(Received for publication, July 31, 1995; and in revised form, November 29, 1995)
From the
Two naturally occurring mutant insulin receptors, Arg-1174
Gln and Leu-1178
Pro, found in patients with dominantly
inherited Type A insulin resistance, showed unusual signaling
properties when stably expressed in Chinese hamster ovary (CHO) cells.
Both mutant receptors were expressed on the cell surface and bound
insulin normally, but showed markedly impaired autophosphorylation in
response to insulin. In addition, the in vitro tyrosine kinase
activity of both mutant receptors toward an artificial substrate was
also severely impaired. Despite these defects of kinase activity,
anti-phosphotyrosine immunoblotting of whole cell lysates and
anti-phosphotyrosine immunoprecipitation of
P-labeled
cells showed insulin-stimulated tyrosine phosphorylation of a protein
of
185 kDa to an extent comparable to that seen in CHO cells
expressing wild-type human insulin receptors. Anti-insulin receptor
substrate-1 (IRS-1) immunoprecipitation followed by
anti-phosphotyrosine immunoblotting confirmed that this
tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells
expressing an insulin receptor mutated at the ATP binding site
(Lys-1030
Arg) showed no insulin-stimulated autophosphorylation
or phosphorylation of IRS-1. Despite exhibiting apparently normal
insulin stimulation of IRS-1 tyrosine-phosphorylation, cells expressing
the Arg-1174
Gln or Pro-1178
Leu receptors showed marked
impairment in insulin stimulation of glycogen synthesis, thymidine
incorporation, and activation of MAP kinase. The inability of these
mutant receptors to signal normally to metabolic and mitogenic
responses suggests that insulin-stimulated tyrosine phosphorylation of
IRS-1 alone is insufficient to fully mediate insulin action.
The insulin receptor is a heterotetrameric transmembrane
tyrosine kinase (Ullrich et al., 1985). Binding of ligand to
the extracellular subunit activates the tyrosine kinase activity
of the receptor, the initial substrate of which appears to be the
receptor itself (Ullrich and Schlessinger, 1990). Three tyrosine
residues in the tyrosine kinase domain of the receptor (1158, 1162, and
1163) are phosphorylated in trans by the reciprocal
half-receptor (Lammers et al., 1990). Phosphorylation of all
three tyrosines appears to be necessary to allow full activation of the
receptor's tyrosine kinase activity toward other substrates and
to allow normal signaling to downstream effectors (Murukami and Rosen,
1991). Unlike many other receptor tyrosine kinases, the phosphorylated
tyrosine residues on the receptor itself do not appear to act primarily
as docking sites for Src homology 2 domain-containing signal
transduction proteins. The insulin and insulin-like growth factor-1
receptors phosphorylate one or more substrate proteins with a molecular
mass of approximately 185 kDa. Further characterization of the
185-kDa substrate(s) resulted in the cloning of insulin receptor
substrate-1 (IRS-1) (
)(Sun et al., 1991). IRS-1
contains multiple tyrosine residues in YMXM or YXXM
motifs suitable for interaction with Src homology 2 domain-containing
proteins (Sun et al., 1993; Shoelson et al., 1992).
Several specific signaling molecules that interact with
tyrosine-phosphorylated IRS-1 have been identified (Backer et
al., 1993; Skolnik et al., 1993; Lee et al.,
1993; Xiao et al., 1994; Yamauchi et al., 1995). The
precise function of each of these signal transduction molecules and
their relationship to the spectrum of biological responses to insulin
is currently the focus of major attention. Evidence is rapidly emerging
to indicate that IRS-1 is not the only protein directly phosphorylated
by the insulin receptor tyrosine kinase (Lavan and Lienhard, 1993;
Pronk et al., 1993; Tobe et al., 1995; Sun et
al., 1995; Patti et al. 1995). The establishment of the
relative importance of IRS-1 vis à vis other
molecular targets to the mediation of insulin's effects on
metabolic and mitogenic responses will be of considerable importance to
the fuller understanding of insulin signaling.
