Site-Directed Mutagenesis and Yeast Two-Hybrid Studies of the Insulin and Insulin-Like Growth Factor-1 Receptors: The Src Homology-2 Domain-Containing Protein hGrb10 Binds to the Autophosphorylated Tyrosine Residues in the Kinase Domain of the Insulin Receptor
Lily Q. Dong,
Sarah Farris,
Jeff Christal and
Feng Liu1
Department of Pharmacology The University of Texas Health
Science Center at San Antonio San Antonio, Texas 78284-7764
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ABSTRACT
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To characterize the structural basis for the
interaction between hGrb10 and the insulin receptor and the
insulin-like growth factor-1 receptor, different mutant receptors
containing a segment of deletion in either the juxtamembrane domain or
in the C terminus of the receptors, or containing
tyrosine-to-phenylalanine point mutations in these regions of the
insulin receptor, were generated. Yeast two-hybrid and in
vitro binding studies of the interaction between the mutant
receptors and hGrb10 revealed that tyrosine residues in these regions
are not essential for the binding of hGrb10. To further identify the
binding site for hGrb10, all conserved tyrosine residues in the kinase
domain of the insulin receptor were replaced with either phenylalanine
or alanine by site-directed mutagenesis. Mutations of all tyrosine
residues in this region, except at positions 1162/1163, did not inhibit
the binding of the receptor to hGrb10. The binding of the Src homology
2 domain of hGrb10 to the receptors was significantly enhanced in the
presence of an intact pleckstrin homology domain. Our findings suggest
that, unlike other Src homology 2 domain-containing proteins, hGrb10
binds to the autophosphorylated tyrosine residues in the kinase domain
of the insulin receptor, and the pleckstrin homology domain plays an
important role in hGrb10/receptor interaction. Because the
autophosphorylated tyrosine residues are critical for the
autophosphorylation and kinase activity of the receptor, the binding of
hGrb10 at these sites may suggest a role for the protein in the
transduction or regulation of insulin receptor signaling.
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INTRODUCTION
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The insulin receptor (IR) and insulin-like growth factor I
receptor (IGF-1R) are heterotetrameric transmembrane proteins that show
a high degree of structural and functional similarities. They both
consist of two extracellular
-subunits and two transmembrane
ß-subunits. The juxtamembrane and the kinase domains of these two
receptors are highly homologous. Both receptors contain a NPXY motif in
their juxtamembrane regions that has been shown to bind IR substrate-1
(IRS-1) (1), Shc (2), and GTPase-activating protein (GAP) (3). Mutation
of the conserved tyrosine residue in the NPXY motif of the IR abolishes
the interaction between the receptor and IRS-1 in vitro (4)
and in the yeast two-hybrid system (5) and impairs insulin-stimulated
metabolic and mitogenic effects in cells (6, 7). Both receptors also
contain three tyrosine residues in close proximity in the kinase
domains (Tyr1158, Tyr1162, and
Tyr1163 in the IR and Tyr1131,
Tyr1135, and Tyr1136 in the IGF-I. [(The
numbering systems used for IR and IGF-1R are Ebina et al.(8)
and Ullrich et al.(9), respectively.] x-Ray crystallography
of the human IR indicates that Tyr1162 is bound to the
active site of the receptor kinase (10). Autophosphorylation of these
residues has been shown to play a critical role in the activation of
the receptor kinases toward their cellular substrates (7, 11, 12).
Despite the high sequence homology in the juxtamembrane and kinase
domains, the C-terminal region of the two receptors has only
limited (44%) homology (9). For example, there are two
autophosphorylation sites (Tyr1328 and Tyr1334)
in the C-terminal domain of the IR, but only one of them
(Tyr1334) is conserved between the IR and IGF-1R. The two
tyrosine residues in the C-terminal of the IR have been shown to be the
binding sites for Src homology 2 (SH2) domain-containing proteins such
as the p85 subunit of phosphatidylinositol (PI) 3-kinase,
syp (3), and Shc (13), and have been suggested to play a
role in modulating mitogenic function (14, 15, 16).
