From the Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, New York 10016
Received for publication, March 10, 2003 , and in revised form, April 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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Three tyrosine residues in the cytoplasmic juxtamembrane region of the insulin receptor, Tyr-965, Tyr-972, and Tyr-984, are conserved to various extents in the insulin receptor subfamily of RTKs (Fig. 1). This subfamily includes the insulin receptor, the insulin-like growth factor-1 (IGF1) receptor, the insulin receptor-related receptor (IRR), and insulin receptor-like proteins in invertebrates such as Daf-2 in Caenorhabditis elegans and DIR in Drosophila melanogaster. Tyr-965, an autophosphorylation site that is not conserved in the invertebrate receptors, appears to be involved in receptor endocytosis (6, 7). Tyr-972, invariant in the insulin receptor subfamily, is an essential autophosphorylation site (NPXY motif) that recruits IRS proteins (4), Shc (8), and Stat-5b (9) via a phosphotyrosinebinding domain in these adapter proteins. Tyr-984 is also invariant in this subfamily, yet its role in insulin receptor signaling has not been determined. Biochemical studies indicate that Tyr-984 is not, to any appreciable extent, a site of autophosphorylation (10, 11).
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Previous crystallographic studies on the tyrosine kinase domain of the
insulin receptor (IRK) have elucidated the mechanism by which
trans-autophosphorylation of tyrosines in the kinase activation loop
stimulates catalytic activity
(12,
13). These studies were
performed using a cytoplasmic domain construct comprising the core tyrosine
kinase domain (residues 9891283) and the juxtamembrane region proximal
to the kinase domain (residues 978988). This original construct
included two amino acid substitutions in the kinase-proximal juxtamembrane
region, Cys-981 Ser and Tyr-984
Phe. Cys-981, which is not
conserved even among mammalian insulin receptors, was substituted with serine
to prevent formation of disulfide-linked dimers in vitro. At the time
of the initial structural studies, it was unclear whether Tyr-984 was an
autophosphorylation site, and to avoid potential autophosphorylation
heterogeneity, this tyrosine was substituted with phenylalanine. Use of the
Tyr-984
Phe IRK protein (IRKY984F) in past structural
studies, and the disordered state of Phe-984 therein, has left obscure the
function of this conserved tyrosine.
To elucidate the role of Tyr-984 in insulin receptor function, we have
determined a crystal structure of unphosphorylated IRK containing wild-type
Tyr-984. Although crystals of wild-type IRK were obtained, the N-terminal
kinase lobe in this crystal form is poorly ordered, and the position of
Tyr-984 could not be ascertained. Crystals of a mutant IRK in which conserved
Asp-1132 in the catalytic loop was substituted with asparagine
(IRKD1132N) diffract to high resolution, and a structure of this
protein has been determined at 1.9 Å resolution. In this crystal
structure, Tyr-984 is involved in several hydrophobic and hydrogen-bonding
interactions with residues in -helix C (
C) and in the
five-stranded
sheet in the N-terminal lobe of the kinase. These
interactions suggest that Tyr-984 serves a structural role in the kinase
activation mechanism. Comparison of the in vitro steady-state
kinetics properties of wild-type IRK and IRKY984F shows that the
wild-type enzyme has a 4-fold lower kcat than
IRKY984F. Moreover, full-length insulin receptors bearing a Tyr-984
Ala mutation are hyperphosphorylated in transiently transfected
mammalian cells, either in the basal (without insulin) state or upon insulin
stimulation, consistent with an autoinhibitory role for Tyr-984. These data,
together with previous structural studies of activated IRK
(13), suggest a novel strategy
for designing small molecule activators of the insulin receptor with potential
application to noninsulin-dependent diabetes mellitus.
