(Received for publication, July 28, 1995; and in revised form, October 2, 1995)
From the
Insulin and insulin-like growth factor (IGF-I) receptors are
heterotetrameric proteins consisting of two - and two
-subunits and members of the transmembrane tyrosine kinase
receptors. Specific ligand binding to the receptor triggers a cascade
of intracellular events, which begins with autophosphorylation of
several tyrosine residues of the
-subunit of the receptor. The
triple cluster in the tyrosine kinase domain of the
-subunit is
the earliest and major autophosphorylation site. Previous studies have
shown that substitutions of these three tyrosines by phenylalanines of
both insulin and IGF-I receptors practically abolish any activation of
cellular signaling pathways. We have studied the effect of double
tyrosine mutations on IGF-I-induced receptor autophosphorylation,
activation of Shc and IRS-1 pathways, and cell proliferation and
tumorigenicity. Substitution of tyrosines 1131/1135 blocks any
detectable autophosphorylation, whereas substitution of tyrosines
1131/1136 or 1135/1136 only reduces autophosphorylation levels in some
clones by
50%. Nevertheless, all the cells expressing IGF-I
receptors with double tyrosine substitutions demonstrated markedly
reduced signaling through Shc and IRS-1 pathways. In addition, they
were unable to respond to IGF-I-stimulated cell growth in culture, and
tumor formation in nude mice was abrogated. These data suggest that the
presence of tyrosine 1131 or 1135 essential for receptor
autophosphorylation, whereas the presence of each of these tyrosines is
necessary for a fully functional receptor.
The multiple physiological actions, including cell growth and
differentiation of the insulin-like growth factors (IGFs) ()are mediated by the IGF-I receptor. While the IGF-I
receptor and the structurally related insulin receptor are members of
the type II receptor tyrosine kinase family, their in vivo biological functions are quite separate. Both the IGF-I and
insulin receptors are heterotetrameric proteins composed of two
extracellular
-subunits and two membrane-spanning
-subunits
linked by disulfide bonds(1, 2, 3) .
Sequences found in the
-subunits of each receptor are important
for determining ligand specificity. The amino-terminal and
carboxyl-terminal portions of the
subunit of the insulin receptor
are critical for high affinity insulin binding, while the cysteine-rich
domain of the IGF-I receptor determines high affinity IGF-I
binding(4, 5, 6) . Likewise, the
-subunits contain a number of structurally distinct domains
including the extracellular, transmembrane, juxtamembrane, tyrosine
kinase, and carboxyl-terminal regions. Binding of ligand to the
-subunit activates the tyrosine kinase activity of the
-subunit resulting in autophosphorylation on distinct tyrosine
residues. The triple tyrosine cluster within the kinase domain (1131,
1135, and 1136 tyrosines in the IGF-I receptor and the equivalent
residues in the insulin receptor; numbering system of Ullrich et
al.(2) ) is the earliest and major site of
autophosphorylation. Phosphorylation of these three tyrosine residues
is necessary for activation of the kinase toward other
substrates(7, 8) . When the triple tyrosine cluster is
substituted by phenylalanine residues, the receptor loses all
ligand-induced biological actions(9, 10) .
Whereas the function of the triple tyrosine cluster in the tyrosine kinase domain of the insulin receptor has been well characterized (11, 12, 13) , much less is known about the corresponding tyrosines of the IGF-I receptor. Single substitutions of tyrosine 1131 or 1135 have relatively small effects on receptor and endogenous substrate phosphorylation or on cell proliferation(14, 15) . In contrast, substitution of tyrosine 1136 apparently has an inhibitory effect on those functions(14) . In addition, it has been demonstrated, at least in the case of the insulin receptor, that in intact cells, bis phosphorylation of the kinase domain at Tyr-1158 and either Tyr-1162 or Tyr-1163 comprises 80% of phosphorylated receptors(7, 16, 17, 18) . Thus, a study involving the substitution of combinations of double tyrosines may be more instructive. To further characterize the role of these tyrosines in IGF-I receptor function, we have performed substitutions of combinations of two tyrosines in the kinase domain of the IGF-I receptor. We transfected NIH-3T3 cells to study receptor autophosphorylation and post-receptor signaling pathways as well as biological functions of the receptor including cell growth and tumorigenicity.
Figure 1:
IGF-I stimulation of receptor
autophosphorylation in intact cells. Cells were stimulated with 100
ng/ml IGF-I for 1 min at 37 °C as described under
``Experimental Procedures.'' Similar amounts of receptors
were separated by 7.5% SDS-PAGE, transferred to nitrocellulose filters,
and blotted with a monoclonal anti-phosphotyrosine antibody (4G10). The
position of protein molecular weight standards as well as the
-subunit of the IGF-I receptor are indicated. The results shown
are representative of at least three individual experiments. WT, wild type.