To identify potential molecules involved in the IR-signaling pathway,
we have recently used the yeast two-hybrid technique with the IR
cytoplasmic domain as bait to find its interacting proteins. We
identified a SH2 domain-containing protein hGrb10 that binds
specifically to tyrosine-phosphorylated IR (17). Unlike other SH2
domain-containing proteins such as p85 and syp, hGrb10 does
not bind to IRS-1 in vivo. Several isoforms of hGrb10, which
differ in their pleckstrin homology (PH) domain and in the N-terminal
region, have been found in skeletal muscle, fat, and HeLa cells
(17, 18, 19). Expression in cells of the isoform containing a deletion in
the PH domain (Grb-IR/hGrb10
) inhibits insulin-stimulated substrate
tyrosine phosphorylation and PI 3-kinase activity, suggesting that this
protein may play a role in the regulation of insulin action (17).
To better understand the mechanisms of hGrb10 involvement in the IR or
the IGF-1R signal transduction pathways, we decided to further
characterize the interaction between hGrb10 and these receptors. The
data presented in this paper show that, unlike other SH2
domain-containing proteins, which bind to either the juxtamembrane
domain or the C-terminal region of the IR or IGF-1R, hGrb10 binds
specifically to the autophosphorylated tyrosine residues in the kinase
domain of the receptors. Because the autophosphorylated tyrosine
residues in the kinase domain of the receptors are critical for
receptor autophosphorylation and kinase activity, the direct binding of
hGrb10 to these residues may provide a mechanism for the regulation of
receptor signaling.
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RESULTS
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The Binding of hGrb10 to the IR and IGF-1R Is through a Direct
Interaction between the SH2 Domain of the Protein and the
Phosphotyrosine Residues on the Receptors
We have previously shown that the binding of the SH2
domain-containing protein hGrb10 to the IR requires the tyrosine
phosphorylation of the receptor (17). Although this finding suggests
that the binding is probably through a direct interaction between the
phosphorylated tyrosine residue(s) of the receptor and the SH2-domain
of hGrb10, it is also possible that the phosphorylation-induced
conformational change of the receptor, rather than the
autophosphorylated tyrosine residue itself, is required for hGrb10 to
bind. To test whether the SH2 domain of hGrb10 is directly involved for
binding to the IR and IGF-1R, we replaced the conserved arginine
residue (Arg474) within this domain to glutamine and
studied the interaction of the mutant SH2 domain to the IR using the
yeast two-hybrid system. We found that the point mutation within the
SH2 domain completely abrogated the binding of hGrb10(SH2) to the
IRcyto and IGF-1Rcyto (data not shown). To
further investigate this interaction, we generated a mutant IR in which
the conserved lysine residue within the ATP-binding site of the IR was
mutated to alanine (IRK1030A). Yeast two-hybrid studies
revealed that this IR mutant, which was unable to autophosphorylate
itself, failed to bind to hGrb10(SH2) (data not shown). These findings
provide further evidence that the binding of hGrb10 to the receptor is
through a direct interaction between the SH2 domain of hGrb10 and the
autophosphorylated tyrosine residues on the IR.
Tyrosine Residues in the Juxtamembrane or the C Terminus of the IR
and IGF-1R Are Not Essential for the Binding to hGrb10
There are a total of 13 tyrosine residues in the cytoplasmic
domain of the human IR (15 in the cytoplasmic domain of the IGF-1R). At
least six of these tyrosine residues, Tyr972 in the
juxtamembrane domain, Tyr1158, Tyr1162 and
Tyr1163 in the kinase domain, and Tyr1328 and
Tyr1334 in the C-terminal region, have been shown to
undergo insulin-stimulated autophosphorylation. To determine which
residue(s) is involved in binding hGrb10, we first mapped the regions
on the receptor involved in the binding. We constructed several yeast
two-hybrid plasmids encoding the cytoplasmic domains of IR or IGF-1R
with deletions at either the juxtamembrane domain or the C-terminal
region (Fig. 1A
). Significant ß-Gal
activity was detected when all of these GAL4BD/IR fusion
proteins, except for GAL4BD/IR
CT, which has
a 47-amino acid deletion in the C-terminal region of the IR, were
coexpressed in yeast cells with GAL4AD/hGrb10(SH2) fusion
protein (Fig. 1A
). Our data suggest that tyrosine residues in either
the juxtamembrane domains of the IR and IGF-1R or in the C-terminal
region of the IGF-1R are not essential for the binding of hGrb10.