Naturally occurring mutations in the insulin receptor have provided considerable insights into the residues and domains of the receptor that are important for function (Taylor et al., 1992). Study of the functional properties of mutations occurring in association with human disease has the advantage that there is often good a priori evidence that altered function caused by the mutation produces a physiologically significant defect in insulin signaling in the whole organism. To date, most studies of both naturally occurring and artificial mutant insulin receptors have confirmed the important role for IRS-1 phosphorylation in the mediation of insulin signaling. In general, mutant receptors that have impaired autophosphorylation also fail to phosphorylate IRS-1 and show impaired insulin-mediated signaling to downstream metabolic and mitogenic events (Moller et al., 1990; Taylor et al., 1992; Cama et al., 1992). Additional evidence for the importance of IRS-1 phosphorylation for insulin signaling comes from studies of artificial insulin receptor mutants, which show a dissociation between receptor autophosphorylation and IRS-1 phosphorylation (White et al., 1988; Backer et al., 1992; Yamamoto-Honda et al., 1993). Studies of these mutant receptors have supported the concept that the phosphorylation of IRS-1, rather than that of the insulin receptor itself, correlates with insulin's activation of downstream effects.
We report on the unique functional properties of two naturally occurring mutants in a region of the insulin receptor tyrosine kinase domain distal to the major sites of tyrosine autophosphorylation. These mutations are the cause of dominantly inherited resistance to insulin-mediated glucose disposal in vivo. Both mutations result in the abolition of insulin-stimulated receptor autophosphorylation but retention of insulin-stimulated IRS-1 tyrosine-phosphorylation. Despite mediating apparently normal IRS-1 phosphorylation, neither mutant insulin receptor was capable of stimulating metabolic and mitogenic events. These results lend support to the notion that IRS-1-independent signaling events are necessary for the mediation of the downstream effects of insulin.
For immunoblotting of total cell lysates, CHO cells were
harvested directly in Laemmli sample buffer containing 100 mM dithiothreitol and boiled for 5 min. After sonication and
centrifugation at 15,000 g for 10 min, aliquots of
supernatants were resolved by 7.5% SDS-polyacrylamide-gel
electrophoresis and Western blot analysis was carried out as described
above.
Figure 1:
Arg-1174 Gln
and Pro-1178
Leu mutant insulin receptors show markedly impaired in vitro tyrosine kinase activity. Insulin receptors obtained
from lysates of mock-transfected CHO cells and cells expressing
wild-type human insulin receptor and Arg-1174
Gln, Pro-1178
Leu, or Arg-1030
Lys mutant receptors, either
unstimulated or stimulated with 100 nM insulin for 5 min, were
captured on an anti-insulin receptor antibody-coated microtiter plate.
After incubation of the captured receptor with reaction mix containing
[
-
P]ATP, the incorporation of
P into an artificial peptide substrate was measured as
described under ``Materials and Methods.'' The results, from
three independent experiments, are presented as the percentage of the
insulin-stimulated activity seen in mock-transfected cells (means
± S.E.).
Figure 2:
Arg-1174 Gln and Pro-1178
Leu mutant receptors show markedly impaired insulin-stimulated
autophosphorylation in vivo.a, cells were grown to
confluence, serum-starved overnight, and then either left untreated or
treated for 2 min with 100 nM insulin as indicated. Cells were
harvested and lysates were immunoprecipitated with an anti-insulin
receptor antibody as described under ``Materials and
Methods.'' After electrophoresis on a 7.5% SDS-polyacrylamide gel,
samples were transferred onto a nylon membrane and immunoblotted with
an anti-phosphotyrosine antibody as described. The position of the
insulin receptor
-subunit is indicated. b, cells were
labeled for 3 h with [
P]orthophosphate and then
stimulated with insulin (100 nM) for 2 min. Cells were
harvested, and lysates were immunoprecipitated with an anti-insulin
receptor antibody. After electrophoresis on a 7.5% SDS-polyacrylamide
gel, samples were analyzed by autoradiography (see ``Materials and
Methods'').
