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J. Biol. Chem., Vol. 278, Issue 34, 31564-31573, August 22, 2003
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¶ **
From the
Division of Endocrinology and the Hallett
Center for Diabetes and Endocrinology, Rhode Island Hospital, Brown Medical
School, Providence, Rhode Island 02903 and
||Department of Emergency and Organ
Transplantation, Internal Medicine, Endocrinology, and Metabolic Diseases,
University of Bari, 70124 Bari, Italy
Received for publication, November 13, 2002 , and in revised form, May 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Grb10 is believed to have a role in regulating cellular responses to
insulin, IGF-I, and possibly other hormones. However, published studies on the
effects of Grb10 overexpression in cultured cells have provided conflicting
evidence for stimulatory versus inhibitory actions on mitogenesis and
other hormone responses. For example, overexpression of human Grb10
in
Chinese hamster ovary cells was shown to reduce both insulin-dependent
phosphorylation of IRS-1 and activation of phosphatidylinositol 3-kinase
(4). Data showing an inhibitory
effect of Grb10
on IGF-I-stimulated mitogenesis, as well as a decrease
in the catalytic activity of both insulin and IGF-I receptors
(19), also have been reported
(20). In contrast, other
published work suggests positive effects of Grb10 on insulin and IGF-I action.
Overexpression of a presumed dominant-negative fragment of Grb10, lacking the
BPS-SH2 domain, partially inhibited cell growth stimulation by insulin and
IGF-I in rat fibroblasts (5).
In another study, a presumed dominant-negative SH2 domain fragment decreased
cell proliferation, whereas overexpression of full-length Grb10 increased DNA
synthesis upon growth factor treatment
(21). The conflicting data do
not appear to be attributable to the effects of different Grb10 isoforms or
the properties of specific cell types and thus leave the physiological role of
Grb10, as well its mechanism of action, unclear.
The SH2 domain of Grb10 has been reported to bind to some intracellular molecules such as mitogen-activated receptor 1, mitochondria-associated Raf (22, 23), and the ubiquitin protein ligase Nedd-4 (24), in addition to its interaction with receptor tyrosine kinases. However, there are no data on the functional significance of these interactions as they may relate to Grb10 effects on cell signaling. In a recent study, an interaction between Grb10 and Akt was demonstrated, suggesting a role for Grb10 in promoting Akt translocation to the plasma membrane in response to c-kit receptor tyrosine phosphorylation, thus augmenting Akt activity (16). The determinants of binding of Grb10 to Akt were not elucidated, although the PH and the SH2 domains were excluded.
Because Grb10 has the structure of an adapter protein, it has the potential to function as a bridge protein, with the C terminus bound to phosphorylated receptors and the N-terminal region bound to one or more other proteins. Using the yeast two-hybrid cloning system with the N-terminal portion of Grb10 as a bait, we have identified two novel proteins that bind to Grb10, which we have designated GIGYF1 (Grb10 interacting GYF protein 1) and GIGYF2. Studies on GIGYF1 demonstrate that it associates with Grb10 in cultured cells, becomes linked to activated IGF-I receptors, and modifies IGF-I signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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full-length cDNA
(6) was digested with
EcoRI-SalI. The resulting 516-bp fragment (Grb10-NT), which
includes the complete proline-rich region of Grb10, was subcloned into the
yeast vector pBTM116.
The L40 yeast strain was transformed with pBTM116 containing the fused
LexA-Grb10-NT coding sequence by a standard lithium acetate protocol
(28,
29). After growth in
Trp– medium, L40/pBTM116/Grb10-NT yeast cells were transformed with a
pool of pVP16/9.5-day and pVP16/10.5-day mouse embryo cDNA libraries and,
separately, with a TA-1 mouse fat cell cDNA library in 
GT11.
Transformation with 500 µg of library plasmid DNA yielded 
12 x
106 primary transformants, as confirmed by colony growth in
Trp–/Leu– medium. The yeast were subsequently grown in
Trp–/Leu– liquid medium for 16 h to allow for the expression and
formation of the transactivating LexA/VP16 complex, and a fraction of the
yeast suspension (corresponding to 4 x 106
transformants) was plated on Trp–/Leu–/His– plates. Library
clones showing activation of both reporter genes (HIS3 and
LacZ) were tested in the two-hybrid system for specificity of
association with the Grb10-NT using a LexA-lamin construct as a negative
control. Plasmids from library clones were subsequently isolated from the
yeast, transferred to Escherichia coli, sequenced, and used to search
for similar sequences in GenBankTM.