Figure 2: IRS-1 signal transduction pathway. Confluent monolayers of cells were made quiescent in serum-free medium overnight and then stimulated with 100 ng/ml IGF-I for 1 min at 37 °C. Cells were lysed and IRS-1 immunoprecipitated as described under ``Experimental Procedures.'' IRS-1 immunoprecipitates were separated by 9% SDS-PAGE, transferred to a nitrocellulose filter, and blotted with the appropriate antibodies. Panel A, immunoblot with polyclonal anti-phosphotyrosine antibody RC20H. Panel B, immunoblot with monoclonal anti-PTP1D antibody. Panel C, immunoblot with monoclonal anti-Grb2 antibody. The positions of protein molecular weight standards are indicated. WT, wild type.
To evaluate Shc pathway activation, cells were stimulated with 100 ng/ml IGF-I for 5 min at 37 °C as described above, and Shc immunoprecipitates were assayed for Shc phosphorylation and Grb2 association. Similar amounts of Shc proteins were detected when the membranes were reblotted with a polyclonal anti-Shc antibody (data not shown). A typical experiment is shown in Fig. 3, A and B, respectively. IGF-I stimulation of tyrosine phosphorylation of the 52- and 46-kDa isoforms of Shc protein (Fig. 3A) was observed in wild-type clones, whereas cells overexpressing the double tyrosine mutant receptors presented very low levels of Shc phosphorylation. Shc-Grb2 association following IGF-I stimulation was similarly diminished in mutant clones with respect to the wild-type clones (Fig. 3B). These results suggest that activation of both the IRS-1 and Shc pathways by IGF-I are at least partially blocked in cells overexpressing double tyrosine mutant receptors as compared with cells overexpressing a similar number of wild-type receptors.
Figure 3: Shc signal transduction pathway. Confluent monolayers of cells were made quiescent in serum-free medium overnight and then stimulated with 100 ng/ml IGF-I for 5 min at 37 °C. Cells were lysed, and Shc proteins were immunoprecipitated as described under ``Experimental Procedures.'' Shc immunoprecipitates were separated by 9% SDS-PAGE, transferred to a nitrocellulose filter, and blotted with the appropriate antibodies. Panel A, immunoblot with monoclonal anti-phosphotyrosine antibody RC20H. Panel B, immunoblot with monoclonal anti-Grb2 antibody. The positions of protein molecular weight standards are indicated. WT, wild type.
Figure 4:
Cell
growth stimulation by IGF-I in culture. Cells were plated at 3
10
cells per well in 96-well plates in DMEM with 10% FBS.
After 24 h, medium was changed to DMEM with 1% FBS and supplemented
with 100 ng/ml IGF-I. All assays were carried out in triplicate. Panel A shows the results representative of three individual
experiments. Panel B shows the mean of three independent
experiments ± S.D., and the data are expressed as percentage of
WT43 cell number at 120 h.
Figure 5:
Tumorigenicity of NIH-3T3 cells
transfected with the double tyrosine mutants and wild-type IGF-I
receptor. 1 10
cells were injected subcutaneously
in nude mice. The animals were examined for tumor formation on the
dorsal region, and tumor volume was calculated as described under
``Experimental Procedures.'' Each point represents the mean
of tumor volume measured in a group of four (DYF12(10) and DYF13(42)),
five (DYF12(4), DYF12(33), DYF23(1), DYF23(46), and WT43), or six
(WT50) mice.
The earliest post-binding event following the interaction of
insulin and IGF-I with their specific receptors is the
autophosphorylation of the triple tyrosine cluster within the
-subunit tyrosine kinase domain(7, 8) .
Mutational analysis of the insulin receptor tyrosine kinase domain has
provided interesting, and at the same time, controversial information
about the regulatory role of these three tyrosines in receptor kinase
activity. The most dramatic effect has been shown with insulin
receptors where these three tyrosine residues (tyrosines 1158, 1162,
and 1163) have been mutated to phenylalanine. Insulin-stimulated
autophosphorylation and cell signaling by these mutant receptors are
impaired(13, 23) . Single substitutions of any one of
these tyrosine residues with phenylalanine (i.e. Y1158F,
Y1162F, or Y1163F) may reduce in vitro autophosphorylation of
the receptor
-subunit on the remaining tyrosines and reduce
tyrosine kinase activity, although, in contrast, Zhang and Roth (6) reported that the Y1158F mutation had no effect on
autophosphorylation or on insulin-stimulated exogenous tyrosine kinase
activity. Data on thymidine incorporation is also controversial. In
general, however, substitutions of any single tyrosine have only slight
effects on insulin-stimulated thymidine
incorporation(13, 23) . A more substantial reduction
in insulin-induced tyrosine kinase activity was observed when double
tyrosine substitutions were studied(23) . Generally, the triple
tyrosine substitution results in even more reduction in thymidine
incorporation, although HTC cells expressing the triple tyrosine mutant
receptor exhibit normal thymidine incorporation(24) . Despite
these discrepancies, the data overall are consistent with the idea that
autophosphorylation of tyrosines 1158, 1162, and 1163 is essential in
autoactivation of the insulin receptor tyrosine kinase toward other
substrates.
