The insulin receptor plays important roles in metabolism and
growth regulation of target
tissues(1, 2, 3) . Upon insulin stimulation
the receptor becomes autophosphorylated on at least six or seven
tyrosine residues located in the juxtamembrane, kinase, and
carboxyl-terminal regions of its
-subunit(4, 5, 6, 7) . Many
signals are relayed through the insulin receptor substrate-1 (IRS-1), (
)which is phosphorylated by the receptor on various
tyrosine residues(8) . Phosphorylation of a tyrosine at
position 960 in the juxtamembrane region of the insulin receptor is
essential for signaling through IRS-1(9, 10) . Among
the proteins that bind to IRS-1 are the p85 subunit of
phosphatidylinositol 3-kinase (PI3-kinase), Grb2, the tyrosine
phosphatase Syp/SHPTP2, and Nck(2) . IRS-1 is required for
insulin-mediated mitogenesis(11, 12) ; however,
studies using homozygous IRS-1 knockout mice predict important
IRS-1-independent insulin signaling pathways(13, 14) .
A promising candidate, the structurally closely related protein IRS-2,
was recently identified, which has signaling functions similar to
IRS-1(15, 16) . The Shc proteins represent additional
receptor tyrosine kinase substrates that mediate p21
activation in the mitogenic pathway of insulin
action(17) . We employed the yeast two-hybrid system to
identify signaling mediators for alternative pathways analogous to
signals emerging from other receptor tyrosine kinases. Here we describe
the identification of Grb10 as an IRS-1-independent interactive protein
of the activated insulin receptor, and we define the sites of
interaction in the receptor and Grb10.
MATERIALS AND METHODS
Antibodies
The antibody against the insulin
receptor
-subunit was from Biodesign and against the insulin
receptor
-subunit from Transduction Laboratories and American
Research Products. Anti-GAL4 and anti-IRS-1 antibodies were from
Upstate Biotechnology, and anti-Tyr(P) antibodies from Transduction
Laboratories and Upstate Biotechnology. Antibodies 121 and 122 were
produced in rabbits against a GST fusion protein containing the Grb10
SH2 domain (Hazelton Research Products). Horseradish peroxidase-coupled
anti-IgG antibodies (Sigma) were used for immunoblotting.
Yeast Two-hybrid System
A BglI-PstI cDNA fragment encoding the
carboxyl-terminal 402 amino acids of the receptor
-subunit (18) was ligated to the EcoRI-PstI sites of
pGBT9 (19) using an EcoRI-BglI adaptor
(5`-AATTCGCGAGCAGAAAGAGGCAGCCAGAT-3` and 5`-TGGCTGCCTCTTTCTGCTCGCG-3`).
The mutation IR K1018A was introduced by exchanging a BglI-PstI fragment of the insulin receptor bait with
the corresponding fragment of the mutated receptor(6) . A ScaI-BamHI fragment encoding the intracellular domain
of the insulin-like growth factor-1 receptor (20) was inserted
into the EcoRI-BamHI sites of pGBT9 with an EcoRI-ScaI adaptor (5`-AATTCGGTGAGT-3` and
5`-ACTCACCG-3`). A XbaI-KpnI cDNA fragment encoding
the carboxyl-terminal 642 amino acids of rat IRS-1 (8) was
inserted into the SalI site of pGBT9 using SalI-XbaI (5`-TCGACTTGGTACCGGT-3` and
5`-CTAGACCGGTACCAAG-3`) and KpnI-SalI
(5`-CGACCTGGAG-3` and 5`-TCGACTCCAGGTCGGTAC-3`) adaptors. A BglI-PstI fragment encoding the carboxyl-terminal 358
amino acids of protein kinase C
(21) was ligated to the EcoRI-PstI sites of pGBT9 with an EcoRI-BglI adaptor (5`-AATTCGCGAGCGGCGGTGC-3` and
5`-CCGCCGCTCGCG-3`). For the screen, yeast strain Y153 (22) was
co-transformed with the insulin receptor bait and a 10.5 days
postconception mouse embryo cDNA library in plasmid pVP16(23) .
The specificity of interaction was tested in mating experiments as
described(23) .