Full-length GIGYF1 and GIGYF2 cDNA Cloning—Approximately 6
x 105 plaques from a 15-day mouse embryo cDNA library in
GT11 (Clontech, Palo Alto, CA) were screened with standard techniques
using as probes partial cDNAs of GIGYF1 and GIGYF2 obtained from the
two-hybrid screening. Clones obtained by this method containing the largest
inserts of GIGYF1 (lacking a portion of the 5' coding region) and GIGYF2
(lacking both 5' and 3' coding sequences) were chosen for further
study. The remaining 5' coding sequence of GIGYF1 was obtained by
reverse transcriptase PCR of mouse lung poly(A)+ RNA (Ambion,
Austin, TX) using the Smart RACE cDNA amplification kit (Clontech, Palo Alto,
CA), a forward adapter primer, and GIGYF1-specific reverse primer. The product
was cloned into the pCR4-TOPO vector (Invitrogen) and sequenced in both
directions. The missing 5' end of GIGYF2 was amplified from mouse liver
Marathon-Ready cDNA (Clontech) using the Smart RACE cDNA amplification kit as
described for GIGYF1. The terminal 3' coding sequence of GIGYF2 was
amplified from mouse liver poly(A)+ RNA (Ambion) by Smart RACE,
cloned into pCR4-TOPO vector, and sequenced in both directions.
Northern Blotting—GIGYF1 and GIGYF2 probes (571 and 388 bp, respectively) were generated by NotI excision of the GIGYF1 and GIGYF2 cDNAs from the pVP16 plasmids obtained from the two-hybrid screening and labeled with [32P] deoxy-CTP by random priming (multiple DNA labeling kit; Amersham Biosciences). A mouse multiple tissue poly(A)+ mRNA Northern blot (mouse MTNTM blot; Clontech, Palo Alto, CA) was hybridized overnight with radiolabeled probes at 65 °C and washed twice with 2x SSC-0.05% SDS (40 min each) and once with 0.1x SSC-0.1% SDS, and specific mRNA bands were identified using a PhosphorImager (Amersham Biosciences).
Site-directed Mutagenesis—Deletion mutations of the proline-rich region of Grb10 and the GYF domain of GIGYF1 were generated using the ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA) with 25–30-bp primers corresponding to the 5' and 3' ends of each of three proline-rich boxes or the GYF domain, respectively. The proline-rich box 1 deletion (aa 78–89), box 2 deletion (aa 94–102), and box 3 deletion (aa 106–113) were amplified using the appropriate site-specific primers with a fragment of Grb10 in pBluescript as a template. The double proline-rich box deletions 1,2 (aa 78–102), 1,3 (aa 78–89 and 106–113), and 2,3 (aa 94–113) were amplified using appropriate combinations of the same primers as the single box mutations and with single-deletion mutants as templates. The 17-aa GYF domain deletion of GIGYF1 was amplified with specific primers using a fragment of GIGYF1 in pBluescript as template. Point mutations of the 17-aa GYF region of GIGYF1 (W489A, G503A, Y504A, F505A) were generated with the QuikChange site-directed mutagenesis kit (Stratagene). The manufacturer's protocol was followed using sequence-specific primers and the GIGYF1/pGilda construct as a template. All constructs were verified by automated sequencing.
Yeast Two-hybrid Interaction Analysis—Yeast two-hybrid
assays were performed using the LexA-based Matchmaker kit (Clontech). GIGYF1
and GIGYF2 gene fragments were cloned into pGilda, and the Grb10 proline-rich
domain (wild type) was cloned into pB42AD. The Grb10 and GIGYF1 mutants in
pBluescript were subcloned into the pGilda yeast vector at
EcoRI/BamHI and, sequentially, into pB42AD at the
EcoRI/XhoI site. These constructs were transformed into the
EGY48 yeast strain according to the manufacturer's protocol. Interactions
between the Grb10 proline-rich domain and GIGYF1 or GIGYF2 were detected on
selective medium agar plates containing
5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal). A liquid
-galactosidase assay was performed on the co-transformed yeast cells,
using o-nitrophenyl--D-galactopyranoside as
substrate.