Although IGF-I and insulin receptors are structurally similar, their in vivo biological actions are different. At which level this divergence of function occurs is not yet defined. Thus, analyses of the structural and functional aspects of the IGF-I receptor is of considerable scientific interest. Studies using mutational analyses of the IGF-I receptor are considerably less complete than of the insulin receptor. We and others have reported previously that substitution of the triple tyrosine cluster with phenylalanine has similar effects as seen with the insulin receptor, i.e. essentially all of the functions of the IGF-I receptor were abrogated(9, 10) . A single substitution of tyrosine residue 1131 reduces autophosphorylation and receptor internalization, whereas IRS-1 phosphorylation, thymidine incorporation, and cell proliferation were unaffected(15) . Li et al.(14) showed that single substitutions of tyrosines 1131 or 1135 did not affect mitogenicity and only slightly reduced autophosphorylation. In contrast, they reported that substitution of tyrosine 1136 abrogated autophosphorylation and cell growth.
In the present study, we show that substitutions of
tyrosines 1131 and 1135 reduced -subunit autophosphorylation to
levels below the level of detectability by our antibody, suggesting
that the presence of either tyrosine 1131 or 1135 is required for full
-subunit autophosphorylation. Substitutions of 1131/1136 and
1135/1136 affect autophosphorylation to a lesser degree, suggesting
that tyrosine 1136 is not crucial for IGF-I-stimulated
autophosphorylation. The crystal structure of the tyrosine kinase
domain of the human insulin receptor has recently been
characterized(25) . Apparently, the phosphorylation of tyrosine
1162 (the second of the three tyrosines) is a key step in the receptor
kinase activation. The apo structure shows that the hydroxyl group of
tyrosine 1162 is bound in the active site autoinhibiting the tyrosine
kinase of the receptor. This finding suggests that phosphorylation of
this tyrosine would disengage it from the active site. Our results, in
the present study, suggest an important role not only for tyrosine 1135
(equivalent to tyrosine 1162 in insulin receptor) but also for tyrosine
1131 in autophosphorylation of the IGF-I receptor.
Of particular
interest in our study is the finding that all the cells expressing
double tyrosine substitutions failed to respond mitogenically to IGF-I
stimulation, and tumor formation was reduced compared to cells
overexpressing wild-type IGF-I receptors, despite the fact that
substitution of tyrosines 1131/1136 or 1135/1136 only slightly reduced
receptor autophosphorylation. These findings suggest that perhaps a
relatively high threshold of autophosphorylation is required to fully
activate the signaling pathways. The inability of all double mutant
receptors to mediate biological activities could be explained by the
absence of IGF-I-induced tyrosine phosphorylation of the two major
IGF-I signaling pathways, IRS-1 and Shc. IRS-1 is considered an adapter
protein between the insulin and the IGF-I receptors and the network of
their signaling
pathways(26, 27, 28, 29) . IRS-1 is
phosphorylated on multiple tyrosine residues upon receptor
stimulation(30) . This provides multiple sites of interaction
for proteins with SH2 (src-homology 2) domains(31) .
Several SH2-containing proteins have been shown to associate with
IRS-1: PI3-kinase(32, 33, 34) ,
Nck(35) ,
Grb2(36, 37, 38, 39, 40, 41) ,
and PTP1D(30, 39) . PTP1D is a tyrosine phosphatase
that binds to IRS-1 at tyrosine 1172; this binding provides a potential
mechanism for its activation(40) . Evidence suggests that PTP1D
is involved in stimulation of mitogenesis, and regulation of Ras and
mitogen-activated protein kinase activation (41, 42) .
Grb2 is thought to stimulate p21 activity through a
monovalent interaction with p21
GDP-GTP exchange factor,
mSOS, resulting in stimulation of a serine/threonine phosphorylation
cascade leading to activation of the mitogen-activated protein kinase
pathway implicated in cell growth and metabolism. Thus, the binding of
the Grb2-mSOS complex to IRS-1 after insulin and IGF-I stimulation
links insulin and IGF-I receptor tyrosine kinases and p21
signaling pathways. An alternative and possible redundant pathway
that links insulin and IGF-I signaling with p21
activation is through
Shc(43, 44, 45, 46, 47) .
Shc is tyrosine phosphorylated upon insulin and IGF-I
stimulation(44, 45, 46) . The phosphorylation
of Shc provides a binding site for Grb2, resulting in the formation of
the Shc-Grb2-mSOS complex and probably leading to activation of the
mitogen-activated protein kinase pathway. Therefore, diminished
stimulation of the IRS-1/PTP1D and IRS-1/Grb2 as well as Shc-Grb2
pathways by IGF-I in our cells expressing receptors with double
tyrosine substitutions is associated with decreased IGF-I-stimulated
cell growth response of cells expressing these receptors. We cannot
exclude the possible involvement of other signaling pathways, such as
Crk, that are also involved in IGF-I-induced signaling(48) .
In summary, we have demonstrated that the presence of both tyrosines
1131 and 1135 of the IGF-I receptor are necessary for full
IGF-I-stimulated autophosphorylation of the -subunit. However, the
absence of any of the tyrosine residues of the triple tyrosine cluster
significantly reduced signal transduction through IRS-1 and Shc
activation, thus abrogating IGF-I-stimulated cell growth and IGF-I
receptor-mediated tumor formation of transfected fibroblasts.