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Diabetes Branch, NIDDK, NIH, Bethesda, Maryland 20892-1770
Address all correspondence and requests for reprints to: Derek LeRoith, DB/NIDDK/NIH, Building 10, Room 8S235A, 10 Center Dr MSC 1770, Bethesda, Maryland 20892-1770. E-mail:derek{at}helix.nih.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits containing the ligand binding domain(s) and
two transmembrane ß-subunits with the ligand-sensitive tyrosine
kinase activity (1, 2). The IGF-IR and IR share 84% amino acid
sequence identity in their kinase domains, whereas the juxtamembrane
and carboxy (C)-terminal domains demonstrate a relatively low homology
(61% and 44%, respectively) (3). The biological functions of these
two receptors differ somewhat. Cells treated with insulin rapidly
increase glucose uptake, and lipid and glycogen synthesis but only
increase DNA synthesis after prolonged stimulation. IGF-I, however,
appears to be a more potent stimulator of DNA synthesis and cell growth
(4). Binding of insulin and IGF-I to their cognate receptors stimulates
ß-subunit tyrosine kinase activity, leading to receptor
autophosphorylation and tyrosine phosphorylation of several cellular
substrates. Major substrates of the IR and IGF-IR are the insulin
receptor substrates (IRS-1 and IRS-2). After insulin or IGF-I
stimulation, these substrates are rapidly phosphorylated on multiple
tyrosine residues. This results in docking of several SH2
domain-containing proteins, including the p85 regulatory subunit of the
phosphatidylinositol (PI) 3'-kinase (5, 6, 7, 8), an upstream element in
insulin-stimulated glucose transport and activation of p70 S6 kinase
(9, 10). In addition, Grb-2, an adapter molecule, binds tyrosine
phosphorylated IRS-1 and leads to activation of the Ras and
mitogen-activated protein (MAP) kinase pathway (11, 12, 13). The tyrosine
phosphatase SHPTP1D (or Syp) also has been shown to bind tyrosine
phosphorylated IRS-1 and IRS-2 (14, 15). The IGF-IR and IR also
phosphorylate other cytoplasmic proteins. These include the isoforms of
SHC, which then bind Grb-2 (16), a p62 protein that associates with
Ras-Gap (17) and a 55–60 kDa protein that associates with PI 3'-kinase
(18, 19). In addition, the IGF-IR phosphorylates the 40-kDa
protooncogene Crk-II (20) that binds mSOS and C3G via its SH3 domains,
thereby activating the Ras/Raf/MAP kinase pathway. Despite the
structural and functional similarities between the IR and IGF-IR, a
number of features suggest that differences exist between these two
receptors that may explain the specific responses characteristic of
each receptor. Results of previous studies suggest that the C-terminal domains of the IGF-IR and IR mediate separate functions, but identification of specific subdomains (or motifs) of the C-termini responsible for various interactions with downstream mediators have not been fully elucidated. Individual and double substitutions of the tyrosine residues in the C-terminus of the IGF-IR resulted in markedly reduced mitogenic and tumorigenic properties of the IGF-IR (21, 22), suggesting that the tyrosine residues may participate in mediating signals important for the biological functions of the IGF-IR. In the present study, we have substituted tyrosines 1250 and 1251 with phenylalanine and histidine and introduced a tyrosine in position 1310 to mimic the tyrosine residues present in the IR C-terminus, thus enabling us to compare the potential role of the IR C-terminal tyrosines with the tyrosine residues in the IGF-IR C-terminal domain. NIH-3T3 cells expressing these mutant IGF-IR were studied to identify the post-receptor signals involved in the mitogenesis and transforming activities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of mutant IGF-I receptor DNAs
The wild-type human IGF-IR expression vector has been previously
described (23). DBY and IsY mutant IGF-IRs were generated by the
site-specific mutagenesis approach using PCR to engineer the DNA (24).