Construction of Fusion Proteins and Protein
Purification
A Grb10 cDNA fragment encoding the
carboxyl-terminal 108 amino acids was ligated to the BamHI and EcoRI sites of plasmid pGEX-1
T (Pharmacia Biotech Inc.)
using the polymerase chain reaction primers
5`-CCGGGATCCATTCACAGGACTCAGCATTG-3` and 5`-GCCGAATTCTTCTATCTATCTAGCG-3`
and was confirmed by sequence analysis. The expressed GST-Grb10 SH2
domain fusion and control GST protein were purified on a
glutathione-agarose column (Pharmacia) and stored at 4 °C in 0.1 M ammonium carbonate, 1 mM EDTA, 10 mM dithiothreitol. Insulin receptors were wheat germ agglutinin
(Vector)-purified and activated as described(24) .
Phosphopeptide Interactions
Phosphopeptides
containing Tyr(P)-953 (LpYASSNPEYLSASDV), Tyr(P)-960 (SSNPEpYLSASD),
Tyr(P)-1146 (DIpYETDYYRKG), Tyr(P)-1150 (DIYETDpYYRKG), Tyr(P)-1151
(DIYETDYpYRKG), Tyr(P)-1316 (KRSpYEEHIPY), and Tyr(P)-1322
(HIPpYTHMNGG) were immobilized on Affi-Gel 10 (Bio-Rad)(25) ,
washed extensively with 20 mM Tris, 100 mM NaCl, pH
7.4, and incubated with 10 µg of GST-SH2 for 2 h at 22 °C in a
total volume of 200 µl. After washing the bound proteins were
separated by SDS-PAGE and visualized by Coomassie Brilliant Blue
staining.
Cell Cultures, Immunoprecipitation, and
Immunoblotting
CHO/IR (
10
receptors/cell),
CHO/IR
(
1-3
10
receptors/cell) (26) , and NIH 3T3/IR cells (
6
10
receptors/cell) (27) were cultured as
described(26, 27) . Prior to experiments, cells were
incubated for 16 h without serum and treated with 100 nM insulin for 15-20 min. Culture dishes were washed twice with
phosphate-buffered saline, and the cells lysed in 50 mM Hepes,
10% glycerol, 137 mM NaCl, 1 mM MgCl
, 1
mM CaCl
, 2 mM EDTA, 10 mM NaF,
100 µM Na
VO
, 10 mM
-glycerophosphate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, pH 7.4, with 1% Triton X-100 (CHO)
or with 1% Brij-97 (NIH 3T3). Proteins were immunoprecipitated
(Pharmacia) in the same buffer. The interaction with purified insulin
receptors was assayed by incubation with 100 mg/ml GST-Grb10 SH2 for 1
h, followed by a 1.5-h incubation with glutathione beads in 50 mM Hepes, 150 mM NaCl, 2 mM Na
VO
, 0.1% Triton X-100, pH 7.4, at 4
°C(24) . After four washes, the precipitates were separated
by SDS-PAGE and analyzed by immunoblotting.
RESULTS
Isolation of Insulin Receptor Interactive Proteins with
the Yeast Two-hybrid System
A two-hybrid library with cDNAs from
10.5 days postconception mouse embryos fused to the
transcription-activating domain of VP16 (23) was screened. The
bait plasmid encoded a fusion protein of the GAL4 DNA-binding domain
with the carboxyl-terminal 402 amino acids of the insulin receptor
-subunit(18) . This includes the complete intracellular
region, which had been shown to be constitutively active when separated
from the extracellular domain (24) . Approximately 10
cells of Saccharomyces cerevisiae strain Y153 (22) were co-transformed with bait and library, and 13 colonies
that grew on medium selective for HIS3 expression were
isolated. Among these, 11 colonies showed expression of the lacZ reporter gene. For 10, growth and color were dependent on the
presence of both plasmids. Based on the intensity of color in the plate
assay, the colonies could be placed into two groups with six stronger
(color development within 15-20 min) and four weaker (color
development within 30-45 min) interacting clones. Sequence
analysis of the plasmid inserts revealed that they all encoded protein
fragments containing Src homology-2 (SH2) domains. Three of the weaker
interacting clones represented the carboxyl-terminal SH2 domain of the
regulatory subunit p85 of PI3-kinase(28) , and one represented
a novel sequence. All of the stronger interacting clones carried
plasmids with distinct inserts that encoded the SH2-containing region
of the recently identified signaling mediator Grb10(29) ,
specifically the carboxyl-terminal 161, 163, 169 (two independent
isolates), 172, and 177 of a total of 621 amino acids (Fig. 1).
Figure 1:
Cloning
of Grb10 fragments and specificity of interaction. The inserts isolated
with the two-hybrid screen are aligned with a map of Grb10 and a
truncated clone that lacks the last 30 amino acids of the SH2 domain.