GIGYF1 Fragment Transfection—A fragment of GIGYF1 containing aa 410–598 with an N-terminal Myc tag was generated by PCR using a forward primer containing the 10-aa sequence EQKLISEEDL recognized by Myc monoclonal antibody (clone 9E10) (Roche Applied Science) and reverse primer containing specific GIGYF1 cDNA sequence with the largest of the initial GIGYF1 two-hybrid clones as template. The PCR product was cloned into the XhoI/XbaI sites of the pAdTrack vector (from Dr. T. C. He, Howard Hughes Medical Institute, Baltimore, MD), sequence-verified, and used for transient transfection in R+ IGF-I receptor overexpressing fibroblasts (30) (provided by Dr. Renato Baserga, Jefferson Cancer Center, Thomas Jefferson University, Philadelphia, PA). Monolayers of R+ cells (30–40% confluent) in 10-cm dishes were incubated for 6 h with 9 µg of purified pAdTrack plasmid and 30 µl of LipofectAMINE (Invitrogen) in 5.5 ml of OptiMem medium (Invitrogen) and then returned to standard growth medium consisting of Dulbecco's modified Eagle's medium with 4.5 gm/liter glucose, 5% fetal bovine serum (Invitrogen), and 2.5 µg/ml hygromycin B (Sigma). The cells were studied 48 h post-transfection.
For stable transfection studies, a fragment of GIGYF1 containing aa 410 to 598 was generated by PCR using a forward primer designed to insert the six-aa DTYRYI N-terminal AU1 tag and reverse primer containing specific GIGYF1 cDNA sequence with the largest of the initial GIGYF1 two-hybrid clones as template. After cloning into the BamHI/XhoI sites of pcDNA 3.1 (Zeocin+) vector (Invitrogen) and sequence verification, the purified vector was transfected into R+ fibroblasts using the LipofectAMINE protocol as described above. After growth for 30 days in the presence of Zeocin (100 µg/ml) (Invitrogen), clones were isolated by limiting dilution, tested for GIGYF1 expression by immunoblotting with AU1 monoclonal antibody (BabCO, Covance, Richmond, CA), and thereafter maintained in the presence of Zeocin (80 µg/ml).
Immunoprecipitation and Immunoblotting—Transfected R+ cells were incubated for 4 h in serum-free Dulbecco's modified Eagle's medium-H (4.5 g/liter glucose) containing 0.5% bovine serum albumin and 25 mM HEPES. IGF-I was added to the medium at a final concentration of 10–7 M, and the cells were incubated for 0, 2, or 10 min at 37 °C. The cells were washed with ice-cold buffer (137 mM NaCl, 1 mM MgCl2,1mM CaCl2, 0.1 mM Na2VO4, 20 mM Tris-HCl, pH 7.6) and lysed in the same buffer supplemented with 1% Nonidet P-40, 10% glycerol, 2 mM EDTA, 10 mM pyrophosphate, 10 mM NaF, 2 mM Na2VO4, 2 mM phenylmethylsulfonyl fluoride, and 8 µg/ml leupeptin. After clearing by centrifugation, protein concentration was determined by Bradford assay. To determine Grb10/GIGYF1 association, 0.5 mg of cell lysate was immunoprecipitated with anti-Grb10 antibody and blotted with anti-Myc antibody (Roche Applied Science). Phosphorylation of IGF-I receptors and IRS-1 was measured by direct blotting using anti-phosphotyrosine antibody (PY20) (Transduction Laboratories, San Jose, CA). Shc phosphorylation was examined by blotting with a polyclonal phosphospecific Shc antibody (UBI, Lake Placid, NY). Phosphorylation of ERK and Akt was measured using phosphospecific ERK antibody (from Dr. Laurie Goodyear, Joslin Diabetes Center, Boston, MA) and phosphospecific Akt antibody (New England Biolabs, Beverly, MA). Specific antibodies were used to measure the content of IGF-I receptors (31), IRS-1, and Shc (Transduction Laboratories, San Jose, CA) by direct immunoblotting. Specific protein bands were identified with 125I-protein A and quantified on a PhosphorImager or identified by ECL (PerkinElmer Life Sciences) and quantified on a densitometer.
Statistical Analysis—Data from densitometric analysis were adjusted to reflect percent stimulation of specific proteins in transfected cells at each time point with respect to empty vector transfected controls. All data are expressed as means ± S.E. and were subjected to a Kolmogorov-Smirnov test for normality, followed by paired t tests for comparisons between transfected cells and empty vector controls at each time point.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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that contains
several proline-rich boxes but lacks the PH, BPS, and SH2 domains. The yeast
vector pBTM116 expressing a fragment of mouse Grb10 (aa 1–172) as
a fusion protein linked to the C terminus of the DNA binding domain of the
transcription factor LexA was introduced into the yeast reporter strain L40.