For this purpose, partially complementary primers 5'-CTC CGC GCC AGC
TAC GAC GAG CGG CAG CCT TAC GCC CAC A-3' (sense) and 5'-TA
AGG CTG CCG CTC GTC GTA GCT GGC GCG GAG GAC C-3' (antisense)
were used to introduce the substitution of phenylalanine 1310 to
tyrosine (sequence italicized). The final PCR product was
sequentially cloned into a plasmid containing hIGF-I receptor
complementary DNA (cDNA) and then the entire cDNA containing the
mutation was subcloned into the pBPV expression vector (Pharmacia,
Piscataway, NJ). Amplified cDNA sequences and junction regions were
sequenced to ensure the presence of mutations and proper in-frame
ligation. To create the DBY mutation, a fragment of cDNA encoding
wild-type IGF-IR was used as a template and the previously created yyFH
mutant cDNA (22) was used to create the IsY mutation.
Cell culture and transfection
All the NIH-3T3 cell lines were cultured in DMEM supplemented
with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2
mM glutamine in a humidified atmosphere of 95% air and 5%
CO2 at 37 C. NIH-3T3 cells were cotransfected with 20 µg
of wild-type or mutant IGF-IR expression vector or insert-less pBPV
plus 1 µg pMC1Neo (Clontech, Palo Alto, CA) using the
Lipofectamine reagent (Life Technologies, Inc., Gaithersburg, MD).
Selection was carried out as described previously (23). Stably
transfected cells were maintained in the medium described above
supplemented with 500 µg/ml G418 (Geneticin, Life Technologies,
Inc.). Serum-free medium (SF-DMEM) containing 0.1% BSA, 20
mM HEPES, pH 7.5, and antibiotics were used in assays
involving receptor autophosphorylation and phosphorylation of
endogenous cellular substrates.
MTT cellular proliferation assay
Each cell line was plated in triplicate for each time point with
seeding densities of 3 x 103 and 5 x
105 cells/well in 96-well plates. The cells were allowed to
recover overnight in DMEM plus 10% FBS. Thereafter, cell growth was
continued in DMEM supplemented with 1% FBS and 10 nM IGF-I
for 7 days. Media were replenished at 48 and 96 h of growth. The cell
density at the varying time points was determined by measuring the
colorimetric change of 10% MTT in DMEM without phenol red after
incubation with the cells for 4 h at 37 C, followed by lysis of
the cells with isopropanol (25, 26). The cell density was determined by
comparing the colorimetric change of the media with that of a standard
curve. Each cell line was tested for cellular proliferation in three
separate experiments (22, 27).
Soft agar assay
Anchorage-independent growth of the various cell lines was
determined by a soft agar assay. One thousand cells in growth medium
(10% FBS plus 10 nM IGF-I) containing 0.2% agarose
(Difco, Detroit, MI) were plated in 35-mm dishes underlaid with 0.4%
agarose-containing growth medium. Each cell line was plated in
triplicate. The cells were allowed to grow in the soft agar for 2 weeks
at 37 C. Anchorage-independent growth was assessed by scoring the
number of colonies larger than 125 µM.
Intact cell tyrosine phosphorylation
Subconfluent cells in 100-mm plates were serum-starved in
SF-DMEM for 16 h and then incubated either without or with IGF-I
(10 nM) for 1 min at 37 C. The cells were then lysed in the
presence of 350 µl of freshly prepared lysis buffer (50
mM HEPES, pH 7.9, 100 mM NaCl, 10
mM EDTA, 1% Triton X-100, 4 mM sodium
pyrophosphate, 2 mM sodium orthovanadate
(Na3VO4), 1 mM
phenylmethylsulfonylfluoride (PMSF), 10 mM sodium fluoride,
2 µg/ml leupeptin, and 2 µg/ml aprotinin). Cell lysates were
cleared by centrifugation. Protein content was determined by the method
of Bradford (28) using a protein assay kit (Bio-Rad, Richmond, CA).
Equal amounts of protein (20 µg) were fractionated by 7.5% SDS-PAGE.
