(Received for publication, February 21, 1995; and in revised form, June 12, 1995)
From the
In a previous analysis, we identified a point mutation that
substituted Pro (CCG) for Leu (CTG) at amino acid 87 in the
-subunit of the insulin receptor (IR) in a Japanese patient with
leprechaunism. In the present study, we transfected either the wild
type (Leu-87) or the mutant (Pro-87) IR cDNA into NIH3T3 cells.
Pulse-chase in nonreducing conditions revealed that the dimerization of
Pro-87 IR was slightly impaired. However, cell surface biotinylation
showed that Pro-87 IR was transported to the cell surface. The Pro-87
IR reduced the insulin binding affinity to about 15% of Leu-87 IR, and
the dissociation of insulin in Pro-87 IR was more rapid than in Leu-87
IR. The autophosphorylation of Pro-87 IR was less sensitive to insulin
than that of Leu-87 IR, suggesting the reduced insulin binding
affinity. Site-directed mutagenesis at amino acid 87 was performed to
substitute Ile or Ala for Leu. Both mutant IRs were transported to the
cell surface and labeled by cell surface biotinylation. The Ile-87 IR
enhanced the insulin binding affinity about 4-fold. The insulin binding
affinity of Ala-87 IR was reduced by 85% relative to that of Leu-87 IR.
In addition, the dissociation of insulin in Ile-87 IR was slower than
in Leu-87 IR, but in Ala-87 IR it was more rapid. These results provide
the first direct evidence for a critical role of Leu-87 in binding
insulin.
Insulin receptor (IR) ()is a cell surface
glycoprotein with heterotetrameric structure consisting of two
-subunits (135 kDa) and two
-subunits (95 kDa) linked with
disulfide bonds. The
-subunit is an extracellular domain including
insulin binding sites. The
-subunit consists of extracellular,
transmembrane and cytoplasmic domains, the last of which has
autophosphorylation
sites(1, 2, 3, 4, 5) .
Autophosphorylation at these sites activates tyrosine kinase in the IR,
triggers phosphorylation of intracellular substrates, and transduces
intracellular signaling (6, 7, 8, 9) . Insulin binding to
the IR is the first step and is essential for inducing many biological
actions, such as mitogenic and metabolic actions, on the target cells.
On cloning human IR cDNA(4, 5) , molecular analyses
of patients with genetic forms of severe insulin resistance, including
type A insulin-resistant syndrome, leprechaunism, and Rabson-Mendenhall
syndrome, have been reported(10) . Most cases of mutations in
the -subunit of the IR affected its intracellular transport by
change of folding and reduced the amount of IR on the cell
surface(10) . Thus, it is difficult to assess the insulin
binding affinity of these mutant IRs.
Although site-directed
mutagenesis, photoaffinity labeling studies, and the construction of an
IR/insulin-like growth factor-1 receptor chimera have identified the
presumptive insulin binding domains (11, 12, 13, 14, 15) , the
exact contact sites of insulin in the IR are still unknown. In
addition, crystallization of the IR has not been done, and its
three-dimensional structure is not clear. Thus, the genetic analysis of
mutation of the -subunit without impairment of the
post-translational processing of the IR and the site-directed
mutagenesis at the sites of amino acid replacement might suggest the
significance of these amino acids in binding insulin.
In a previous
study, we identified a point mutation that substituted Pro (CCG) for
Leu (CTG) at amino acid 87 of the IR in a Japanese girl with
leprechaunism. ()Another mutation in her case was a
1.3-kilobase pair deletion in the region between exon 4 and exon 6 on
the genomic DNA, which induced a skipping-out of exon 5 of the IR and
produced a premature stop codon at amino acid 356 in exon 6. We
reported that this deletion of maternal allele reduced the amount of IR
on the cell surface. Leprechaunism is a rare congenital syndrome
associated with severe insulin resistance and multiple anomalies
including intrauterine growth retardation (16) . Previous
studies on molecular analyses of the human IR gene in leprechaunism
disclosed that most cases of leprechaunism are homozygotes or compound
heterozygotes of the mutant IR gene(10) . Therefore, to
investigate how the substitution of Pro for Leu at amino acid 87
impairs the biological actions of insulin on the target cells, we
constructed an expression vector of human IR cDNA and transfected
either the wild type (Leu-87) or the mutant type (Pro-87) IR cDNA into
NIH3T3 cells, obtained stably expressing clones, and assessed the
biosynthesis and insulin binding affinity of these receptors. In
addition, because amino acid 87 is located in the presumptive insulin
binding
domain(11, 12, 13, 14, 15) ,
the assessment of its effect on IR might give a clue to the insulin
contact sites of IR. Thus, to investigate the significance of Leu at
amino acid 87, we performed site-directed mutagenesis at this position,
substituting Ile or Ala for Leu, and assessed the effects of these
substitutions on the insulin binding affinity of NIH3T3 cells
expressing these mutant IRs in a stable manner. We present evidence
supporting the possibility that Leu at amino acid 87 may play an
essential role in binding insulin.