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Figure 1. Interaction of hGrb10 with the IR and IGF-IR in the
Yeast Two-Hybrid System
A, Mapping the interacting domains of the IR and IGF-1R with hGrb10.
Yeast two-hybrid plasmids containing deletions in either the
juxtamembrane domain (JM) or the carboxyl-terminal (CT) of the
receptors were constructed by PCR and subcloned as described in
Materials and Methods. Six individual colonies of yeast
transformants were streaked onto a Trp-Leu-
plate and incubated at 30 C overnight. The interaction was determined
by ß-galactosidase filter assays. (++) indicates high binding
affinity (blue), and (-) indicates no apparent binding
(white) in the assays. B, Construction of GAL4
activation (AD)/hGrb10 yeast two-hybrid plasmids. The PH domain and the
SH2 domain are indicated.
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The observation in the two-hybrid system that a deletion in the
C-terminal region of the IR abolished the binding of the receptor to
hGrb10 suggests that either tyrosine residues in this region or a
certain conformation of the receptor, which was abolished by the
truncation, are required for the binding. To test whether the two
tyrosine residues in the C-terminal region of the IR are involved in
binding hGrb10, two experiments were carried out. First, we studied the
in vitro interaction between the GST/hGrb10(SH2) fusion
protein and a mutant IR in which the C-terminal 69-amino acid residues
were deleted (IR
69). Lysates from insulin-treated
Chinese hamster ovary (CHO) cells overexpressing IR
69
(CHO.IR
69) were incubated with immobilized
glutathione-S-transferase (GST) or GST/hGrb10 fusion
proteins or with wheat germ agglutinin (WGA) agarose to precipitate the
total IR
69 in the lysate. The hGrb10-associated proteins
were separated by SDS-PAGE, transferred to a nitrocellulose membrane,
and detected by immunoblotting using the antibody against
phosphotyrosine (Fig. 2A
) or the
-subunit of the IR (Fig. 2B
). As shown in Fig. 2
, GST/hGrb10(SH2)
precipitated a significant amount of the mutant IR
69 in
insulin-stimulated cells (lane 4). No IR
69 was
precipitated by the GST control (Fig. 2
, lanes 1 and 2) or by the
GST/hGrb10(SH2) fusion protein from non-insulin-treated cells (Fig. 2
, lane 3). The conclusion that Tyr1328 and
Tyr1334 are not the binding sites for hGrb10 was further
confirmed by site-directed mutagenesis and yeast two-hybrid studies. As
shown in Fig. 3A
, substitution of the two
tyrosine residues with phenylalanine did not inhibit the binding of the
mutant receptors to hGrb10 in the yeast two-hybrid system, suggesting
that these residues were not essential for the binding to hGrb10. On
the other hand, mutation of Tyr1334 to phenylalanine
abolished the interaction between the receptor and the p85 subunit of
PI 3-kinase (Fig. 3B
). Our results are consistent with the data of
Staubs et al. (3), who found that the interaction between
p85 and the IR was inhibited by the peptide containing a
phosphotyrosine at position 1334. These findings also provide evidence
that the two SH2 domain-containing proteins, hGrb10 and p85, bind to
the IR at different sites.

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Figure 3. Test of hGrb10 (A) or p85 (B) Interaction with the
IR-Containing Tyrosine to Phenylalanine Mutations at the C-Terminal of
the Receptor
The interaction was determined by ß-galactosidase liquid assays as
described in Materials and Methods. The
ß-galactosidase (ß-Gal) values are means ± SD of
two to six independent assays, and each assay is an average of
triplicate determinations. The data were analyzed by one-way ANOVA and
the post hoc analysis was conducted using Fishers
protected least-significant difference (PLSD) to determine the
significance of difference between the wild type and the individual
mutants. **, P < 0.01.