Proteins were transferred to nitrocellulose membrane for 4 h at
0.2 A in a Tris-glycine buffer with 20% methanol. The nonspecific
binding to the nitrocellulose was reduced by preincubating the filter
in blocking buffer (3% insulin-free BSA in 1x PBST made from 10
mM NaPO4, pH 7.2, 140 mM NaCl and
0.1% Tween 20) for 60 min. The amounts of IGF-IR present on the
nitrocellulose membrane was determined by immunoblotting with Ab53
(1:2000 dilution), a polyclonal antibody that detects the triple
tyrosine cluster of the IGF-IR. This antibody was detected with
horseradish peroxidase-conjugated antirabbit immunoglobulin (1:2000
dilution). The blots were then developed using an enhanced
chemoluminesense (ECL) system. Based on these immunoblots,
approximately equal number of receptors were fractionated by SDS-PAGE
as described above. Blots for tyrosine phosphorylation were
immunoblotted with monoclonal antiphosphotyrosine antibody (clone 4G10)
(1:1000 dilution) and detected with horseradish-peroxidase conjugated
antimouse immunoglobulin (1:5000 dilution) using the ECL system. The
level of phosphorylation of the ß-subunit was quantitated by
digitalizing the signal from x-ray film and analyzing the signal using
NIH Image version 1.55 software. The phosphorylation level was
normalized to receptor density assessed using the Ab53 antibody.
IRS-1, SHC, and Crk immunoprecipitations
Subconfluent cells in 100-mm plates were serum-starved overnight
and then incubated either without or with IGF-I (10 nM) at
37 C for 3 min. Cleared cell lysates were prepared as described above.
For immunoprecipitation, 600 µg of protein were incubated either with
3 µg of polyclonal anti-IRS-1 antibody (a kind gift from J. Pierce,
NCI, NIH), or 3 µg of polyclonal anti-SHC antibody or monoclonal
anti-Crk II antibody at 4 C overnight. Then, 50 µl of 10% (wt/vol)
protein A-Sepharose in 50 mM Tris-HCl buffer, pH 7.0, was
added and incubated at 4 C for 4 h. The precipitates were washed
three times with ice-cold immunoprecipitation buffer (10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1
mM EGTA, 0.2 mM Na3VO4,
0.2 mM PMSF, 1% Triton and 0.5% Nonidet P-40). All of the
immunoprecipitated samples were then boiled for 5 min in sample buffer
containing 50 mM Tris (pH 6.7), 2% SDS, 2%
ß-mercaptoethanol and bromophenol blue as a marker. Samples were then
run on 9% SDS-PAGE. Resolved proteins were electrophoretically
transferred to nitrocellulose membrane. Blots were incubated with
antiphosphotyrosine RC20H antibody (1:2500 dilution) for
phosphorylation studies and then were stripped and reprobed with
polyclonal anti-IRS-1 antibody (1:1000 dilution), monoclonal anti-SHC
antibody (1:250 dilution) or monoclonal anti-Crk antibody (1:1000
dilution). Blots were stripped by incubation for 30 min at 50 C in a
solution containing 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and
0.7% ß-mercaptoethanol. The antibodies used for detecting
coimmunoprecipitated proteins were either monoclonal anti-Grb2 (1:500
dilution), monoclonal anti-PTP1D (Syp) (1:1000 dilution) or monoclonal
anti-p85 (1:2500 dilution). Anti-Grb2, anti-PTP1D (Syp), anti-p85,
anti-SHC and anti-Crk were detected by horseradish
peroxidase-conjugated antimouse immunoglobulin (1:5000 dilution).
Anti-IRS-1 were detected by horseradish peroxidase-conjugated
antirabbit immunoglobulin (1:2000 dilution). The blots were then
developed using the ECL system.
MAP kinase phosphorylation
Subconfluent cells in 100-mm diameter dishes were serum starved
for 16 h and then incubated either without or with IGF-I (10
nM) at 37 C for 8 min. Cleared cell lysates were prepared
as described for the intact cell tyrosine phosphorylation experiment.