Figure 3:
CHO cells expressing Arg-1174 Gln
and Pro-1178
Leu mutant receptors mediate insulin-stimulated
tyrosine phosphorylation of pp185. Cell lysates from mock-transfected
CHO cells and cells expressing wild-type human insulin receptor,
Arg-1174
Gln, or Pro-1178
Leu mutant receptors were
either untreated or treated with 100 nM insulin for 2 min and
analyzed by 7.5% SDS-polyacrylamide gel electrophoresis, transferred
onto a nylon membrane, and immunoblotted with an anti-phosphotyrosine
antibody (see ``Materials and Methods''). The positions of
the insulin receptor
-subunit and pp185 are
indicated.
Figure 4:
CHO cells expressing Arg-1174 Gln
and Pro-1178
Leu mutant receptors mediate insulin-stimulated
tyrosine phosphorylation of IRS-1. Cells were grown to confluence,
serum-starved overnight, and then either left untreated or treated for
2 min with 5 nM insulin as indicated. Cells were harvested and
lysates were immunoprecipitated with an anti IRS-1 antibody as
described under ``Materials and Methods.'' After
electrophoresis on a 7.5% SDS-polyacrylamide gel, samples were
transferred onto a nylon membrane and immunoblotted with an
anti-phosphotyrosine antibody as described.
Figure 5:
Arg-1174 Gln and Pro-1178
Leu mutant receptors fail to mediate normal insulin signaling to
glycogen synthesis and mitogenesis. a, insulin-stimulated CHO
cells (expressing wild-type, Arg-1174
Gln, Pro-1178
Leu
insulin receptors, or mock-transfected) were grown until confluent,
serum-starved overnight, and then incubated with insulin at the
indicated concentration for 1 h before the addition of
[
C]glucose for another 90 min. After this cells
were harvested, glycogen was precipitated, and incorporated
radioactivity counted as described under ``Materials and
Methods.'' Each point was carried out in triplicate, and the
relative stimulation of three independent experiments is reported
(means ± S.E.). b, CHO cells (expressing wild-type,
Arg-1174
Gln, Pro-1178
Leu insulin receptors, or
mock-transfected) were grown until confluent, serum-starved overnight,
and then incubated with insulin at the indicated concentrations. After
incubation with [
H]thymidine (1 µCi/well) for
90 min, trichloroacetic acid-precipitable counts were measured as
described under ``Materials and Methods.'' Each point was
carried out in triplicate, and the result of three independent
experiments is reported (means ±
S.E.).
At lower
(10-100 nM) insulin concentrations, the stimulation of
glycogen synthesis through the Arg-1174 Gln and Pro-1178
Leu receptors was impaired relative to that seen in mock-transfected
CHO cells, suggesting that the mutant receptors were interfering with
signaling through endogenous CHO insulin receptors. This
dominant-negative effect is consistent with the dominant inheritance of
insulin resistance seen in the families with these mutations.
Similarly, insulin-stimulated thymidine incorporation was also markedly
defective in cells expressing the Arg-1174
Gln or Pro-1178
Leu mutant receptors (Fig. 5B). Cells expressing
wild-type receptors achieved 65% of maximal response at 1 nM insulin concentration, whereas at that insulin concentration there
was no measurable response in the cells expressing either of the mutant
receptors. In contrast to insulin-stimulated glycogen synthesis, there
was no evidence of dominant-negative interference with signaling
through the endogenous CHO insulin receptors with respect to
insulin-stimulated thymidine incorporation into DNA.
Figure 6:
Arg-1174 Gln and Pro-1178
Leu mutant receptors fail to mediate normal insulin signaling to MAP
kinase activation. MAP kinase activity was measured in anti-MAP kinase
immunoprecipitates from mock-transfected CHO cells, cells expressing
wild-type human insulin receptor, Arg-1174
Gln, Pro-1178
Leu, or Arg-1030
Lys mutant receptors that were either untreated
or treated with 10 nM insulin for 10 min. The assay was
performed as described under ``Materials and Methods.'' The
results, from three independent experiments, are presented as the
percentage of the insulin-stimulated activity seen in mock-transfected
cells (means ± S.E.)