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Identification of Tyr1162/1163 on the IR as
the Binding Site for hGrb10
Having found that tyrosine residues in either the juxtamembrane
domain or the C-terminal region of the IR or IGF-1R are not essential
for the binding of hGrb10, we focused our attention on the tyrosine
residues in the kinase domain of the receptors. Sequence comparison
between IR and IGF-1R indicated that seven of the eight tyrosine
residues in the kinase domain of the IR, including Tyr1011,
Tyr1087, Tyr1122, Tyr1158,
Tyr1162, Tyr1163, and Tyr1210, are
conserved between the two receptors (Fig. 4
). To determine which tyrosine residues
are involved in the binding of hGrb10, we replaced all the conserved
tyrosine residues, individually or in combination, with either
phenylalanine or alanine by site-directed mutagenesis. Yeast two-hybrid
studies of these mutant IRs with hGrb10 showed that mutation of
tyrosine residues at position 1011, 1087, 1122, 1158, and 1210 of the
IR had almost no effect on the binding of the receptor to hGrb10 (Fig. 5
). In contrast, substitution of tyrosine
residues at position 1162 and 1163 with alanine or phenylalanine
significantly decreased the binding (Fig. 5
).

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Figure 4. The Alignment of the Kinase Domain (KD) of the IR
and IGF-1R
The conserved tyrosine residues are shown in bold
characters.
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Figure 5. Yeast Two-Hybrid Studies of the Interaction between
hGrb10 and the IR Kinase Domain Mutants
The interaction was determined by ß-galactosidase liquid assays as
described in Materials and Methods. The
ß-galactosidase (ß-Gal) values are means ± SD of
three to six independent assays, and each assay is an average of
triplicate determinations. The data were analyzed by one-way ANOVA, and
the post hoc analysis was conducted using Fishers PLSD
to determine the significance of difference between the wild type and
the individual mutants. **, P < 0.01.
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The Involvement of the PH Domain in hGrb10/Receptor Interaction
We have previously shown that there are at least two isoforms of
hGrb10: one has a 46-amino acid deletion in the PH domain (hGrb10) and
another contains an intact PH domain (17). Using a 0.9-kb hGrb10a cDNA
(17) as probe, we screened a human skeletal muscle cDNA library and
cloned the isoform containing an intact PH domain (hGrb10
, GenBank
accession number AF001543 and Fig. 1B
). To test whether the difference
in the PH domain plays a role for hGrb10 binding to the IR or IGF-1R,
we studied the interaction between the IR or IGF-1R and the full-length
hGrb10
isoforms with or without an intact PH domain by the yeast
two-hybrid system. As shown in Fig. 5
, the presence of an intact PH
domain significantly enhanced the binding of hGrb10 to both the IR and
IGF-1R. The interaction, however, was abolished when the conserved
arginine residue in the SH2 domain of hGrb10
was mutated to a
glutamine or Tyr1162/1163 of the IR were replaced with
phenylalanine (Fig. 6
). On the other
hand, truncation mutation at the juxtamembrane domain or point
mutations at tyrosine residues 1011, 1087, 1122, 1158, 1210, and
1328/1334 of the IR had similar or increased activities compared with
that of the wild type IR in the ß-gal liquid assays (data not shown),
suggesting that these tyrosine residues are not directly involved in
the binding. These data provide further evidence that the interaction
between hGrb10 and the IR is through a direct interaction between the
SH2 domain of the protein and Tyr1162/1163 of the IR.

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Figure 6. Interaction of hGrb10 Isoforms with the IR and
IGF-1R in the Yeast Two-Hybrid System
The interaction was determined by ß-galactosidase liquid assays as
described in Materials and Methods. The
ß-galactosidase (ß-Gal) values are means ± SD of
four to six independent assays, and each assay is an average of
triplicate determinations. The data were analyzed by one-way ANOVA
using the SPSS computer program (Prentice Hall, Upper Saddle River,
NJ).
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DISCUSSION
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hGrb10 is a newly identified SH2 domain-containing protein that
binds with high affinity to the IR and IGF-1R. Our previous studies
showed that expression of hGrb10 in cells expressing the IR inhibited
insulin-stimulated GAP-associated p60 protein tyrosine phosphorylation
and PI 3-kinase activity (17). However, the underlying mechanism of the
inhibition has not been elucidated.