Equal amounts of protein (12 µg) were fractionated by 15% SDS-PAGE.
The proteins were transferred to nitrocellulose and nonspecific binding
to the nitrocellulose was reduced as described above. The
nitrocellulose blot was incubated overnight at 4 C with anti-Erk1
antibody (1:2000 dilution) or phospho-specific MAP kinase antibody
(1:1000 dilution). The immunoblotted proteins were detected using the
ECL system.
MAP kinase assay
Subconfluent cells in 100-mm plates were serum-starved for
16 h and then incubated either without or with IGF-I (10
nM) at 37 C for 8 min. The cells were then lysed in the
presence of 400 µl of freshly prepared ice-cold lysis buffer (10%
glycerol, 137 mM NaCl, 25 mM
ß-glycerophosphate, pH 7.3, 20 mM Tris, pH 7.5, 1%
Triton X-100, 1 mM PMSF, 2 mM EDTA, 1
mM Na3VO4 and 2 mM
sodium phosphate, pH 7.0). MAP kinase was assayed as described by Seger
et al. (29). Briefly, 400 µg of protein were
immunoprecipitated with 0.5 µg of anti-Erk2 polyclonal antibody at 4
C for 5 h. Then 50 µl of 10% (wt/vol) protein A-Sepharose in 50
mM Tris-HCl buffer, pH 7.0, was added and incubated at 4 C
overnight. The precipitates were washed twice with ice-cold lysis
buffer followed by two washes with kinase buffer (25 mM
MgCl2, 25 mM ß-glycerophoshate, pH 7.3, 20
mM HEPES, pH 7.6, 2 mM DTT and 0.1
mM Na3VO4). Each pellet was then
incubated in assay buffer (6 µM protein kinase inhibitor
(PKI), 25 mM ß-glycerophosphate, pH 7.3, 2 mM
DTT, 0.1 mM Na3VO4, 25
mM MgCl2, 100 µM ATP and 1.6
µM 
-32P ATP) using 2 mg/ml myelin basic
protein (MBP) as substrate. Reactions were terminated after 30 min at
30 C by adding 5x Laemmli buffer. Samples were then fractionated by
15% SDS-PAGE. The gel was fixed in 7.5% acetic acid/25% isopropanol
for 10 min, dried and exposed to film. Dried gels were quantitated on a
phosphoimager. Nonspecific -32P ATP incorporation was
determinated in identical assays lacking the MBP substrate.
PI 3'-kinase assay
Phosphorylation of phosphatidylinositol was measured as
previously described (23) with some modifications. Cells were
stimulated with IGF-I for 3 min at 37 C and cell lysates were incubated
with a monoclonal antiphosphotyrosine antibody overnight at 4 C. Immune
complexes were precipitated with protein-A Sepharose and PI 3'-kinase
activity was measured as previously described (23). The spots on the
autoradiograph that comigrated with phosphatidyl inositol-4-phosphate
(Sigma) were quantitated by using densitometer and using NIH Image
version 1.55 software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The results presented are the
mean of at least two measurements. Individual clones expressing mutant
IGF-I receptors were matched to NIH-3T3 cells overexpressing similar
numbers of wild-type IGF-IRs. Furthermore, the relative affinities of
IGF-I for the overexpressed mutant IGF-IRs were of the same magnitude
as the affinities of IGF-I for both endogenous mouse and overexpressed
human wild-type IGF-IR (Table 1).
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, A and B. These
experiments were optimized because the recovery of cells following
trypsinization varied depending upon that IGF-IR was expressed. Within
any one experiment, cells were plated at different densities and
cellular proliferation rates were compared between those plates that
had approximately 3 x 103 cells/well after recovery
following transfer of medium from 10% to 1% FBS. In each experiment,
the cells overexpressing the wild-type receptors grew at a faster rate
than did the control cells (pNeo1) and the density of pNeo1 cells at
120 h was statistically different (P < 0.005) from the
density of cells expressing the wild-type receptors (Fig. 1C). The
growth rates of the IsY clones were not different from pNeo1 cells
(Fig. 1, B and C). The DBY clones showed a growth rate not
significantly different from the NWT clones (Fig. 1, A and C).