Because p13-1 had one more translation-initiation sequence ATG in the 5` region of the true translation-initiation site, pBSIIKSHIR was digested with SalI to delete the 5` end of the true translation-initiation site. After ligation of SalI linker 5`-TCGACAATCGATTG-3` containing a ClaI site (underlined) with ClaI-digested pBSKSIIHIR and digestion with ClaI and SpeI, the 4.4-kilobase pair fragment containing the whole human IR cDNA was ligated into ClaI/SpeI-digested pCMV (PCMVHIR).
For the construction of expression vector of Pro-87 IR cDNA, the patient's IR cDNA was amplified with the following primers: 5`-TCCCGGCATGGATATCCGGAACAACC-3` (nucleotides 243-268 of the sense strand) and 5`-CTTCGCGGTACCCGGACAGAT-3` (nucleotides 711-691 of the antisense strand). Amplified cDNA was digested with EcoRV and KpnI to yield a 449-base pair fragment (nucleotides 256-704). This EcoRV/KpnI fragment was substituted for the comparable segment of pCMVHIR. The correct structure of the exchanged fragment was confirmed by DNA sequencing using Sequenase R Version 2.0 (U. S. Biochemical Corp.).
NIH3T3 cells
expressing either Leu-87 or Pro-87 IR cDNA were incubated for 1 h at 37
°C in methionine- and cysteine-free Dulbecco's modified
Eagle's medium (6 ml; Life Technologies, Inc.) including 10%
fetal bovine serum. Thereafter, cells were pulse-labeled for 1 h with
ExpreS
S (100 µCi/ml
[
S]methionine +
[
S]cysteine; DuPont NEN). In the media used in
the subsequent chase periods (0-20 h), the radioactive amino
acids were omitted and replaced by complete Dulbecco's modified
Eagle's medium. At each time point, cells were washed twice with
ice-cold phosphate-buffered saline and solubilized in 50 mM HEPES (pH 7.2) solution containing 150 mM NaCl, 1.5
mM MgCl
, 1 mM EDTA, 10% glycerol, 1%
Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 200
units/ml aprotinin for at least 60 min at 4 °C. After
centrifugation at 12,000 rpm at 4 °C, supernatants were
immunoprecipitated with an anti-IR monoclonal antibody (Ab-3; Oncogene
Science Inc., Uniondale, NY) raised against the
-subunit of human
IR at a dilution of 1:100 for at least 2 h at 4 °C. Immune
complexes were precipitated with protein A-agarose (Life Technologies,
Inc.) overnight at 4 °C and washed three times with 20 mM HEPES (pH 7.2) solution containing 150 mM NaCl, 0.1%
Triton X-100, and 10% glycerol. Thereafter, the pellets were dried up
and boiled for 10 min in the reducing condition in Laemmli's
sample buffer (18) containing 10%
-mercaptoethanol and 100
mM dithiothreitol. In the nonreducing condition, samples were
not boiled, and sample buffer contained 0.1 M Tris-HCl (pH
6.8), 2% SDS, 9 M urea, and 0.05% bromphenol blue. Samples
were electrophoresed in 6.5% SDS-PAGE in reducing conditions. In
nonreducing conditions samples were electrophoresed in 3-10%
gradient gel followed by autoradiography for 2-3 weeks and
estimation of radioactivity with Fuji BAS 2000 Image Analyzer.
Figure 1:
Pulse-chase study of IR with
[S]methionine in reducing condition. NIH3T3
cells expressing either wild type (Leu87IR) or mutant type (Pro87IR) receptor were pulse-labeled with
[
S]methionine for 1 h, followed by chase periods
(0-20 h) in the absence of [
S]methionine.
Thereafter, receptors were immunoprecipitated with Ab-3. The immune
complexes were analyzed by 6.5% SDS-PAGE followed by
autoradiography.
Figure 2:
Pulse-chase study of IR with
[S]methionine in nonreducing condition. NIH3T3
cells expressing either wild type (Leu87IR) or mutant type (Pro87IR) receptor were pulse-labeled for 1 h, followed by
chase periods (0-20 h) in the absence of
[
S]methionine. Thereafter, receptors were
immunoprecipitated with Ab-3. The immune complexes were analyzed by
3-10% gradient SDS-PAGE followed by
autoradiography.
Figure 3: Cell surface biotinylation of wild type and mutant IRs. Confluent monolayers of NIH3T3 cells expressing Leu-87 (lane 1), Pro-87 (lane 2), Ile-87 (lane 3), or Ala-87 (lane 4) IR and nontransfected NIH3T3 cells (lane 5) were biotinylated as described under ``Experimental Procedures.'' Each IR was immunoprecipitated and electroblotted onto nitrocellulose filter and detected with horseradish peroxidase-labeled streptavidin.