The specificity of interaction of the different protein fragments was
analyzed by comparing different baits, and the development (+) or
lack(-) of blue color in colonies on plates with a particular
bait is indicated. The relative strength of interaction was
independently quantified in a liquid color assay and is given
underneath the first row. PPP, proline-rich domain; aa, amino acids.
Specificity of the Grb10 SH2 Domain
Interaction
Bait constructs derived from various proteins were
employed to test the specificity of the interaction between the Grb10
fragment and the insulin receptor bait. We found that not only
transformants with the insulin receptor but also those that expressed a
fusion with the intracellular domain of the IGF-1 receptor showed a
phenotype characteristic for protein interaction with the Grb10 protein (Fig. 1). Results with all other control transformants,
expressing a variety of unrelated baits, such as fusions with the 642
carboxyl-terminal amino acids of IRS-1, the catalytic domain of the
serine/threonine protein kinase C
, or the commonly used test
protein lamin C were negative. The interaction between Grb10 and the
insulin receptor was dependent on a catalytically active tyrosine
kinase. This was demonstrated with an insulin receptor point mutation
(K1018A), which rendered the kinase catalytically inactive(6) .
Immunoblotting experiments with antibodies against the GAL4 DNA-binding
domain confirmed expression of identical amounts of normal or mutated
insulin receptor bait proteins (not shown). In contrast to the normal
receptor, interaction of the K1018A insulin receptor with the Grb10
protein was not detectable (Fig. 1). The isolated Grb10 clones
contained an SH2 domain as the only discernible structural feature. We
identified a truncated sequence of Grb10 lacking 30 amino acids of the
SH2 domain at the carboxyl terminus when we randomly analyzed
transformants from the two-hybrid screen. This truncation exhibited
activities only marginally above the experimental background in
interaction experiments with the insulin and the IGF-1 receptor enzyme (Fig. 1). In contrast, amino-terminal variations outside of the
SH2 domain in other clones did not affect the strength of association
to the receptors (Fig. 1). The interaction was thus found to be
specific for the insulin/IGF-1 receptors and to be dependent on an
active receptor kinase and the intact SH2 domain.
Interaction of Grb10 SH2 Domain Fusion Proteins with
Insulin Receptors from Cell Lysates
CHO cells expressing about
10
human insulin receptors/cell (CHO/IR) (26) were
lysed with detergent and incubated with a GST fusion containing a
minimal Grb10 SH2 domain. Glutathione-Sepharose beads, which bound
equal amounts of GST-Grb10 SH2 or GST (not shown), co-precipitated the
insulin receptor in experiments with the GST-Grb10 SH2 fusion but not
with control GST protein alone (Fig. 2A). The
association between the insulin receptor and the Grb10 SH2 domain was
strictly dependent on insulin stimulation of the receptor, and the
amounts of co-precipitated receptor correlated with the amount of
fusion protein in the interaction assay (Fig. 2A).
These results are in agreement with the findings from the two-hybrid
experiments and emphasize both the role of an active tyrosine kinase
and of the Grb10 SH2 domain in the interaction with the insulin
receptor.
Figure 2:
Interaction of the insulin receptor with
the Grb10 SH2 domain in vitro. Purified GST fusion proteins
with the Grb10 SH2 domain or control GST were incubated with CHO/IR
cell lysates (A) or purified insulin receptors (B)
that were either incubated with 100 nM insulin or were not
stimulated. Proteins were precipitated with glutathione-Sepharose beads
and were analyzed by SDS-PAGE and immunoblotting with antibodies
against the
-subunit of the insulin receptor. As indicated in A, different amounts of GST proteins were used in the lysate
experiments.
Interaction between Purified Insulin Receptors and the
Grb10 SH2 Domain
To demonstrate a direct interaction between
Grb10 and the insulin receptor, affinity-purified receptor from CHO/IR
cells was stimulated with insulin in vitro and incubated with
GST-Grb10 SH2 protein or control GST protein. The insulin receptor
co-precipitated with glutathione-Sepharose beads as detected in
immunoblots (Fig. 2B). As in the experiments with cell
lysates, the interaction was dependent on an activated tyrosine kinase
and on the Grb10 SH2 domain, whereas the control GST protein did not
associate (Fig. 2B). This suggests that Grb10 binds
directly to the insulin receptor without the involvement of an
intermediate protein.
