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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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69). Lysates from insulin-treated
Chinese hamster ovary (CHO) cells overexpressing IR69
(CHO.IR69) were incubated with immobilized
glutathione-S-transferase (GST) or GST/hGrb10 fusion
proteins or with wheat germ agglutinin (WGA) agarose to precipitate the
total IR69 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 IR69 in
insulin-stimulated cells (lane 4). No IR69 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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). 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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 O’Neill 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 0.5–1.0.
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 IR69
with GST/hGrb10 Fusion Protein
CHO.IR69 cells were grown in Ham’s 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 IR69 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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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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