Figure 4:
A, Scatchard plot analysis of insulin
binding to each clone. Insulin binding to the surface of NIH3T3 cells
expressing either Leu-87 IR-1 () or Leu-87 IR-2 (
) was
measured as described under ``Experimental Procedures.'' B, Scatchard plot analysis of NIH3T3 cells expressing either
Leu-87 IR-1 (
) or Pro-87 IR (
). Data were presented as the
means of three replicate experiments.
Figure 5: Insulin-stimulated phosphorylation of IRs in intact cells. After incubation in serum-free Dulbecco's modified Eagle's medium at 37 °C for 2 h, NIH3T3 cells expressing either Leu-87 IR (WT(Leu87)) or Pro-87 IR (Pro87) were left untreated or stimulated with insulin (100 nM) at 37 °C for 1 min. The number of NIH3T3 cells expressing Pro-87 IR was about 4-fold of NIH3T3 cells expressing Leu-87 IR. The cells were solubilized and immunoprecipitated with Ab-3. After being separated in 6.5% SDS-PAGE, the samples were electroblotted onto nitrocellulose filters. The filters were probed with anti-phosphotyrosine monoclonal antibody and analyzed according to ECL Western blotting protocol (Amersham Corp.).
Figure 6:
A, Scatchard plot analysis of Ile-87
() and Leu-87 IR-2 (
). B, Scatchard plot
analysis of Ala-87 (
), Leu-87 IR-1 (
), and nontransfected
NIH3T3 cells (
). [
I]Insulin binding assay
was performed as described under ``Experimental Procedures.''
Data were presented as the means of three replicate
experiments.
Insulin binding to the IR is the first essential step for inducing the biological actions of insulin. However, the mechanism of binding of the IR to insulin and the precise structure of the IR are not known. The investigation of the structure and function of transmembrane receptor glycoproteins is largely dependent on the genetic analysis of naturally occurring mutations and site-directed mutagenesis.
In a previous study we identified a point mutation of
the IR gene in a Japanese girl with leprechaunism whose Epstein-Barr
virus-transformed lymphoblasts showed extremely reduced insulin
binding. This point mutation was the substitution of C for T at
nucleotide 479. It resulted in the substitution of Pro for Leu at amino
acid 87. We considered that this substitution reduced the insulin
binding affinity. However, most mutations in the
-subunit of the IR impair the intracellular transport to the cell
surface and reduce the number of IRs on the cell surface(10) .
Generally, IR is translated as a 190-210-kDa proreceptor that
is glycosylated with N-linked oligosaccharides, dimerized in
endoplasmic reticulum, and transported to the Golgi apparatus, in which
its glycosylated chains are matured and the proreceptor is cleaved into
the - and
-subunits. The IR is then transported to the cell
surface(24) . The Pro-233 mutation inhibits the transport of
the proreceptor from intracellular sites to the cell surface due to the
inhibition of cleavage of the proreceptor and of the addition of sialic
acid(25) . The Val-382 mutation inhibits the final processing
of the N-linked oligosaccharides of the proreceptor in the
Golgi apparatus and their transport to the cell surface(26) .
The Arg-209 mutation impairs dimerization of the proreceptor into a
disulfide-linked structure, proteolytic cleavage of the proreceptor
into the
- and
-subunits and terminal processing of the high
mannose form of N-linked oligosaccharide impairs dimerization
of the proreceptor into a complex carbohydrate(27) . The Arg-31
mutation inhibits the transport of the proreceptor to the Golgi
compartment, where proteolytical processing occurs(28) . These
mutations all result in a decreasing amount of IR on the cell surface.
In the present study, the glycosylation in Pro-87 IR was normal, for
the apparent molecular size of the proreceptor and the
-and
-subunits of Pro-87 IR were the same as those of Leu-87 IR.
However, the post-translational processing of Pro-87 IR was slightly
impaired, for the disappearance of the proreceptor and the appearance
of the
-and
-subunits of Pro-87 IR were slower than in the
case of Leu-87 IR in a pulse-chase study using
[
S]methionine in a reducing condition. In
addition, the dimerization of the monomeric proreceptor of Pro-87 IR
was also slightly impaired, for the appearance of the dimeric form of
the proreceptor was slower than that of Leu-87 IR in a pulse-chase
study using [
S]methionine in nonreducing
conditions. The delayed disappearance of the proreceptor in the
pulse-chase study in reducing condition may be due to the impaired
dimerization of the monomeric proreceptor. The conformational change
resulting from the substitution of Pro for Leu at amino acid 87 may
impair the intracellular transport and especially the dimerization of
the proreceptor (monomer) in the endoplasmic reticulum. But this
impairment of the intracellular transport of Pro-87 IR may be
incomplete, because Pro-87 IR can be cleaved into the
- and
-subunits and transported to the cell surface. In addition,
Scatchard plot analysis of Epstein-Barr virus-transformed lymphoblasts
of the patient's father showed that the amount of IR on the cell
surface was almost normal (data not shown). Thus, the substitution of
Pro for Leu does not reduce the amount of IR on the cell surface.