Mapping of the Interacting Domain of the Insulin
Receptor
Synthetic phosphopeptides, each representing one of the
seven major tyrosine autophosphorylation sites (30, 31, 32) (see ``Materials and
Methods'' for sequences) were compared for their interaction with
the GST-Grb10 SH2 domain. We detected binding to Tyr(P)-1322 with high
affinity and at a significantly reduced level to Tyr(P)-1316 (Fig. 3A). These assays were complemented by
precipitation experiments with the GST-Grb10 SH2 protein,
glutathione-Sepharose beads, and lysates from cells that express about
1-3
10
copies of a truncated insulin
receptor, IR
, per cell. The receptor lacks 43 amino
acids of the carboxyl terminus including tyrosine residues 1316 and
1322(26) . In contrast to the normal receptor, the mutant did
not associate with the Grb10 SH2 domain independent of insulin
stimulation (Fig. 3B). Only a small amount of
co-precipitated receptor was detected in immunoblots after prolonged
exposure in the detection system, which could potentially reflect low
affinity binding to any of the other phosphotyrosines in the kinase or
the juxtamembrane domain. Densitometric scans of the immunoblot
resulted in pellet to lysate ratios of 0.8 for the normal receptor and
not more than 0.05 for the mutant. Taken together with the finding that
Grb10 binds to the IGF-1 receptor, which lacks a tyrosine homologous to
position Tyr-1316 in the insulin receptor, these experiments suggest
the most carboxyl-terminal phosphotyrosine of the receptor
-subunit as the major site of interaction between the insulin
receptor and Grb10.
Figure 3:
The insulin receptor site of interaction
with Grb10. Seven peptides with the major phosphotyrosine motifs of the
insulin receptor (A) or lysates from CHO/IR or
CHO/IR
cells (B), treated like the cell
cultures described in Fig. 2, were incubated with GST-Grb10 SH2
fusion protein. Fusion proteins bound to the immobilized peptides were
eluted, separated by SDS-PAGE, and visualized by Coomassie Brilliant
Blue staining (A); proteins from lysates co-precipitated with
GST-Grb10 SH2 fusion protein (P) were analyzed together with
aliquots from cell lysates (L) by SDS-PAGE and by
immunoblotting with antibodies against the insulin receptor
-subunit or against IRS-1 (B).
Binding Properties of Grb10
Other proteins that
bind to the insulin receptor carboxyl terminus, p85 PI3-kinase and the
phosphatase Syp, associate preferentially with IRS-1(2) . When
we examined co-precipitation experiments with the GST-Grb10 SH2 fusion
protein, we did not observe this association for Grb10. IRS-1 was
neither detected in protein precipitates from cell lysates containing
wild-type insulin receptors nor in those with the carboxyl-terminal
truncation, although the ability of the insulin receptor mutant to bind
and activate IRS-1 is not
impaired(9, 26, 33) , and the pool of Grb10
SH2 fusion protein that is available for potential binding to IRS-1 is
undepleted (Fig. 3B).
Interaction of Grb10 with Insulin Receptors in Cultured
Cells
Grb10 has been immunologically detected in NIH 3T3 cells
and is represented on SDS-polyacrylamide gels by several bands ranging
from 65 to 80 kDa(29) . We used insulin receptor-overexpressing
NIH 3T3/IR cells (27) to determine if Grb10 can be found in a
complex with the insulin receptor in intact cells. For this purpose
cells were incubated with and without insulin and solubilized, and
proteins were immunoprecipitated from the lysates with antibodies
directed against either the insulin receptor or the Grb10 SH2 domain.
As shown in Fig. 4A, two different antibodies against
the Grb10 SH2 domain co-immunoprecipitated the insulin receptor upon
insulin stimulation of the cells. In a complementary experiment, two
different antibodies against the insulin receptor (one directed against
the
-subunit and another against the
-subunit)
co-precipitated Grb10. Following SDS-PAGE and immunoblotting, the Grb10
antibody, but not preimmune serum (not shown), recognized in both cases
the same 75-kDa band in insulin-stimulated lysates (Fig. 4B). This suggests that, at least in NIH 3T3
cells, predominantly one form of Grb10 is bound to the receptor. It
remains to be determined whether other forms of Grb10 are also able to
associate. On prolonged exposure of the immunoblot in Fig. 4B (not shown), a band of slightly greater
mobility was faintly visible, which showed the same pattern of
insulin-dependent association with the receptor. The association of
endogenous Grb10 with an insulin-stimulated insulin receptor in intact
cells supports a role for Grb10 in insulin action.