Two naturally occurring insulin receptor mutations (Arg-1174
Gln and Pro-1178
Leu) associated with dominantly
inherited insulin resistance displayed unusual signaling properties
when expressed in CHO cells. Both mutant receptors were found to
mediate IRS-1 tyrosine phosphorylation in response to insulin but
showed markedly impaired receptor autophosphorylation and in vitro tyrosine kinase activity toward an artificial substrate. Despite
the tyrosine phosphorylation of IRS-1, both mutant receptors were
unable to mediate insulin's effects on glycogen synthesis and
mitogenesis.
The lack of insulin signaling to downstream effectors
in the face of normal IRS-1 tyrosine phosphorylation is in marked
contrast to several previous studies of mutant insulin receptors, which
have suggested that an insulin receptor's ability to signal to
downstream biological effects correlates well with the tyrosine
phosphorylation of IRS-1 (Cama et al., 1992; Moller et
al., 1990). For example, White et al. demonstrated that a
Tyr-972 Phe mutant receptor was capable of normal
autophosphorylation but did not phosphorylate IRS-1 (White et
al., 1988). Intact cells expressing this receptor show marked
impairment of insulin-stimulated glycogen synthesis, thymidine
incorporation (White et al., 1988), and phosphatidylinositol
(PI) 3-kinase activation (Kapeller et al., 1991). A deletion
mutant lacking 12 amino acids in the juxta-membrane region of the
receptor similarly displayed normal insulin-stimulated
autophosphorylation but severely impaired phosphorylation of IRS-1 and
parallel impairment of insulin-stimulated downstream effects (Backer et al., 1990, Backer et al., 1992). These studies
suggest that the juxtamembrane region of the receptor may be important
in allowing access of IRS-1 to the catalytic domain of the receptor
tyrosine kinase, a concept that is further supported by experiments
using the yeast two-hybrid system, which demonstrate that residues
within this region directly interact with IRS-1 (Gustafson et
al., 1995). Conversely, a mutant receptor lacking the 82
C-terminal amino acids did not show any detectable autophosphorylation
upon insulin stimulation but tyrosine phosphorylation of IRS-1 was
normal (Yamamoto-Honda et al. 1993). CHO cells expressing the
82 receptors show normal insulin stimulation of thymidine
incorporation and PI 3-kinase activation compared to cells expressing
wild-type human insulin receptors. Thus, previous studies of mutant
insulin receptors suggest a key role for IRS-1 phosphorylation in the
mediation of the biological effects of insulin. Despite exhibiting a
pattern of behavior in terms of receptor autophosphorylation versus IRS-1 phosphorylation comparable to that seen with the
82
mutant, both the Arg-1174
Gln and Pro-1178
Leu mutant
receptors differed markedly from the
82 receptor in their failure
to mediate insulin-stimulated mitogenic and metabolic effects.
It is possible that we did not detect a more subtle defect in IRS-1 phosphorylation. Thus time points beyond 5 min were not studied, and it is conceivable that mutant receptors might be capable of initiating phosphorylation of IRS-1 yet be defective in maintaining further rounds of tyrosine phosphorylation. It is also possible that qualitative aspects of IRS-1 phosphorylation, perhaps related to the specific sites of tyrosine phosphorylation, may be defective in cells expressing the mutant receptors. Notwithstanding these caveats, it is of interest that despite apparently normal IRS-1 tyrosine phosphorylation at physiologically relevant concentrations of insulin, downstream signaling remained significantly impaired.