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). The cells overexpressing wild-type
IGF-IRs demonstrated transforming ability in the presence of 10% FBS.
The control cells (pNeo1) and IsY clones did not form colonies in soft
agar (Table 2) even when further stimulated with IGF-I (data not
shown). The DBY clones had a reduced cell-transforming activity
(
50%) as compared with NWT clones but formed significantly more
colonies than the pNeo1 control cells (Table 2).
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IRS-1, SHC and Crk-II tyrosine phosphorylation in vivo
We studied the IGF-I-induced phosphorylation of IRS-1, SHC, and
Crk-II in control cells and cell lines expressing mutant or wild type
IGF-IRs. Tyrosine phosphorylation of IRS-1 in cleared whole cell
lysates was detected after IGF-I stimulation of intact cells and
blotting of the SDS-PAGE resolved proteins with RC20H (data not shown).
The IGF-IR mutations did not affect the overall level of tyrosine
phosphorylation of IRS-1. In experiments to evaluate the association of
substrates with tyrosyl-phosphorylated IRS-1, cells were stimulated
with 10 nM IGF-I for 3 min at 37 C. Cleared whole cell
lysates were immunoprecipitated for IRS-1 as described and assayed for
tyrosine phosphorylation of IRS-1 and the association of Grb2 and PTP1D
(Syp) with IRS-1. A typical result of these experiments is presented in
Fig. 2. The upper panel shows
tyrosyl-phosphorylated IRS-1 detected by immunoblotting with the
antiphosphotyrosine antibody RC20H. Confirming the results of
tyrosyl-phosphorylated IRS-1 in whole cell lysates, the levels of
phosphorylation of tyrosine residues in the immunoprecipitated IRS-1
from cells expressing mutant IGF-I receptors (DBY and IsY) were similar
to that observed in cells expressing wild-type IGF-I receptors (compare
lanes 8, 10, 12, and 14 with lanes 4 and 6). The blot was then stripped
and reprobed with a polyclonal anti-IRS-1 antibody confirming that
similar amounts of IRS-1 protein were immunoprecipitated in all samples
(data not shown). The middle and lower panels show IGF-I-stimulated
association of PTP1D (Syp) and Grb2 with IRS-1, respectively.
Equivalent amounts of Grb2 and Syp associated with
tyrosyl-phosphorylated IRS-1 in cells expressing mutant receptors (IsY
and DBY) when compared with cells expressing wild-type IGF-I
receptors.
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). SHC was phosphorylated as
efficiently in the DBY and IsY cell lines (lanes 8, 10, 12, and 14) as
in the NWT cell lines (lanes 4 and 6). Grb2 association with SHC was
increased following IGF-I stimulation in all cell lines tested.
Furthermore, the amount of Grb2 associated with SHC was similar in the
cell lines expressing mutant IGF-I receptors (IsY and DBY) and cells
expressing wild-type receptors (NWTb3 and NWTc52).
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, A and B, stimulation of all cell lines with IGF-I
increased the phosphotyrosine content of Crk-II in a representative
experiment. The ability of IGF-I to stimulate Crk-II tyrosine
phosphorylation was similar in cells expressing wild-type IGF-IR and in
cells expressing mutant receptors (IsY and DBY). These blots were than
stripped and reprobed with anti-Crk-II antibody (lower panels of Fig. 4, A and B). In unstimulated cells, the majority of Crk-II
immunoreactivity was localized in the lower 40-kDa band. With IGF-I
stimulation, Crk-II immunoreactivity was shifted into a more slowly
migrating band from cell expressing mutant or wild-type receptors. In
addition to studying the effect of the mutant receptors with the
addition of tyrosine 1316 and replacement of tyrosines 1250 and 1251
(IsY), two cell lines expressing mutant receptors with only tyrosines
1250 and 1251 replaced (yyFHb1 and yyFHb16) were studied for their
ability to phosphorylate Crk-II in response to IGF-I. As shown in Fig. 4A, IGF-I stimulated tyrosine phosphorylation of Crk-II in these cells
(lanes 14 and 16) that correlated with the appearance of a slower
migrating band detected by the anti-Crk-II antibody (lower
panel). Thus, it appears that replacement of tyrosines 1250 and
1251 or substitution of phenylalanine 1310 with tyrosine does not
affect the IGF-I-induced tyrosine phosphorylation of Crk-II.