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EXPERIMENTAL PROCEDURES |
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Crystallographic StudiesCrystals of IRKD1132N
were grown at 4 °C by vapor diffusion in hanging drops containing 1.5
µl of protein stock solution (15 mg/ml IRKD1132N, 50
mM Tris-HCl, pH 7.5, and 170 mM NaCl) plus 1.5 µl of
reservoir buffer (20% polyethylene glycol 8000, 100 mM Tris-HCl, pH
7.5, 30 mM NaCl, and 5 mM dithiothreitol). The crystals
belong to the orthorhombic space group
P212121 with unit cell dimensions a
= 57.47 Å, b = 69.48 Å, and c = 88.81 Å
when frozen. There is one molecule in the asymmetric unit, and the solvent
content is 51%. The crystals were transferred stepwise (in ethylene glycol
content) into a cryo-solvent consisting of 20% polyethylene glycol 8000, 100
mM Tris-HCl, pH 7.5, 200 mM NaCl, and 15% ethylene
glycol. Data from a single crystal were collected at Beamline X12C at the
National Synchrotron Light Source (Brookhaven National Laboratory) and were
processed using DENZO and SCALEPACK
(14). Because the unit cell of
the IRKD1132N crystals was very similar to the unit cell of
crystals of an Asp-1161 Ala mutant IRK
(15) (Protein Data Bank code
1I44
[PDB]
), 1I44
[PDB]
was used as the starting model for building and refinement of the
IRKD1132N structure. Rigid body, positional/B-factor refinement and
simulated annealing were carried out with CNS
(16), and model building was
performed with O (17).
Steady-state Kinetics MeasurementsSteady-state kinetics values for wild-type IRK and IRKY984F, both the unphosphorylated (0P) and tris-phosphorylated (3P) forms, were derived using a continuous spectrophotometric assay (18, 19). All of the experiments were carried out at 30 °C in 75 µl of buffer containing 100 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM phosphoenolpyruvate, 0.2 mg/ml NADH, 111 units/ml pyruvate kinase, and 156 units/ml lactate dehydrogenase (Sigma). For determinations of Km,peptide, reactions contained either 500 nM (0P) or 20 nM (3P) enzyme, 5 mM (0P) or 1 mM (3P) ATP, and 012 mg/ml poly-Glu-Tyr peptide (Sigma-Aldrich). For determinations of Km,ATP, reactions contained either 500 nM (0P) or 20 nM (3P) enzyme, 12 mg/ml poly-Glu-Tyr peptide, and 05 mM (0P) or 01 mM (3P) ATP. Kinetic parameters were determined by fitting data to the Michaelis-Menten equation. The conditions of the continuous assay, initial rate measurements and no buildup of ADP, limit ADP-dependent autodephosphorylation of the enzyme (20). Rates of ATP consumption for enzyme alone (<10% of rates with peptide) were subtracted before data fitting.
Transient Transfection of Wild-type and Tyr-984 Ala
Insulin Receptors in HEK 293T CellsA mammalian expression vector
encoding the full-length insulin receptor (pEF-IR) was kindly provided by Dr.
R. Kohanski. Mutations in the insulin receptor were first introduced into the
vector pX-CKD, which encodes the cytoplasmic domain of the receptor
(21), using the QuikChange
system (Stratagene). The mutated pX-CKD insert was cloned into pEF-IR as a
1.6-kb BglI-XbaI fragment as described by Frankel et
al. (22). Wild-type and
mutant pEF-IR were purified using the Endofree Maxi Kit (Qiagen). HEK 293T
cells were seeded in 10-cm tissue culture dishes and cultured in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum, reaching
60%
confluence on the day of transfection. 60 µl of LipofectAMINE 2000 reagent
(Invitrogen) and 10 µg of DNA were used to transfect each 10-cm dish
according to the protocol supplied by the manufacturer. The cells were split
into two 6-well plates 24 h post-transfection and grown for another 24 h in
complete medium (Dulbecco's modified Eagle's medium with 10% fetal bovine
serum and antibiotics). The cells were then serum-starved for 3 h at 37 °C
in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin
(RIA grade, Sigma) prior to insulin stimulation. Bovine insulin (Sigma) was
diluted with 10 mM HCl to various concentrations and applied to the
cells for 10 min at 37 °C. The cells were then placed immediately on ice
and washed once with ice-cold phosphate-buffered saline.
The cells were lysed by the addition to each well of 200 µl of RIPA buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM Na3VO4, 1% Triton X-100, 1% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 10 min. The cell lysates were collected and then clarified by centrifugation at 12,000 x g for 10 min at 4 °C. Proteins in the lysate were separated on 10% SDS-PAGE gels. Lysates containing wild-type and mutant insulin receptors were run on the same gel to ensure accurate comparison. After SDS-PAGE, the proteins were transferred to nitrocellulose membranes (NitroBond, Osmonics) that were blocked overnight in 5% bovine serum albumin in TBST (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.5% Tween 20). Polyclonal rabbit antibodies against phosphotyrosine (Upstate Biotechnology, Inc.) and against the kinase domain of the insulin receptor (provided by Dr. R. Kohanski) were used to detect insulin receptor phosphorylation and protein levels, respectively. Goat anti-rabbit IgG horseradish peroxidase (Santa Cruz Biotechnology) was used as the secondary antibody. Enhanced chemiluminescence (Lightening; Perkin Elmer) was used for signal detection. The phosphorylation content of insulin receptors was determined by scanning the developed films on a Personal Densitometer (Amersham Biosciences), using ImageQuant (version 1.2, Amersham Biosciences) for quantification.