Yeast expressing the pLexA-Grb10 fragment fusion protein were then transformed
with combined 9.5- and 10.5-day mouse embryo or differentiated TA-1 fat cell
cDNA expression libraries in fusion with the VP16 transcriptional activation
domain (25). With this system,
positive clones are identified by detecting the activation of two reporter
genes, HIS3 and LacZ, inserted into the L40 yeast genome with multiple LexA
upstream activating sequences. A total of 64 and 78 yeast transformants were
obtained from the embryo and fat cell libraries, respectively, representing 15
independent Grb10-interactive clones from the two libraries. Five of these
clones consisted of partial sequences of two novel related proteins, which we
have designated GIGYF1 and GIGYF2. GIGYF1 was encoded by three independent
clones from the embryonic libraries and one clone from the fat cell library,
whereas GIGYF2 was encoded by a single clone from the fat cell cDNA library.
The GIGYF1 cDNA defined by the yeast two-hybrid clones represented a 571-bp
ORF lacking a translation start site and stop codon. Similarly the GIGYF2 cDNA
represented a 388-bp ORF lacking 5' and 3' terminal coding
sequence. The remaining 10 clones were represented in single copy and were
unrelated to GIGYF1 and GIGYF2. Full-length Cloning and Structural Analysis of GIGYF1 and GIGYF2—An extended 2473-bp cDNA encoding an additional 594 bp of 5' sequence and the complete 3' sequence was obtained by screening a mouse 15-day embryo cDNA library using the largest GIGYF1 cDNA from the two-hybrid cloning as a probe. An overlapping clone containing the remaining 659 bp of 5' coding sequence was generated from mouse lung poly(A)+ RNA by 5'-RACE PCR, and full-length GIGYF1 cDNA containing 3132 bp of coding sequence was assembled in pBluscript by restriction digestion-ligation of the two overlapping clones. The full-length GIGYF2 cDNA (3876 bp of coding sequence) was obtained in a similar manner. Screening of the mouse 15-day embryo cDNA library yielded a 2677-bp clone lacking both 5' and 3' terminal coding sequence. These segments were generated by 5'- and 3'-RACE PCR using mouse liver poly(A)+ RNA, and the fragments were assembled by restriction digestionligation of overlapping clones.
Analysis of nucleotide and amino acid sequences revealed that GIGYF1 and
GIGYF2 are homologous proteins with common structural elements encoded by
distinct genes (Fig. 1, A and
B). Structural motifs present in both GIGYF1 and GIGYF2
include three potential tyrosine phosphorylation sites and candidate binding
sites for protein phosphatase-1 (RVPF), 14–3-3 proteins (RSpSXP), and
phospholipase C
(V(F)DDY). In addition, there is a proline-rich region
in both proteins, a consensus bipartite nuclear localization motif, and a
region of clathrin light chain homology
(Fig. 1B). A striking
feature is multiple stretches of Gln and Glu residues in the C-terminal half
of both proteins (Fig.
1A).
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Using the cloned cDNAs, mouse genomic sequences corresponding to both
GIGYF1 and GIGYF2 were identified in GenBankTM. The GIGYF1 gene mapped to
mouse chromosome 5 as a relatively small gene (7000 bp) with a coding
sequence of 3 kb composed of 24 exons and very short introns. Our cloned
GIGYF1cDNA corresponds to an ORF that has been designated Perq1 (XM_124667)
based on its high abundance of Pro, Glu, Arg, and Gln
(32). There are several single
nucleotide differences in the cloned GIGYF1 in comparison with the predicted
ORF, three of which would result in amino acid sequence substitutions. The
GIGYF1 cDNA that we have cloned includes an additional 30 aa extending
N-terminally from the putative start site of the Perq1 protein proposed on the
basis of genomic sequence. This region is encoded by an ORF that includes an
addition 5' exon and starts with a strong Kozak consensus ATG (ANNatgG)
(33). The cloned cDNA also
includes a run of 12 CAG triplets in the C-terminal region, whereas only nine
CAG repeats are noted in the comparable region of genomic sequence. Although a
homologous Perq1 gene product (NM_022574
[GenBank]
) has been predicted by the analysis
of human chromosome 7q22 (34)
and both human (AK001739
[GenBank]
and AB014542
[GenBank]
) and chicken (U90567
[GenBank]
) EST sequences,
there are no published data on the expression or functional properties of
Perq1.