One possible explanation for the inhibition of insulin action by hGrb10
is that hGrb10 binds to a site on the IR that blocks the binding of
other downstream signaling molecules. To test this hypothesis, we
attempted to identify the binding site of hGrb10 to the receptor. Our
results show that, unlike other SH2 domain-containing proteins such as
p85, Grb2, and Shc, which bind either to the juxtamembrane domain or
the C-terminal of the IR, hGrb10 binds to the autophosphorylated
tyrosine residues in the kinase domain of the IR. This finding is
consistent with the result from the recent study of ONeill et
al. (18), who showed that the binding of the hGrb10
splice
variant Grb10/IR-SV1 (hGrb10ß) to the IR and IGF-1R was independent
of the juxtamembrane domain and the C-terminal region of the receptors
and was significantly reduced when the tyrosine residues at positions
1162 and 1163 of the IR were changed to phenylalanines, although they
could not exclude the possibility that other tyrosine residues in the
region may be involved in the binding. Data presented in this study
showed that substitution of all the conserved tyrosine residues at
positions 1011, 1087, 1122, and 1210 in the kinase domain of the IR did
not inhibit the binding of the receptor to hGrb10, suggesting that
these tyrosine residues are not the binding site for hGrb10. On the
other hand, mutation of Tyr1162 and Tyr1163
significantly inhibited the binding. The binding of the SH2 domain of
hGrb10 to the autophosphorylated tyrosine residues in the kinase domain
was supported by the observation that a phosphopeptide corresponding to
the sequence of the IR containing Tyr1158/1162/1163 bound
to the GST-hGrb10(SH2) but not to the GST fusion protein in
vitro (our unpublished observations). This conclusion is also
consistent with the recent finding that the binding of the SH2 domain
of hGrb10ß was inhibited by the same IR activation loop
phosphopeptide (19). This finding, however, is contradictory to that of
Hansen et al. (20), who reported that the hGrb10 mouse
homolog mGrb10, whose SH2 domain sequence is 99% identical to that of
hGrb10, binds to the phosphotyrosine residue at 1334 in the C terminus
of the IR. The reason for the discrepancy between our results and those
of Hansen et al. (20) is unclear. However, a recent study
has shown that Tyr1316 of the human IGF-1R (equivalent to
Tyr1334 of the human IR) is not the site for mGrb10 to bind
(21).
Our data have shown that replacement of the two tyrosine residues at
the C-terminal regions resulted in a 2-fold gain of function for
binding of the receptor to hGrb10 (Fig. 3A
). It is interesting to note
that this same mutant was 2-fold more active than the wild type for
poly(Glu/Tyr) phosphorylation in vitro (22). These data
suggest that the two C-terminal tyrosine residues may play a role in
modulating affinities of the IR, probably by blocking the critical
residues in the activation loop of the receptor from their downstream
substrates or binding proteins. These findings are consistent with the
results from many studies that show that the C-terminal region of the
IR plays an important role in insulin action (23, 24).
The observation that the interaction between hGrb10 and the IR or
IGF-1R was significantly increased in the presence of an intact PH
domain suggests that the PH domain may play a role in the interaction,
either due to a direct interaction of this motif to the IR or to the
generation of a PH-domain-induced conformation of hGrb10 that assisted
the interaction. The finding that a single amino acid mutation in the
SH2 domain of hGrb10 completely abolished the binding of the protein to
the receptors suggests that the latter hypothesis is more likely. This
conclusion is consistent with our findings that either an intact PH
domain, or the full-length protein of hGrb10
with a 180-amino acid
deletion at the C terminus [which includes the SH2 domain and the
insert between the PH and the SH2 domain (IPS)] did not interact with
the IR or IGF-1R in the yeast two-hybrid system (S. Farris and L.
Q. Dong, unpublished data). These results suggest that neither the PH
domain nor the N-terminal region of hGrb10 isoforms was sufficient to
bind to the IR and that the SH2 domain of hGrb10 is directly involved
and is sufficient to bind to the phosphotyrosine residues on the
receptors. It is possible that a new binding site, which recognizes the
autophosphorylated IR, may be generated in hGrb10 after the protein
binds to the IR. This hypothesis is consistent with the recent finding
that the IPS region of hGrb10 can bind to the IR in the yeast
two-hybrid system (24a, 24b).
