(Received for publication, July 26, 1994; and in revised form, November 16, 1994 )
From the
The amino acid sequence of the tyrosine kinase domain of the
insulin-like growth factor-I (IGF-I) receptor is 84% identical to the
sequence of the analogous region of the insulin receptor. A naturally
occurring mutation of the tryptophan residue at position 1200 of the
insulin receptor to serine results in impaired subunit
autophosphorylation of wheat germ agglutinin-purified receptors,
severely impaired thymidine incorporation and moderately reduced
glycogen synthesis; however, glucose uptake was unaffected. To study
the importance of this residue in IGF-I receptor function, we mutated
the analogous tryptophan residue at position 1173 of the IGF-I receptor
to serine and overexpressed the mutant receptor in NIH-3T3 cells. In
cell lines overexpressing this mutant IGF-I receptor,
subunit
autophosphorylation was severely reduced. Additionally, the
overexpressed mutant receptors exhibited a dominant-negative effect on
IGF-I-stimulated autophosphorylation of endogenous mouse IGF-I
receptors. Phosphorylation of insulin receptor substrate (IRS)-1 in
intact cells by the mutant IGF-I receptors was similar to the level of
IRS-1 phosphorylation seen in the parental NIH-3T3 cells, but there was
no obvious dominant-negative effect on IRS-1 phosphorylation. Wheat
germ agglutinin-purified mutant receptors were as active in
phosphorylating poly(Glu,Tyr) 4:1 as wild-type IGF-I receptors,
suggesting that, in intact cells, additional factors are necessary in
order for the IGF-I receptor to phosphorylate IRS-1. Thymidine
incorporation was severely reduced in one clone overexpressing the
mutant IGF-I receptor and abolished in a second clone. Glucose uptake
in both clones was reduced to about half of that seen in a cell line
overexpressing wild-type IGF-I receptors. Thus, we propose that the
tryptophan residue at position 1173 of the IGF-I receptor is important
in the regulation of autophosphorylation in vivo. This study
again confirms that high levels of autophosphorylation are not required
for mediation of all of the biologic activities of the IGF-I receptor.
The insulin-like growth factor-I (IGF-I) ()receptor
is a transmembrane tyrosine kinase that mediates the biological effects
of IGF-I (Yarden and Ullrich, 1988) and many of the actions of IGF-II.
The IGF-I receptor is structurally homologous to the insulin receptor
and the insulin receptor-related receptor. The members of this receptor
family are hetero-tetrameric glycoproteins consisting of two
extracellular ligand-binding
subunits and two
subunits with
extracellular, transmembrane, intracellular juxtamembrane, tyrosine
kinase, and carboxyl-terminal domains (Ullrich et al., 1985,
1986; Ebina et al., 1985; Shier and Watt, 1989).
After ligand binding, the IGF-I and insulin receptors may mediate both similar and distinct physiological functions. The mechanisms by which the IGF-I and insulin receptors mediate these distinct functions remain to be fully elucidated, and it has been difficult to reconcile the specificity of function with the structural homology of these receptors (Adamo et al., 1991). In each case, binding of ligand results in receptor autophosphorylation, association with intracellular proteins, phosphorylation of intracellular substrates, and activation of specific signaling processes that are involved in growth regulation or metabolism (Lowe, 1991). Mutants of both the IGF-I and insulin receptors that are incapable of binding ATP, or that lack the triple tyrosine cluster that constitutes the major autophosphorylation site in the tyrosine kinase domain, are not autophosphorylated and are not able to initiate the normal cascade of intracellular events that occurs subsequent to receptor activation (Ebina et al., 1987; Kato et al., 1993, 1994; Gronborg, 1993). Mutations outside the tyrosine kinase domain also modulate the ability of the IGF-I and insulin receptors to mediate mitogenic and metabolic functions. Replacement of the poorly conserved carboxyl-terminal domain of the insulin receptor by the carboxyl terminus of the IGF-I receptor resulted in decreased insulin stimulation of intact cell autophosphorylation, phosphorylation of insulin receptor substrate-1 (IRS-1), c-fos expression, ornithine decarboxylase activity, and thymidine incorporation. Insulin-stimulated glucose uptake was increased as compared to cells overexpressing wild-type insulin receptors. The reciprocal mutation (replacement of the carboxyl terminus of the IGF-I receptor with that of the insulin receptor) had only minimal effects on functions as compared to cells overexpressing the wild-type IGF-I receptor (Faria et al., 1994). These findings are consistent with the notion that the relatively nonconserved COOH-terminal domains of the IGF-I receptor and insulin receptor are important for receptor-specific signal transduction.