The data obtained from
study of both the Arg-1174 Gln and Pro-1178
Leu mutant
receptors suggests that IRS-1 tyrosine phosphorylation may not be
sufficient for full activation of insulin signaling. This is consistent
with a growing body of evidence indicating the existence of other
signal transduction pathways from the insulin receptor to downstream
events. Recent evidence suggests that the main route of
insulin-stimulated GTP loading of p21
occurs through the
direct phosphorylation by the insulin receptor of Shc (Sasaoka et
al., 1994; Yamauchi and Pessin, 1994; Pronk et al.,
1994). Thus, insulin receptors in which two of the three major tyrosine
autophosphorylation sites have been replaced by phenylalanine show
marked impairment of IRS-1 phosphorylation but signal normally to
insulin-stimulated Shc phosphorylation and activation of p21
(Ouwens et al. 1994). Studies of mice lacking IRS-1 have
indicated the existence of another IRS-1-related molecule (Araki et
al., 1994; Tamemoto et al., 1994). This molecule (IRS-2)
has recently been characterized and shows considerable homology to
IRS-1 (Tobe et al., 1995a). CHO cells express both IRS-1 and
IRS-2, (
)and pp185 appears to be composed of both molecules.
As tyrosine phosphorylation of pp185 appears to be preserved in cells
expressing the Arg-1174
Gln and Pro-1178
Leu mutant
receptors, it is likely that IRS-2 phosphorylation is also normal in
these cells, although definitive confirmation that this is the case
will require direct study of IRS-2.
Thus it appears that at least three substrates of the insulin receptor kinase (IRS-1, IRS-2, and Shc) can act as intermediates between the receptor and downstream events. Other tyrosine-phosphorylated proteins have been identified in insulin-stimulated cells but have not yet been precisely characterized (Lavan and Lienhard 1993). The fact that the p85 subunit of PI 3-kinase has been shown to be capable of direct interaction with the autophosphorylated insulin receptor (Ruderman et al., 1990; Levy-Toledano et al., 1994) also provides a potential mechanism for insulin-mediated signal transduction that is independent of the phosphorylation of adapter molecules. It has recently become apparent that the interleukin 4 (IL-4) receptor is capable of stimulating the tyrosine phosphorylation of IRS-1. This observation has led to experiments that also suggest the requirement of IRS-1-independent events for the mediation of normal insulin action. Thus, phosphorylation of IRS-1 by IL-4 receptors expressed in L6 myoblasts was not sufficient to promote mitogenesis (Pruett et al., 1995). Using the same system, phosphorylation of IRS-1 by IL-4 was also incapable of stimulating glucose uptake (Isakoff et al., 1995).
Despite the evidence for multiple routes from the insulin receptor to downstream pathways, there is a considerable body of evidence that indicates the importance of IRS-1 phosphorylation to insulin signaling. Down-regulation of IRS-1 expression in CHO cells, via antisense RNA, impaired insulin-stimulated mitogenic signaling (Waters et al., 1993). Micro-injection of IRS-1 into Xenopus oocytes permits insulin mediation of germinal vesicle breakdown (Chuang et al., 1993) Insulin-responsive mitogenesis in 32D cells is seen after co-transfection of these cells with the insulin receptor and IRS-1 but not with the insulin receptor alone (Wang et al., 1993). Consistent with these observations is the fact that in IRS-1 knockout mice, despite normal phosphorylation of IRS-2 and Shc, modest impairment of insulin-stimulated growth and glucose metabolism is found (Araki et al., 1994;,Tamemoto et al., 1994). However, mice with a complete deletion of the insulin receptor have a substantially more severe defect in insulin action than that seen in the IRS-1 knockout experiments (Accili et al., 1995), highlighting the importance of other, non-IRS-1-mediated pathways from the insulin receptor.
Thus, although IRS-1 is apparently required
for the full activation of insulin-stimulated biological events, an
emerging body of evidence supports the hypothesis that IRS-1
phosphorylation alone is not sufficient for normal insulin signaling.
Our findings with the Arg-1174 Gln and Pro-1178
Leu
insulin receptor mutants indicate that additional signals, other than
that of IRS-1 phosphorylation, must be activated to permit normal
insulin signaling to downstream events.