The direct binding of hGrb10 to the activation loop of the IR may show
its physiological relevance. x-Ray crystallography of the IR showed
that the autophosphorylated tyrosine residues are part of the active
site of the receptor tyrosine kinase (10). Numerous studies have also
shown that the autophosphorylated tyrosine residues in the kinase
domain of the IR or IGF-1R play a critical role in receptor
autophosphorylation and receptor kinase activity (1). The binding of
hGrb10 to this region may suggest a role for the protein in the IR and
other growth factor receptor signaling. For example, the binding of
hGrb10 at the active site of the receptor tyrosine kinase may prevent
some downstream substrates from binding to the receptor or to be
phosphorylated by the receptor tyrosine kinase and thus plays a
regulatory role in signaling. This hypothesis is consistent with the
findings that overexpression of Grb-IR/hGrb10
or microinjection of
the GST fusion protein containing the SH2 domain of hGrb10 in cells
inhibits insulin-stimulated PI 3-kinase activity (17) or mitogenesis
(18), respectively. On the other hand, the binding of these hGrb10
isoforms to the autophosphorylated tyrosine residues in the kinase
domain of the receptors may bring certain other substrates closer to
the active site so that specific signaling cascades will continue.
hGrb10 isoforms may thus function as a molecular switch to control
specific signaling pathways. As hGrb10 contains multiple functional
domains, including the SH2 domain, the PH domain, and a proline-rich
sequence at its N terminus, it is capable of binding different
signaling molecules in cells. Identification and characterization of
hGrb10 downstream interacting proteins should provide a better
understanding of the physiological role of of the protein in signaling
processes initialized by insulin or other growth factors.
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MATERIALS AND METHODS
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Plasmids and Cell Lines
Yeast two-hybrid plasmids pGBT9, pGAD424, and pGADGH and the
host strain SFY526 were from CLONTECH (Palo Alto, CA). Restriction
enzymes, DNA ligase, and T4 DNA polymerase are from Life Technologies
(Gaithersburg, MD) and New England Biolabs (Beverly, MA). All
site-directed mutagenesis primers and sequencing primers were
synthesized by Life Technologies. Human IR and IGF-1R cDNAs and
CHO.IR
69 cells, a Chinese hamster ovary cell line
overexpressing the mutant IR with a 69-amino acid deletion in the
C-terminal region (25), were gifts of R. A. Roth (Stanford
University, Stanford, CA). The pGAD/p85 two-hybrid plasmid was provided
by J. Koland (University of Iowa, Iowa City, IA) and was described
previously (26). The construction of plasmids pGBD9/IR,
pGAD/hGrb10(SH2), and pGEX/hGrb10(SH2) and the expression of the
GST/hGrb10(SH2) fusion protein were described previously (17).
Site-Directed Mutagenesis
A 2.4-kb BamHI-XbaI cDNA fragment encoding
the cytoplasmic domain of the human IR was subcloned into plasmid
pBluescript (Stratagene, La Jolla, CA) and used as a template for
site-directed mutagenesis. Mutagenesis was carried out according to the
protocol as described by Kunkel et al. (27) using customized
primers. Complementary DNA fragments encoding different mutant IRs were
generated by PCR and fused to the sequence encoding the Gal4
DNA-binding domain in the plasmid pGBT9. Complementary DNAs encoding
IRY1162/1163A and IRY1158/62/63A were provided
by Dr. B. Zhang (Merck Research Laboratories, Rahway, NJ). All PCR and
site-directed mutagenesis products were confirmed by restriction
mapping and DNA sequencing (detailed mutagenesis and cloning strategies
are available upon request).