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). There was little to no phosphorylation of
tyrosine 185 of the MAP kinases Erk1 (arrowhead) and Erk2
(arrow) in unstimulated cells (upper panel, odd
numbered lanes). Phosphorylation of the MAP kinases of control
cells (pNeo1) was not detectable after IGF-I stimulation (lane 2). The
levels of IGF-I-induced phosphorylation of the MAP kinases were similar
in the cells overexpressing mutant IGF-IRs (DBY, lanes 6 and 8; and IsY
lanes, 10 and 12) to that observed in cells overexpressing wild-type
IGF-IRs (lane 4) (Fig. 5, upper panel). The blot was then
stripped and reprobed with rabbit polyclonal anti-Erk1 antibody
confirming that approximately equal amounts of Erk1 and Erk2 proteins
were loaded in all lanes (Fig. 5, lower panel). Addition of
tyrosine 1310 or replacement of tyrosines 1250 and 1251 did not reduce
IGF-I-induced phosphorylation of tyrosine 185 in Erk1 or Erk2.
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). Control cells (pNeo1) had no increase in MAP kinase
activity in response to IGF-I stimulation, whereas cells expressing
wild-type IGF-IRs (NWTb3 and NWTc43) had a significant 3-fold increase
in activity. The DBY cell clones also had a significant increase (about
2.5-fold) in MAP kinase activity in response to IGF-I. The two IsY
clones demonstrated varied responses (IsY44 with a 2-fold increase and
IsY45 with a 3.5-fold increase) in response IGF-I, however both had
significantly greater MAP kinase activity than did the pNeo1 control
cells.
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. In all cell lines, there was minimal
activity of PI 3'-kinase in unstimulated cells. After IGF-I stimulation
for 3 min, there was an enhancement of PI 3'-kinase activity in cells
expressing wild-type receptors (NWTb3) or mutant receptors (DBY9, DBY
12, IsY44 and IsY45). The mean fold-stimulation of PI 3'-kinase
activity in all cell lines is shown in Table 3. Whereas
cells expressing only endogenous receptors had less than a 2-fold
response in activity following IGF-I stimulation, cells expressing
wild-type receptors had over a 6-fold increase in activity in response
to IGF-I. The DBY and IsY clones had even greater increases in PI
3'-kinase activity in response to IGF-I (approximately 8- and 11-fold,
respectively). Thus, substitutions of tyrosines 1250 and 1251 and
phenylalanine 1316 do not affect the ability of the IGF-IRs to mediate
a signal that results in enhancement of PI 3'-kinase activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Autophosphorylation of the IGF-IR ß-subunit was unaffected by replacement of the C-terminal tyrosine residues (1250, 1251) and the contemporaneous mutation of phenyalanine 1310 to tyrosine (IsY) or the single mutation of phenyalanine 1310 to tyrosine (DBY). Autophosphorylation was similarly unaffected by the substitutions of only tyrosines 1250 and 1251 as reported previously by our laboratory and others (21, 22). It is most likely that the major tyrosine autophosphorylation sites of the IGF-IR are homologous to the insulin receptor, being tyrosines 960, 1131, 1135, and 1136 of the IGF-IR transmembrane and tyrosine kinase domains. Furthermore, because these tyrosine moeities remain in the mutant IGF-IRs we have studied here, it is not surprising that the total level of tyrosine phosphorylation observed is unaffected by replacement of two tyrosine residues in the C-terminus of the IGF-IR. Phosphorylation of IRS-1 and SHC, known substrates of the activated IGF-IR, was unaffected by the mutated IGF-IRs. Furthermore Grb-2 association with phosphorylated IRS-1 and SHC was similar in cells expressing the wild type or the mutated IGF-IRs. Rather, the amount of Grb-2 association was directly proportional to the amount of phosphorylated IRS-1 or SHC. These findings are consistent with results from other laboratories