The GIGYF2 gene is located on mouse chromosome 1 (contig NW_000169),
although the exact chromosomal location is still ambiguous. GIGYF2 is encoded
by 27 exons and 26 large introns. Despite a high degree of homology in
sequence and exon structure in GIGYF1 and GIGYF2, the GIGYF2 gene spans a much
larger region of gene sequence (90,000 versus 7000 bp of
GIGYF1). The GIGYF2 cDNA that we have cloned encodes a protein identical to a
predicted gene product described as similar to TNRC15, which has been
identified in mouse genomic contig NW_ 000169. Partial cDNAs encoding this
sequence have been identified from at least two different mRNA sources
(BC027137
[GenBank]
and BC030845
[GenBank]
). The homologous human GIGYF2 gene is located on
chromosome 2q36.1 and was designated TNRC15
(trinucleotide repeat containing
15) (locus identification 26058). Evidence for mRNA expression has
been obtained through a study screening for large proteins in human brain
(35).
Tissue Distribution of Mouse GIGYF1 and GIGYF2—Northern
blotting experiments on multiple mouse tissues were performed to examine the
distribution of GIGYF1 and GIGYF2 mRNA. As shown in
Fig. 2A, a single band
of 6 kb corresponding to the GIGYF1 message was detectable in all of the
tissues tested. The highest levels of mRNA were seen in brain and lung, with
heart, spleen, and kidney also exhibiting comparatively high levels. GIGYF1
mRNA is poorly expressed in liver and testis and very low in skeletal muscle,
where only a faint band was detected. When the same tissues were probed for
GIGYF2, a 7-kb transcript was detected. Expression levels are highest in heart
and liver, somewhat lower in kidney and brain, and barely detectable in lung
(Fig. 2B). In
proportion to the 7-kb band, there was a second 1.5-kb band in these same
tissues. The significance of this mRNA species, which is not large enough to
encode the full GIGYF2 coding sequence, is not known. GIGYF2 mRNA was not
measurable in spleen and skeletal muscle. In testis a single GIGYF2 mRNA band
with a distinct size of 4 kb was identified.
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Analysis of Structural Requirements for Grb10 Binding— The only candidate protein-protein interaction motif identified in the fragment of Grb10 used in the two-hybrid cloning of the GIGYF proteins consisted of three blocks of proline-rich sequence. Analysis of the GIGYF1 and GIGYF2 sequences revealed a region with high homology to the GYF domain described recently (Fig. 3A) (36). This 17-aa motif in T-lymphocyte CD2-binding protein 2 (CD2BP2) was shown to mediate association of two proline-rich boxes in CD2 with CD2BP2 (37). To determine its role in GIGYF binding to Grb10, a pGilda yeast two-hybrid vector was prepared containing sequence equivalent to the initial GIGYF two-hybrid clone from which the entire 17-aa GYF was deleted by excision mutagenesis. This resulted in a total loss of binding to the proline-rich region of Grb10, as determined by two-hybrid plaque assays (not shown) and liquid-phase assays (Fig. 3B). To further investigate the association of the GYF domain of GIGYF1 with Grb10, four mutant constructs were generated with Ala substituted for a single aa shown previously (37) to be critical for the interaction of CD2BP2 with CD2. As shown in Fig. 3B, the W489A, G503A, and Y504A substitutions within the GYF domain resulted in a marked decrease in binding of GIGYF1 to Grb10. Substitution of the C-terminal aa of the GYF motif (F505A) resulted in complete inhibition of binding.
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The role of the three proline-rich boxes of Grb10 in GIGYF binding was investigated in two-hybrid assays using Grb10 constructs with different proline-rich domain deletions (Fig. 4). Elimination of proline-rich box 2 (PGSPPSVAP) had no effect on Grb10 binding to GIGYF1 or GIGYF2 (Fig. 4, B and C). Deletion of box 1 (SLPAIPNPFPEL) resulted in a modest but significant decrease in GIGYF2 binding to Grb10 but no change in GIGYF1 binding. Deletion of box 3 (PPPPSQPP) resulted in a significant decrease in binding of Grb10 to GIGYF1 and a suggestive decrease in GIGYF2 binding, which did not reach statistical significance. Excision of any two proline-rich boxes led to an essentially complete loss of Grb10 binding to GIGYF1 and GIGYF2. The data indicate distinct influences of individual proline-rich domains on Grb10-GIGYF binding and a critical requirement for the presence of at least two of the three proline-rich sequences.