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RESULTS |
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In the original crystal structure of unphosphorylated IRK
(12), the activation loop
traverses the cleft between the N- and C-terminal lobes of the kinase, and
Tyr-1162 in the activation loop functions as a pseudosubstrate inhibitor,
hydrogen-bonded to conserved Asp-1132 and Arg-1136 in the catalytic loop. This
activation loop conformation, which occludes the active site, is
representative of a "gate-closed" conformation
(22). In the
IRKD1132N structure, the substitution of asparagine for Asp-1132 in
the catalytic loop disrupts hydrogen bonding between Asp-1132 and Tyr-1162,
leading to a "gate-open" conformation in which the activation loop
is disengaged from the active site and mostly disordered
(Fig. 2B).
IRKD1132N has negligible catalytic activity, however, because of
the critical role of Asp-1132 in the phosphoryl transfer mechanism
(23). The overall structure of
IRKD1132N is similar to the structure of another IRK mutant,
Asp-1161 Ala, in which loss of four hydrogen bonds mediated by Asp-1161
in the activation loop is responsible for switching the activation loop
conformation from gate-closed to gate-open
(15). The gate-open
conformation observed in the IRKD1132N crystal structure is in
agreement with solution studies that monitored the accessibility of the active
site in this and other IRK mutants
(23).
The conformation of the juxtamembrane region proximal to the kinase domain
(residues 978988) in the IRKD1132N structure differs
appreciably from those in IRK structures reported previously. In the
IRKD1132N structure, this segment lies along C in the
N-terminal kinase lobe (Figs.
2B and
3A). Tyr-984 is
partially buried in a hydrophobic pocket formed at the junction between the
five-stranded anti-parallel
sheet and
C in the N-terminal lobe
(Fig. 3A). In addition
to hydrophobic contacts between Tyr-984 and Leu-1045 and Val-1065, the
hydroxyl group of Tyr-984 is hydrogen-bonded to Glu-990, another invariant
residue in the insulin receptor subfamily. Glu-990 is also hydrogen-bonded to
invariant Ser-1067.
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In fact, a cluster of conserved residues in the insulin receptor subfamily
is located in the junction between the juxtamembrane region and the kinase
domain: Tyr-984, Asp-987, Trp-989, Glu-990, Ala-1048, Lys-1052, and Ser-1067
(Fig. 3C). In the
IRKD1132N structure, Lys-1052 (C) participates in three
hydrogen bonds with juxtamembrane residues, two with the side chain of Asp-987
and one with the main chain of Val-985
(Fig. 3, A and
C). This conformation of the juxtamembrane region is
further stabilized by hydrogen bonds between the side chain of Asn-1046
(
C) and main chain atoms of Ser-981 (Cys-981 in wild-type IRK) and
Val-983 in the juxtamembrane region. Asn-1046 is conserved in insulin and IGF1
receptors and is a lysine in IRR.
In the recent crystal structures of the IGF1 receptor kinase domain in its unphosphorylated (24) or bis-phosphorylated state (25), the tyrosine equivalent to Tyr-984, Tyr-957, is situated in the same hydrophobic pocket as observed for Tyr-984 in the IRKD1132N structure. Of note, Tyr-957 is hydrogen-bonded to conserved Lys-1025 (Lys-1052 in IRK) in the IGF1 receptor kinase structures, whereas in the IRKD1132N structure, Tyr-984 is hydrogen-bonded to conserved Glu-990. This subtle difference in the positioning of Tyr-984/957 in the insulin/IGF1 receptor kinase structures could be a manifestation of the five-residue insertion in the insulin receptor versus the IGF1 receptor just prior to Tyr-984 (Fig. 1).