In addition to mutants of the tyrosine kinase domain deficient in ATP binding or autophosphorylation, other naturally occurring mutations in the tyrosine kinase domain of the insulin receptor have been identified and have been shown to alter receptor-mediated functions. One of these, a tryptophan to serine substitution at position 1200 of the insulin receptor (numbering system of Ebina et al.(1985)) was identified in a patient with the type A syndrome of insulin resistance (Moller et al., 1990). In Chinese hamster ovary cells overexpressing this mutant insulin receptor, insulin binding and receptor internalization were normal, but autophosphorylation and phosphorylation of IRS-1 were severely impaired (Moller et al., 1990). Insulin-stimulated glucose uptake and glucose incorporation into glycogen measured in cells expressing mutant insulin receptors were increased as compared to control neomycin-resistant Chinese hamster ovary cells. However, there was no increase in insulin-stimulated thymidine incorporation over those seen in control cells (Moller et al., 1991).
In this study, the homologous tryptophan at position 1173 (numbering system of Ullrich et al., 1986) in the IGF-I receptor was mutated to serine. This tryptophan is found in a highly conserved 12-amino acid segment in the carboxyl-terminal portion of the tyrosine kinase domain. The function of this mutated IGF-I receptor was studied in NIH-3T3 cells to determine if this mutation affected subsequent intracellular signaling and, in particular, if it had a differential effect on these functions.
Figure 1: Tyrosine phosphorylation in intact cells. Whole cells were stimulated with IGF-I (0 nM or 100 nM) as described under ``Experimental Procedures.'' Protein content in cleared whole-cell lysates was determined. All samples were separated by 7.5% SDS-PAGE. The positions of protein molecular weight standards are indicated in each panel. Each panel is representative of three individual experiments. Panel A, equal amounts of IGF-I receptors from WT, b13, and b14 cells were applied to the gel. pNeo1 was loaded as the amount of protein similar to that loaded for b13 and b14 extracts. The transferred proteins were immunoblotted with antiphosphotyrosine antibody (monoclonal, mouse) and detected by a second antibody conjugated to horseradish peroxidase. Panel B, equal amounts of cleared whole cell lysate were separated by SDS-PAGE. After transfer to a nitrocellulose membrane the proteins were immunoblotted with RC20H (monoclonal antiphosphotyrosine antibody conjugated to horseradish peroxidase).
Figure 2: Autophosphorylation of WGA-purified IGF-I receptors. Equal amounts of WGA-purified receptors, as determined by binding assays, were unstimulated or stimulated for 1 h at 24 °C. The activated receptors were then treated with 50 µM ATP and 200 µM CTP for 15 min at 24 °C. The tyrosylphosphorylated proteins were detected by Western blotting with a monoclonal antiphosphotyrosine antibody following resolution through a 7.5% SDS-PAGE and electrotransfer to a nitrocellulose membrane. One of two experiments is shown. Each experiment used independently prepared WGA-purified receptors. The positions of protein molecular weight standards are indicated in Panel A. Panel A, Kodak X-AR film was exposed for 1 min against the Western blot. Panel B, Kodak X-AR film was exposed for 15 min against the same nitrocellulose filter as in panel A.
Figure 3: In vitro tyrosine kinase activity. Equal amounts of WGA-purified receptors, as determined by binding assays, were unstimulated or stimulated overnight with 3 or 100 nM IGF-I. The activated receptors were then assayed for their ability to phosphorylate the exogenous substrate poly(Glu,Tyr) 4:1. One of the two experiments is shown. Each data point was done in triplicate and represents the mean ± S.E. after subtraction of nonspecific filter-bound counts. The receptors were unstimulated (hatched bars), stimulated with 3 nM IGF-I (solid bars), or stimulated with 100 nM IGF-I (shaded bars).
Figure 4:
IGF-I stimulation of thymidine
incorporation. Subconfluent monolayers of cells were made quiescent in
serum-free media for 24 h and then stimulated with the indicated
concentration of IGF-I or 10% fetal bovine serum for 16 h at 37 °C.