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Grb10-GIGYF1 Interaction in Intact Cells—To assess the
interaction of GIGYF1 with Grb10 in intact cells, a Myc-tagged fragment of
GIGYF1 corresponding to the sequence originally cloned by two-hybrid screening
was generated in the pAdTrack vector and transiently transfected into R+
fibroblasts, a cell line expressing a high number of IGF-I receptors
(approximately 1 x 106/cell). Grb10 association with
GIGYF1 was assessed by immunoprecipitation with Grb10 antibody and
immunoblotting with Myc antibody. Cells transfected with empty vector were
used as control. Binding of the transfected Myc-GIGYF1 fragment to Grb10 was
evident in the basal state, increased markedly 2 min after IGF-I stimulation,
and then became essentially undetectable 10 min after IGF-I
(Fig. 5A).
Quantitative analysis of three independent experiments indicated that the
fraction of total cellular GIGYF1 fragment associated with Grb10 increased
3.5-fold after IGF-I treatment from 0.9% of the total GIGYF1 fragment in
the basal state to 3.4% at 2 min after IGF-I. Because partial dissociation of
GIGYF1 from Grb10 likely occurred during the immunoprecipitation procedure,
this represents a minimum estimate of the fraction of GIGYF1 associated with
Grb10 as a consequence of IGF-I stimulation. Co-precipitation of GIGYF1 with
IGF-I receptors was demonstrated 2 min after IGF-I stimulation but
undetectable in the basal state and 10 min after IGF-I
(Fig. 5B). As
described previously in our laboratory
(6), the co-precipitation of
Grb10 with IGF-I receptors was absent in the basal state, rapidly stimulated
by IGF-I, and persisted for >20 min (data not shown). These data are
consistent with the following: 1) GIGYF1 binding to Grb10 in the basal state,
2) increased GIGYF1-Grb10 binding and recruitment to the GIGYF1-Grb10 complex
to the activated IGF-I receptor, and 3) dissociation of GIGYF1 from a
persistent Grb10-IGF-I receptor complex after several minutes.
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GIGYF1 Effects on IGF-I Signaling—To evaluate the effects of
GIGYF1 on IGF-I signaling, the same GIGYF1 fragment used in the
co-precipitation studies described above was AU1-tagged and cloned into the
pcDNA 3.1 vector. After transfection into R+ fibroblasts, multiple cell clones
stably overexpressing similar levels of GIGYF1 were obtained
(Fig. 6A). Signaling
responses to IGF-I were compared in these cells and R+ fibroblasts transfected
with empty pcDNA 3.1 vector. Overexpression of the GIGYF1 fragment had no
effect on basal IGF-I receptor tyrosine phosphorylation but resulted in an
60% increase in receptor phosphorylation 2 min after IGF-I stimulation
and a less but still significant (41%) increase 10 min after IGF-I
(Fig. 6, B and
C). A similar increase in tyrosine phosphorylation of two
cellular signaling proteins immediately downstream from the IGF-I receptor
also was observed with GIGYF1 fragment overexpression. IRS-1 tyrosine
phosphorylation was unaffected in the basal state and increased 50% at 2 min
and 40% at 10 min after IGF-I in GIGYF1 overexpressing cells
(Fig. 7, A and
B). Tyrosine phosphorylation of the p52 Shc isoform was
unaltered under basal conditions and increased by 62% at 2 min and 38% at 10
min after IGF-I stimulation in cells overexpressing GIGYF1
(Fig. 8, A and
B).
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Signaling intermediates downstream from IRS-1 and Shc were assessed to evaluate the propagation of the GIGYF1-augmented IGF-I responses. A significant increase in the Thr phosphorylation (activation) of Akt was evident 2 min after IGF-I stimulation (50% higher in GIGYF1-transfected cells) but not at 10 min after IGF-I (Fig. 9A). A similar effect on ERK1 and ERK2 was observed (significant 39% increase at 2 min after IGF-I in GIGYF1 transfected cells and no effect of GIGYF1 10 min after IGF-I) (Fig. 9B). The effects of overexpression of a full-length construct of GIGYF1 on IGF-I signaling were not investigated because of extensive inclusion body formation (38) in cells transfected with the full-length cDNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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extending from the N
terminus to just upstream from the PH domain, thus excluding the
receptor-binding BPS and SH2 domains, we screened mouse embryo and fat cell
expression libraries. This resulted in the identification of a novel family of
two proteins, GIGYF1 and GIGYF2, which are encoded by distinct genes but share
a 17-aa GYF motif that mediates their binding to a proline-rich region in
Grb10.