In the unphosphorylated IRKY984F structure
(12), the proximal
juxtamembrane region is disposed along C in approximately the same
manner as in the IRKD1132N structure, but the side chain of Phe-984
is exposed to solvent and disordered, and the hydrophobic pocket between
C and the
sheet is instead filled with Val-985. In this
juxtamembrane configuration, conserved Trp-989 and Lys-1052 are poorly
ordered, and hydrogen bonding between Lys-1052 and conserved Asp-987 is not
observed.
The conformation of the proximal juxtamembrane region in the
tris-phosphorylated, activated IRK structure
(13) is markedly different
from the conformations in the unphosphorylated IRKD1132N and
IRKY984F structures. In the activated kinase, the juxtamembrane
segment is disengaged from the sheet-
C cleft
(Fig. 3B). This
juxtamembrane conformation is similar to that observed in the structure of the
tris-phosphorylated form of the IGF1 receptor kinase domain
(11), which contained
wild-type tyrosine at residue 957 (Tyr-984 equivalent) and crystallized in a
different space group. These structural observations indicate that the
phosphorylation state of the activation loop dictates the conformation of the
proximal juxtamembrane segment.
Steady-state Kinetics Measurements of IRKWT Versus
IRKY984FTo determine whether the observed interactions
between the proximal juxtamembrane region, specifically Tyr-984, and residues
in the N-terminal kinase lobe affect catalytic activity, the steady-state
kinetics parameters (kcat,
Km,ATP, and
Km,peptide) were determined for the purified
soluble kinase domains, wild-type IRK (IRKWT), and
IRKY984F. Kinetics measurements were performed using a continuous
spectrophotometric assay (18,
19) on both the
unphosphorylated (basal) and tris-phosphorylated (activated) forms of the
enzymes. The data presented in Table
II demonstrate that IRKWT is less active than
IRKY984F in the basal state. The kcat value for
IRKWT is 4-fold lower than for IRKY984F, with minor
differences in the Km values for ATP and
substrate peptide. For tris-phosphorylated IRKWT and
IRKY984F, the kcat and
Km values are essentially equivalent. The
increases in kcat upon phosphorylation of IRKWT
and IRKY984F are 15- and 4-fold, respectively, accompanied by a
6-fold decrease in Km,ATP.
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Transient Transfection of Wild-type and Tyr-984 Ala
Insulin Receptors in Mammalian CellsHaving established from in
vitro kinetics studies that Tyr-984 represses catalytic activity in the
soluble kinase domain, we tested whether full-length insulin receptors bearing
the substitution Tyr-984
Ala are hyperphosphorylated in cells. HEK 293T
cells were transiently transfected with either wild-type or Tyr-984
Ala
insulin receptors, and the level of tyrosine phosphorylation of the receptors
prior to and following stimulation with insulin was examined. Multiple
independent transfection experiments were performed and gave consistent
results. The data shown in Fig.
4 demonstrate that tyrosine phosphorylation levels in the Tyr-984
Ala insulin receptors are considerably elevated over the levels in
wild-type receptors, both in the basal state and upon insulin stimulation. The
extent of phosphorylation in the mutant receptors is particularly striking in
the absence of insulin, with an average (n = 5) fold increase over
wild type of
30. The degree of hyperphosphorylation of mutant
versus wild-type receptors diminished as the dose of insulin was
increased, yet was still
2-fold at 100 nM insulin
(Fig. 4B).
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DISCUSSION |
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To determine the mechanism by which wild-type IRK is catalytically
repressed in the basal state relative to IRKY984F, we determined
the crystal structure of unphosphorylated IRK containing Tyr-984. In this
structure, the juxtamembrane region proximal to the kinase domain (residues
979988) lies along C in the N-terminal kinase lobe, with Tyr-984
situated in a hydrophobic pocket between
C and the
sheet in the
N-terminal lobe (Fig.
3A).
The interactions of Tyr-984 in the sheet-
C cleft and a
comparison of the
C positions in the unphosphorylated and
tris-phosphorylated IRK structures suggest the structural mechanism by which
Tyr-984 represses catalytic activity in the basal state. One of the
consequences of IRK activation loop autophosphorylation and reconfiguration is
the unrestrained rotation of the N-terminal lobe toward the C-terminal lobe
(lobe closure) to bind ATP productively
(13). In addition to an
overall hinge movement of the N-terminal lobe,
C pivots downward toward
the C-terminal lobe (Fig.