[H]Thymidine (1 µCi/ml) was added and the
incubation was continued for 1 h. Incorporated counts were measured as
described under ``Experimental Procedures.'' All assays were
carried out in triplicate and the standard error for each cell line in
one experiment was within 5%. The data shown are the means ±
S.E. of three independent experiments with the exception of Neo, which
was an average of four experiments. Where not shown, S.E. bars are
smaller than the size of the symbol. For each cell line, the relative
stimulation with respect to stimulation by serum was calculated as
follows: percent of serum stimulation = (incorporation in the
presence of IGF-I - basal incorporation)/(incorporation in the
presence of serum - basal incorporation)
100. Although
absolute responses varied between experiments, maximal responses when
corrected for protein showed less variability. Values for serum
stimulation were: Neo (
, 3176, 3400, 3126, and 2851 cpm/µg
protein), WT (
; 2919, 3537, and 3113 cpm/µg protein), b13
(
; 6117 and 2455 cpm/µg protein), and b14 (
; 4031 and
2368 cpm/µg protein). Within the same experiment, counts
incorporated in the presence of serum/µg of protein for each mutant
cell line as compared to counts incorporated in the presence of
serum/µg of protein for WT ranged from 96 to
149%.
Figure 5:
IGF-I
stimulation of 2-deoxyglucose uptake. Confluent monolayers of cells
were incubated in the presence of the indicated concentration of IGF-I
for 20 h and washed as indicated under ``Experimental
Procedures.'' Uptake was measured after a 10-min incubation in the
presence of 2-deoxy-D-[C]glucose (0.5
µCi/well) and 0.2 mM 2-deoxy-Dglucose. The
results shown are the means ± S.E. of three experiments, with
the exception of WT which is an average of four experiments, and b14,
which was from two experiments. Where not shown, S.E. bars are smaller
than the size of the symbol. Additionally, each assay point for each
experiment was done in triplicate with a standard error for each cell
line within one experiment less than 5%. The results shown are for Neo
(
), WT (
), b13 (
), and b14
(
).
The molecular basis for the differential physiologic
functions of the insulin and IGF-I receptors has not been fully
elucidated. Although the two receptors share remarkable structural
homology and are capable of associating with some of the same proteins
in the tyrosine kinase signal transduction cascade, ligand stimulation
of these receptors can elicit different effects on cellular metabolism
and growth in vivo. In the present study, we have investigated
the regulatory function of an amino acid residue in the tyrosine kinase
domain of the IGF-I receptor. The tryptophan residue at position 1173
in the IGF-I receptor is highly conserved in other members of the
protein-tyrosine kinase family, particularly the insulin, epidermal
growth factor, and platelet-derived growth factor receptors, and the abl oncogene (Hanks et al., 1988). In addition, in
both tyrosine and serine/threonine kinases, there are two invariant
amino acids NH-terminal to this tryptophan. Aspartic acid
is 9 amino acids away and glycine is 4 amino acids to the
NH
-terminal side. Replacement of tryptophan 1173 with
serine severely reduced IGF-I-stimulated autophosphorylation of the
subunit in intact cells. This autophosphorylation was
significantly less than that seen in parental cells (Neo) with 20-fold
fewer IGF-I receptors, indicating that these mutant receptors exert a
dominant-negative effect on autophosphorylation of the endogenous mouse
IGF-I receptors. In vitro autophosphorylation of the mutant
receptors was also significantly less than that exhibited by wild-type
receptors. Phosphorylation of an endogenous substrate, IRS-1, was
impaired, but not to the same extent as autophosphorylation;
presumably, this degree of phosphorylation is sufficient to mediate
certain of the biological responses studied. Furthermore, there is no
evidence of the mutant receptors exerting a dominant-negative effect on
phosphorylation of IRS-1 by the endogenous mouse IGF-I receptors. The in vitro kinase activity of the mutant receptors, measured as
the ability to phosphorylate poly(Glu,Tyr) 4:1, was equivalent to that
of the wild-type receptor, suggesting that, in intact cells, additional
factors are involved in the ability of the receptor to phosphorylate
endogenous substrates such as IRS-1. The tryptophan residue at position
1173 in the IGF-I receptor is homologous to tryptophan 1200 in the
insulin receptor. A naturally occurring mutation in the insulin
receptor at position 1200 (numbering system of Ebina et al.,
1985) has been described (Moller et al., 1990). Substitution
of the homologous tryptophan in the insulin receptor in a patient with
type A insulin resistance illustrated that this residue is also
important for insulin-stimulated autophosphorylation in transformed
lymphocytes (Iwanishi et al., 1993). Phosphorylation, using
radiolabeled ATP, of insulin-stimulated WGA-purified receptors was
significantly diminished in the mutant insulin receptor. It has not yet
been determined if this loss of autophosphorylation is exclusively on
tyrosine residues or if serine/threonine kinase activity is also
affected.