Construction of the IR, IGF-1R, and hGrb10 Truncation Mutants
cDNAs encoding the cytoplasmic domain of the IR or IGF-1R
with truncation mutations in either the juxtamembrane domain or in the
C-terminal region were generated by PCR using human IR or IGF-1R cDNAs
as templates, respectively (Fig. 1
). The PCR primers used were: 1)
5'-GCGAATTCGATGGGCCGCTGGGA-3'; 2)
5'-GCGAATTCGTGCCGGACGAGTGGG-3'; 3)
5'-CAGCGTCGACAGTGCGAGGAACG-3'; 4)
5'-CAGCGTCGACATGGTAGAGTCGT-3'; 5)
5'-GCGAATTCAGCAGGCTGG-GGAATG-3'; 6)
GCGAATTCGTTCCTGATGAGTGG-3'; 7)
GAGCGTCGACAGGCTGTCTCTCGTCG-3'; 8)
5'-GAGCGTCGACAGATTCAGGATCCA-3', with the added restriction
sites underlined. After restriction digestion with EcoRI and
SalI, the cDNA fragments were subcloned into the yeast
two-hybrid plasmid pGBT9 to generate different GAL4 DNA binding
domain/IR mutant fusion protein constructs (Fig. 1A
). To generate
different hGrb10 yeast two-hybrid constructs, the following PCR primers
were used: 9) 5'-GCGAATTCCTTTTTGCACCATCC-3'; 10)
GCGAATTCTCGACGCCAGTG-3'; 11)
5'-GCGGATCCATTGCCACGAGG-3'; 12)
5'-GACCTCGAGAGGACATCTGCG-3', with the added restriction
sites underlined (Fig. 1B
).The full-length hGrb10
cDNA
(GenBank accession number AF001543) was obtained by screening a human
muscle cDNA library (Stratagene) using a 0.9-kb Grb-IR/hGrb10
cDNA
as probe (17). To generate the SH2-domain mutant hGrb10
, we replaced
the conserved arginine residue within the SH2-domain
(FLLR529DS) with a glutamine residue. The cDNA encoding the
wild type or mutant hGrb10
was amplified by PCR and subcloned into
the plasmid pGADGH (CLONTECH). Full-length or different truncated
versions of hGrb10 were generated by PCR using cDNAs encoding for
Grb-IR/hGrb10
or its PH domain-containing isoform hGrb10
as
templates. After digestion with the corresponding restriction enzymes,
the cDNA fragments were subcloned into plasmids pGAD GH or pGAD424 to
generate GAL4 activation domain (AD)/hGrb10 constructs (Fig. 1B
).
Transformation of SFY526 yeast cells was carried out by
electroporation.
ß-Galactosidase Filter and Liquid Assays
The recombinant pGBT9/hGrb10 plasmids were used to transform the
yeast host strain SFY526 with plasmids pGAD/hGrb10 by electroporation.
Single colonies of transformants were picked, selected in minimal
medium lacking tryptophan and leucine, and grown in yeast pepton
dextrose (YPD) medium to OD600 of 0.51.0.
ß-Galactosidase activity (Miller unit) was assayed using
o-nitrophenyl ß-D-galactopyranoside as substrates (28).
The values are the means ± SD of three to six
independent assays.
In Vitro Binding of IR
69
with GST/hGrb10 Fusion Protein
CHO.IR
69 cells were grown in Hams F12 medium
containing 10% newborn calf serum to 90% confluence in 100-mm plates.
After being serum starved for 1 h at 37 C, the cells were treated
with 10-8 M insulin for 8 min and lysed in
lysis buffer containing 50 mM HEPES, (pH 7.6), 1
mM EDTA, 150 mM NaCl, 1% Triton-X-100, 10
mM sodium fluoride, 20 mM sodium pyrophosphate,
1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin. Cell lysates were incubated with 15 µg GST or
GST-hGrb10(SH2) coupled to glutathione agarose or with 25 µl of WGA
agarose to precipitate the total IR in the cells. After incubation at 4
C for 4 h, the agarose beads were washed three times with WGA
buffer (50 mM HEPES, pH 7.6, 150 mM NaCl and
1% Triton X-100) and then boiled in SDS sample buffer. The
precipitated proteins were separated by SDS-PAGE and blotted to a
nitrocellulose membrane. The hGrb10-associated IR
69 were
detected by antibodies to either the phosphotyrosine (RC20,
Transduction Laboratories, Lexington, KY) or to the
-subunit of the
IR (3B11, gift of Dr. K. Shii).
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ACKNOWLEDGMENTS
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We thank Dr. Bei Zhang for the IRY1158F and
IRY1162/1163A cDNAs, Dr. Jeffrey E. Pessin for permission
to use, and Dr. John G. Koland for circulating, the p85 yeast
two-hybrid plasmid, and Dr. Kozui Shii for the monoclonal antibody
(3B11) to the IR. We would also like to thank Dr. Richard A. Roth for
the IR and IGF-1R cDNAs and his continuous support and helpful
suggestions.
 |
FOOTNOTES
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Address requests for reprints to: Feng Liu, Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7764.
This research was supported in part by a Grant-in-Aid from the American
Heart Association, Texas Affiliate, Inc., and by a Research Grant from
the Juvenile Diabetes Foundation International.
1 Recipient of the Lyndon Baines Johnson Research Award from the
American Heart Association, Texas Affiliate. 
Received for publication May 13, 1997.
Revision received July 1, 1997.
Accepted for publication July 30, 1997.
 |
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