with downstream signaling molecules. It should be pointed out that the tetramerization may not be unique for Grb10 but may occur in other adaptor molecules as well. For example, the COOH-terminal region of Grb10 has recently been shown to bind to the full-length Grb7 in the yeast two-hybrid system.2 It would be interesting to see whether oligomerization is a general mechanism for other adaptor proteins in the regulation of cellular signaling processes.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK52933 (to F. L. and L. Q. D.) and by a Research Grant from the South Texas Health Research Center for Diabetes Research (to F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
UTHSCSA, 7703 Floyd Curl Dr., San Antonio, TX 78284-7764. Tel.:
210-567-3097; E-mail: liuf{at}uthscsa.edu.
1 The abbreviations used are: SH2, Src homology 2; GST, glutathione S-transferase; IR, insulin receptor; IGF-1, insulin-like growth factor-1; IPS, insert between the PH domain and the SH2 domain; PAGE, polyacrylamide gel electrophoresis; PH, pleckstrin homology; WGA, wheat germ agglutinin; CHO, Chinese hamster ovary; Ni-NTA, nickel-nitrilotriacetic acid.
2 J. Cooper, personal communication.
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REFERENCES |
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