GIGYF1 and GIGYF2 exhibit extensive regions of high aa sequence homology
and an overall aa homology of 40%. The 17-aa GYF domain was described
initially as a proline-rich interactive motif in CD2BP2
(37), and, based on crystal
structure data, it has been proposed that a bulge-helix-bulge conformation of
the GYF domain interacts with paired proline-rich sequences
(36). Using mutant constructs
of GIGYF1, we have shown that its interaction with Grb10 in two-hybrid assays
exhibits sequence requirements similar to those essential for the binding of
the CD2BP2 GYF domain to CD2. Although the specific Grb10 proline-rich
sequences are distinct from the GYF-interactive sequences in CD2, our
two-hybrid analyses of Grb10 deletion mutants have shown a similar requirement
for two proline-rich motifs separated by a six- to seven-aa segment. Thus, our
data support the general interaction model proposed by Reinherz and co-workers
(36) but define additional
proline-rich sequences capable of GYF domain binding.
In an earlier report on the CD2BP2 protein, partial human sequences corresponding to GIGYF1 and GIGYF2 were recognized in GenBankTM (36). Using the complete mouse cDNAs that we have cloned, analysis of the human genome sequence and available DNA sequences from multiple other species indicate that GIGYF1 and GIGYF2 are the only two members of this protein family. Based on multi-tissue poly(A)+ Northern blots, GIGYF1 and GIGYF2 mRNAs have distinct patterns of expression in specific mouse tissues. For example, GIGYF1 mRNA is most abundant in brain, spleen, lung, and kidney, whereas GIGYF2 has highest abundance in heart and liver. Grb10 has been shown previously (3) to have broad tissue expression in the mouse, and, in general, mRNAs for either GIGYF1, GIGYF2, or both of these proteins are present in all tissues expressing Grb10. One exception appears to be skeletal muscle, which has abundant Grb10 mRNA but very low levels of GIGYF1 and GIGYF2 mRNA.
Interactions of a transfected Myc-tagged GIGYF1 fragment containing the GYF domain with Grb10 and IGF-I receptors were investigated in the R+ IGF-I receptor overexpressing fibroblast cell line. In cells deprived of serum overnight and incubated in the absence of IGF-I a modest level of Grb10 co-precipitation with GIGYF1 was evident, but there was no detectable co-precipitation of either Grb10 or GIGYF1 with the IGF-I receptor. Within 2 min of IGF-I stimulation, there was rapid recruitment of Grb10 to the IGF-I receptor, a marked increase of the amount of GIGYF1 associated with Grb10, and a coordinate co-precipitation of GIGYF1 with the receptor. These data are consistent with the previously described recruitment of Grb10 to activated (tyrosine-phosphorylated) IGF-I receptors through receptor interactions with the Grb10 BPS domain (11). The co-precipitation of GIGYF1 with activated IGF-I receptors most probably reflects the formation of a trimeric complex, with the C-terminal region of Grb10 linked to the receptor and the N-terminal region bound to GIGYF1, because the GYF domain is the only apparent protein interaction motif in the transfected fragment of GIGYF1, and the IGF-I receptor lacks paired proline-rich motifs, which might function as direct GIGYF1 binding sites. We speculate that increased binding of GIGYF1 to Grb10 occurs as a consequence of Grb10 recruitment to the activated receptor, because effects of IGF-I on Grb10 must be receptor-mediated. It is not yet clear whether the observed level of Grb10 association with GIGYF1 in the basal state is a consequence of GIGYF1 overexpression or a characteristic of the endogenous proteins.