Functional studies of IGF-I receptors with mutated tryptophan residues showed that thymidine incorporation was severely reduced in one mutant clone and abolished in the second clone. Glucose uptake in both clones was only reduced to about half of that seen with NIH-3T3 cells overexpressing the wild-type IGF-I receptor. Functional studies performed on Chinese hamster ovary cells transfected with the mutant insulin receptor (Trp-1200 to Ser) revealed severely impaired thymidine incorporation and moderately reduced glycogen synthesis but unaffected glucose uptake when compared to wild-type insulin receptors (Moller et al., 1991).
The importance of tyrosine autophosphorylation has been studied in both the insulin receptor and the IGF-I receptor. A cluster of 3 tyrosines located 21 amino acids downstream of the COOH terminus of the catalytic loop are the first tyrosine residues to be phosphorylated following ligand binding (Yarden and Ullrich, 1988). Mutation of all 3 tyrosines in the triple tyrosine cluster of the insulin or IGF-I receptor results in severe reduction of the tyrosine kinase activity (Ellis et al., 1986; Zhang et al., 1991; Murakami and Rosen, 1991; Wilden et al., 1990, 1992a, 1992b; Debant et al., 1988; Rafaeloff et al., 1991; Gronborg et al., 1993; Kato et al., 1994). Thymidine incorporation and glucose uptake were diminished in Chinese hamster ovary cells expressing mutant insulin receptors and NIH-3T3 cells expressing mutant IGF-I receptors, suggesting an essential requirement of autophosphorylation of the triple tyrosine cluster in biological activities (Ellis et al., 1986; Zhang et al., 1991; Murakami and Rosen, 1991; Gronborg et al., 1993; Kato et al., 1994). Mutation of only 2 of the 3 tyrosines in the cluster within the insulin receptor renders measurable but significantly reduced insulin-stimulated kinase activity in intact cells (Yonezawa and Roth, 1991). Recently it has been suggested that the autophosphorylation domain contains sequences that inhibit autophosphorylation of tyrosine residues (Filipek and Soderling, 1993). Consistent with this hypothesis, mutation of a conserved amino acid (Arg-1152 to Gln) just downstream from the last tyrosine in the autophosphorylation cluster resulted in increased insulin-independent kinase activity with diminished insulin-stimulated tyrosine kinase activity (Formisano et al., 1993).
Unlike our results in
the IGF-I mutant receptor, the solubilized mutant insulin receptor was
incapable of phosphorylating an exogenous substrate, and
phosphorylation of IRS-1 in intact cells was undetectable (Moller et al., 1991). The difference in these results may be due to
the use of a different exogenous substrate in the first case and the
sensitivity of antibodies used to detect phosphorylated proteins in the
second, or these findings may suggest that the IGF-I and insulin
receptor subunits are inherently different.
Our observations on the substitution of Trp-1173 suggest that the functional defect in the receptor is similar to those previously reported in the tyrosine kinase domain; that is, incomplete autophosphorylation. However, unlike mutations of the glycine-rich segment with its proximal triad of charged amino acids and mutations of the ``catalytic loop,'' some biologic function is maintained despite severely reduced tyrosine autophosphorylation. The retention of certain biologic functions is more akin to the pattern seen in mutations of the triple tyrosine cluster in the autophosphorylation site. The mechanism by which this differentiation of function occurs may be of at least two types. First, a mutation causing disruption of conformation in the unstimulated state or conformational change after ligand stimulation may block specific signal transduction. Second, phosphorylation of specific residues may be essential for specific signal transduction.
In conclusion, based on our present study and observations on mutations in this region of the insulin receptor, we propose that the region surrounding Trp-1173 of the tyrosine kinase domain of the IGF-I receptor is an important regulator of autophosphorylation in vivo. Furthermore, the phenotype of cells expressing this mutation demonstrates once again that high levels of autophosphorylation are not required for mediation of all the biologic activities of the IGF-I receptor.