Several researchers have tried to identify the insulin binding
domain. A consensus that at least two separate regions of the IR are
involved in binding insulin has recently emerged. They are mapped
within the amino-terminal domain encoded by exon 2 (residues
1-119) and the carboxyl-terminal domain encoded by exon 6 and 7
(residues 311-428) of the extracellular
domain(11, 12, 13, 14, 15, 29, 30) .
However, the exact contact sites of insulin on the IR are not known. A
few cases that showed decreased insulin binding without impaired
post-translational processing of the IR have been reported. The Lys-15
mutation causes a 5-fold reduction in the capacity of the IR to bind
insulin. However, this mutation also retards the post-translational
processing of the IR and impairs transport of the receptor to the cell
surface(31) . Recently, the Leu-323 mutation has been reported
to cause decreased insulin binding with normal post-translational
processing and cell surface expression of IR(32) . These
naturally occurring mutations have suggested that the sites of amino
acid replacement may play an important role in binding insulin.
However, to confirm that these amino acids are the insulin contact
sites, site-directed mutagenesis is needed, because these substitutions
may only cause conformational change of the true insulin contact sites.
De Meyts et al. showed that an aromatic side chain of Phe at
amino acid 89 seemed necessary but not sufficient for high affinity
interaction with insulin, but Phe-88 was not involved in binding
insulin by site-directed mutagenesis(12) . In addition,
site-directed mutagenesis at amino acids 85 and 86 showed that these
positions were probably not directly involved in ligand
binding(22) . Leu at amino acid 87 is placed in the insulin
binding domain described above and is also adjacent to Phe at amino
acid 89. In addition, Pro is often found at sites of -turn of
polypeptides, and the introduction of Pro does bend the polypeptides
and induce the conformational change of the protein. Several naturally
occurring mutations in patients with insulin resistance, site-directed
mutagenesis, photoaffinity cross-linking studies, and the construction
of IR/insulin-like growth factor-1 receptor chimeras give insight into
the mechanism of insulin binding and the insulin binding domains.
Indeed, both Scatchard plot analysis and dissociation kinetics reveal
that the substitution of Pro for Leu at amino acid 87 results in
reduced binding affinity. This substitution may thus induce
conformational change of the insulin binding domain and reduce the
insulin binding affinity without gross impairment of post-translational
processing of the IR. In addition, the capacity for autophosphorylation
of the
-subunit of Pro-87 IR is depressed compared with that of
Leu-87 IR, probably because of decreased insulin binding affinity.
However, Pro-87 IR preserves the capacity for autophosphorylation in a
dose-dependent manner.
Furthermore, Leu at amino acid 87 is
conserved among other kinds of receptors, such as human insulin-like
growth factor-1 receptor, Drosophila epidermal growth factor
receptor, and c-erb-B2 receptor(29) . Thus, Leu at amino acid
87 may play an important role in binding insulin. Site-directed
mutagenesis at amino acid 87 also suggests the importance of Leu at
amino acid 87 in binding insulin. The substitution of Ile for Leu
increases the insulin binding affinity. In contrast, the substitution
of Ala for Leu decreases the insulin binding affinity. These findings
identify the substitution of Ile for Leu at amino acid 87 of the IR as
being of special significance and suggest that at amino acid 87 the
- or
-branched side chain is needed for binding insulin and
that the
-branch of Leu results in an ineffectively disturbed side
chain mass at amino acid 87 as opposed to the
-branch of Ile.
Similarly, the replacement of the
-branched side chain of Val with
the
-branched side chain of Leu at position A3 of human insulin
results in a naturally occurring insulin variant (Insulin Wakayama)
possessing the lowest receptor binding potency of any abnormal human
insulin studied to date(33) . Because the side chain is
important for the interaction between the ligand and its receptor,
these modifications at amino acid 87 may be associated with a change of
insulin binding affinity. These findings suggest that at amino acid 87
of the IR, the
-branched side chain is more suitable than the
-branched side chain for binding insulin.
In conclusion, the substitution of Pro for Leu at amino acid 87 of the IR reduces the insulin binding affinity without gross impairment of intracellular transport. In addition, Leu at amino acid 87 may play an important role in binding insulin, such as providing the insulin contact site. Crystallization of the IR may reveal this in the future.