Because activated IGF-I receptors are uniformly located on the cell surface 2 min after IGF-I stimulation, with ligand-induced receptor internalization occurring at somewhat later time points (39), it can be concluded that Grb10 and GIGYF1 are recruited initially to the cell surface by the activated IGF-I receptors. In a recent published study (16), Grb10 was shown to associate with Akt in basal (serum-deprived) Mo7e and K562 cell lines and to recruit Akt to the plasma membrane as a consequence of binding between the SH2 domain of Grb10 and activated c-kit receptors following stimulation with stem cell factor. Although the specific domains mediating the Grb10-Akt interaction were not defined, this appeared to require Grb10 sequence located N-terminally from the PH and SH2 domains. On analyzing the aa sequence of Akt, we have not observed paired proline-rich motifs, and thus we speculate that the Grb10-Akt interaction occurs through a mechanism distinct from Grb10-GIGYF1 binding. It remains to be determined whether Grb10 has a broader role in recruiting additional proteins to cell surface tyrosine phosphoproteins.
When cell extracts are studied 10 min after IGF-I stimulation, there is sustained association of Grb10 with IGF-I receptors, but co-precipitation of GIGYF1 with Grb10 or IGF-I receptors becomes undetectable. The dissociation of GIGYF1 from the receptor-Grb10 complex may result from a time-dependent modification in Grb10 and/or GIGYF1 or, alternatively, as a consequence of events specifically associated with receptor internalization. Although the data reported in this study were obtained using a Myc-tagged fragment of GIGYF1 containing the GYF domain, we have observed generally similar time-dependent interactions of transfected full-length GIGYF1 with Grb10 and IGF-I receptors. These data are not shown, because overexpression of the full-length GIGYF1 construct resulted in the extensive formation of inclusion bodies, which may be cytotoxic (38, 40). Although the cells expressing inclusion bodies are not suitable for functional studies, the occurrence of a similar transient association of full-length GIGYF1 with Grb10 and IGF-I receptors indicates that this is not a phenomenon limited to the Myc-tagged fragment that we have utilized. In future studies, it will be important to determine the cellular location and functional significance of the GIGYF1 released from receptor-Grb10 complexes. Because the GIGYF1 and GIGYF2 proteins both contain a consensus bipartite nuclear localization sequence and multiple Gln repeat domains, it will be particularly important to determine whether these proteins undergo nuclear targeting and interact with transcriptional regulatory complexes (41, 42).
Overexpression of the GYF domain fragment of GIGYF1 results in augmented IGF-I-stimulated tyrosine phosphorylation of IGF-I receptors, IRS-1, and Shc, and consequent increased activation of Akt, ERK1, and ERK2 in R+ cells, which are fibroblasts expressing abundant IGF-I receptors. All of these actions of GIGYF1 are decreased 10 min after IGF-I in comparison with GIGYF1 effects 2 min after IGF-I. Thus, the augmentation of IGF-I receptor signaling may be manifested by Grb10-GIGYF1-bound receptors but does not characterize receptors linked to Grb10 in the absence of GIGYF1. This concept is consistent with a previously published study (20) demonstrating binding of transfected Grb10 to IGF-I receptors in the same receptor overexpressing fibroblast cell line but no effect of Grb10 transfection on IGF-I signaling.
Further studies will be required to determine the mechanism of GIGYF1 effects on IGF-I receptors. In initial studies with NIH-3T3 fibroblasts expressing lower levels of GIGYF1 and IGF-I receptors, augmented IGF-I signaling was not evident (data not shown). It therefore will be important to examine the roles of receptor, Grb10, and GIGYF1 abundance in the regulatory effects of GIGYF1 on IGF-I receptor signaling. However, the observed augmentation of IGF-I signaling by the GIGYF1 fragment in R+ fibroblasts indicates a potentially important role for the GIGYF proteins in the transmission of intracellular signals from IGF-I receptors and possibly from other receptor tyrosine kinases that bind Grb10.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01
DK43038 (to R. J. S.). The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
¶ Supported by an American Diabetes Association mentor-based fellowship.
** To whom correspondence should be addressed: The Hallett Center for Diabetes and Endocrinology, Rhode Island Hospital, One Hoppin St., Suite 200, Providence, RI 02903. Tel.: 401-444-3420; Fax: 401-444-4921; E-mail: rsmith4{at}lifespan.org.
1 The abbreviations used are: PH, pleckstrin homology; IGF-I, insulin-like
growth factor; SH2, Src homology 2; IRS-1, insulin receptor substrate 1; ERK,
extracellular signal-regulated kinase; ORF, open reading frame; aa, amino
acids; RACE, rapid amplification of cDNA ends; CD2BP2, CD2-binding protein
2.
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Contributed equally.