Figure 4:
Association of Grb10 with stimulated
insulin receptors in intact cells. NIH 3T3/IR cells were incubated with
and without 100 nM insulin, and proteins were
immunoprecipitated from detergent cell lysates either with two
antibodies against the Grb10 SH2 domain (A) or with antibodies
directed against either one of the two insulin receptor subunits (B). Immunoprecipitates were separated by SDS-PAGE and
characterized by immunoblotting with anti-IR
-subunit (A)
or anti-Grb10 SH2 domain (B)
antibodies.
DISCUSSION
We used the complete intracellular region of the insulin
receptor as a bait to screen a two-hybrid library. We identified one
known and two previously unknown interacting proteins, including a
novel SH2 domain that is being characterized. All sequences also
interact with a comparable IGF-1 receptor bait, which is not unexpected
given the close structural and functional relationship between these
receptors(3) . Several isolated clones encode different
fragments of the regulatory subunit p85 of PI3-kinase, most frequently
of the
-isoform, all of which comprised the more carboxyl-terminal
of the two SH2 domains of p85. The interaction of both SH2 domains of
p85 with the insulin receptor has been shown
before(28, 34) .
The screen retrieved multiple,
independently cloned inserts of the 3` end of the Grb10 coding region.
Grb10 had been cloned earlier from
gt11 expression libraries based
on an association with a carboxyl-terminal epidermal growth factor
receptor fragment, but the interaction appeared rather weak, and a role
of Grb10 in epidermal growth factor action remains
questionable(29, 35) . Recently, the RET receptor has
been shown to be a putative binding partner for Grb10 (36) .
Our results provide several independent lines of evidence that Grb10 is
a novel insulin receptor interactive protein, including specific
binding not only in the two-hybrid system but also in vitro with a GST-Grb10 SH2 fusion protein and in intact NIH 3T3 cells.
The Grb10-insulin receptor interaction depends on an active receptor
kinase and the intact SH2 domain. The interaction appears to be direct,
based on experiments with purified insulin receptors.
Phosphopeptides of each of the seven autophosphorylation motifs in
the insulin receptor
-subunit showed that the highest interaction
affinity with the Grb10 SH2 domain occurs through Tyr(P)-1322.
Consistent with this result, an insulin receptor mutant lacking the
carboxyl-terminal 43 amino acids, including tyrosines 1316 and 1322
(IR
), no longer binds to the Grb10 SH2 domain. A role
for Tyr-1322 as the Grb10 binding site is also consistent with the
Grb10 interaction with the IGF-1 receptor, in which Tyr-1322 is
conserved but Tyr-1316 has been replaced by phenylalanine(3) .
Although the structural requirements for the interaction are not known,
the SH2 domain belongs to group 1, based on a phenylalanine in position
5 of the structural element
D. The composition of Tyr-1322
conforms to the binding properties predicted for this group
(Tyr(P)-hydrophilic-hydrophilic-hydrophobic)(37) .
The p85
regulatory subunit of PI3-kinase and Syp bind through SH2 domains to
the same carboxyl-terminal motif in the receptor(38) . However,
both of these proteins associate preferentially with IRS-1, which
questions the physiological relevance of their direct association with
the insulin receptor(38, 39, 40) . We could
neither detect interaction of the Grb10 SH2 domain with IRS-1 from
cells expressing normal insulin receptors nor from those expressing the
carboxyl-terminally truncated IR
, although this
mutant binds and activates IRS-1
normally(9, 26, 33) . At present we cannot
rule out the possibility that Grb10 might be capable of associating
with the recently identified IRS-2, although this protein is
structurally very similar to IRS-1 and binds to the same motif in the
insulin receptor as IRS-1(15) .
It appears that Grb10
belongs to a family of related proteins, not unlike the members of the
IRS signaling system, which depending on the context and physiological
condition of the cell may have both shared and specific functions as
signaling mediators and may play a role in insulin action. This is
supported by the recent report of an insulin receptor interactive
protein, termed Grb-IR, for which a role in insulin action has been
suggested(41) . Compared with Grb10 a distinct tissue
distribution and a smaller size has been reported for Grb-IR, but both
proteins have, like IRS-1 and IRS-2, similar domain structures of
distinct regions with generally high but varying degrees of amino acid
similarities(41) .
There is increasing evidence that the
insulin receptor carboxyl terminus plays an important role not only in
mitogenesis (42) but especially in the metabolic functions of
the insulin receptor(43) . Since our findings imply a role for
Grb10 in insulin signaling that is distinct from that of IRS-1 and
IRS-2, the investigation of Grb10 may help to clarify the role of the
carboxyl-terminal region in insulin signaling.