demonstrating that both IRS-1 and SHC bind to the juxtamembrane domain of the IGF-IR (43). Similarly, it is not surprising that replacment of the tyrosine residues in the C-terminal domain of the IGF-IR did not affect IGF-I-induced PI 3'-kinase activity because the regulatory subunit (p85) of PI 3'-kinase binds to activated IRS-1 (44). It had been shown that while IGF-I stimulated Crk-II tyrosine phosphorylation in 293 kidney embryonic cells, insulin did not (20). Given that the IGF-I and insulin receptors are least homologous in the C-terminal domains it was an intriguing possibility that the C-terminal domain of the IGF-IR interacted with Crk-II, thereby mediating the phosphorylation and presumably the activation of Crk-II. However, the mutated IGF-IRs studied here with replacement of the tyrosine residues in the C-terminal domain did not affect Crk-II phosphorylation. In fact, it was subsequently shown that Crk binds to IRS-1, and IRS-2 (45). Thus, substitution of the tyrosine residues of the C-terminus of the IGF-IR might not affect phosphorylation of Crk-II because IRS-1 interaction with the IGF-IR is primarily with the juxtamembrane domain (43). More recently our laboratory has shown that Crk-II also interacts directly with the IGF-IR through the juxtamembrane domain (Koval, personal communication), providing evidence that Crk-II interaction with the receptor is not limited to the C-terminus.
Despite being unable to show alterations in interactions with proximal substrates of the IGF-IR with replacements of residues in the C-terminal domain, we considered the possibility that activation of known downstream pathways could have been altered by these mutant receptors. We tested the activation of the MAP kinase pathway because there is strong evidence that this pathway is involved in growth factor-mediated cellular proliferation and tumorigenesis. However, neither IGF-I-induced specific tyrosine phosphorylation of the MAP kinases Erk1 or Erk2 nor stimulation of MAP kinase activity was reduced by replacement of tyrosine residues in the C-terminus of the IGF-IR. Thus, it seems unlikely that we have affected, to any significant extent, the activation of any of the adapter molecules or proximal kinases that converge on and activate the MAP kinase pathway. Furthermore, we have no evidence of an alteration of the IGF-I-stimulated PI 3'-kinase pathway when mutated IGF-IRs are expressed. We have considered that the relatively intact signaling through the MAP kinase and PI 3'-kinase pathways may have occurred via the formation of heterodimers of the expressed mutant receptors and endogenous mouse receptors. Because the majority of mouse receptors would occur in the form of heterodimers, the effects seen on signaling pathways, mitogenesis, and tumorigenesis are most likely mediated by the mutant expressed IGF-IRs. We have shown that replacement of tyrosines 1250 and 1251 significantly affects the IGF-I-induced phenotype of NIH-3T3 cells expressing these receptors, such that mitogenesis and tumorigenesis are significantly reduced. Based on the results of these studies of the signal transduction pathways of the IGF-IR, we conclude that the tyrosine residues in the C-terminus of the receptor do not significantly mediate signals that use the MAP kinase or PI 3'-kinase pathways. The exact IGF-I-stimulated pathways perturbed by these mutant IGF-IRs remains to be elucidated.
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Footnotes |
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Received February 7, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) NWTb3, (•) NWTc52, (
) DBY9, (
) DBY12,
(
) IsY44, and (
) IsY45. C, Mean of three independent
experiments ± SE and the data are expressed as
percentage of NWTb3 cell number at 120 h. *, Statistical
significance (P < 0.005) as compared with NWTb3
and NWTc52 cell lines.