5A). This independent (from the
sheet) movement of
C facilitates formation of a catalytically important salt bridge
between conserved Lys-1020 (
3) and conserved Glu-1047 (
C).
Importantly, in the activated IRK structure, the proximal juxtamembrane region
is disengaged from the
sheet-
C cleft (Figs.
3B and
5). These observations indicate
that, in addition to the activation loop, the proximal juxtamembrane segment,
anchored by Tyr-984, provides steric restraints preventing
C from
assuming its catalytically competent position.
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In many protein kinases, C is a key structural element in the
regulation of catalytic activity. For example, in cyclin-dependent kinase 2,
C (or PSTAIRE helix) is not positioned properly to form the conserved
lysine-glutamic acid salt bridge
(26). Cyclin A binding to
cyclin-dependent kinase 2 pushes
C toward the
sheet, resulting
in significant stimulation of kinase activity, even in the absence of
activation loop phosphorylation
(27). In the nonreceptor
tyrosine kinase c-Src, intramolecular interactions between the SH3 domain and
the SH2-kinase linker stabilize a noncatalytically productive position of
C, contributing to repressed kinase activity
(28,
29).
A structural interplay between C and the activation loop has been
documented for c-Src (30).
Autophosphorylation of Tyr-416 and concomitant rearrangement of the activation
loop disrupts the interactions that stabilize the mispositioning of
C.
Conversely, mutations in the
3-
C loop partially activate c-Src
irrespective of Tyr-416 autophosphorylation. Similarly, the structural and
kinetic data presented here indicate that partial activation of IRK in the
basal state results from disengagement of the proximal juxtamembrane region
from the
sheet-
C cleft (via mutation of Tyr-984). The
observation that, once phosphorylated, wild-type IRK and IRKY984F
have very similar catalytic efficiencies
(Table II) implies that the
influence of the activation loop on
C positioning is
"dominant" over that of the proximal juxtamembrane region.
To determine whether autoinhibition by Tyr-984 as detailed here for the
soluble kinase domain is necessary for maintaining low basal-level
phosphorylation in the full-length insulin receptor, we transiently
transfected HEK 293T cells with either wild-type insulin receptors or mutant
insulin receptors in which Tyr-984 was replaced by alanine. These studies
demonstrated that in the absence of insulin, the tyrosine phosphorylation
levels of Tyr-984 mutant receptors were significantly elevated (30-fold)
relative to that of wild-type receptors and were
2-fold elevated at
maximal insulin stimulation (Fig.
4). The elevated tyrosine phosphorylation levels in the Tyr-984
Ala mutant were not due to a decreased susceptibility to
dephosphorylation; the wild-type and mutant receptors displayed comparable
dephosphorylation kinetics (data not shown).
Interestingly, the activating effect of the Tyr-984 Ala substitution
in the full-length insulin receptor is considerably more robust than for the
Asp-1161
Ala substitution described previously
(22). The Asp-1161
Ala
substitution in the activation loop partially relieves activation loop
autoinhibition (15), resulting
in a sizable increase in catalytic efficiency for the soluble kinase domain in
the unphosphorylated state; kcat increases 10-fold, and
Km,ATP decreases 15-fold
(22). Yet in the context of
the full-length receptor transfected into C2C12 cells
(22) or HEK 293T cells (data
not shown), a modest effect (
3-fold) on basal-level or insulin-stimulated
phosphorylation is observed. In contrast, mutation of Tyr-984 in the soluble
kinase domain yields a comparatively small increase in catalytic efficiency
(4-fold higher kcat) but results in a substantial increase
(
30-fold) in the phosphorylation level of the full-length receptor in
unstimulated cells.
The difference in the basal-level phosphorylation states of these two
mutant insulin receptors, Tyr-984 Ala and Asp-1161
Ala, suggests
that, in addition to influencing the position of
C in the kinase
domain, the proximal juxtamembrane region plays a key role in the mechanism by
which insulin binding to the extracellular
subunits induces a
structural rearrangement in the cytoplasmic
subunits. It is conceivable
that the interactions between the proximal juxtamembrane segment and
C
in the N-terminal kinase lobe are important for maintaining a spatial
arrangement of the two kinase domains that limits
trans-autophosphorylation in the basal state. Loss of these
interactions (via mutation of Tyr-984) might partially relieve the
steric constraints imposed by the extracellular domains (devoid of insulin) on
the cytoplasmic domains. The observation that insulin stimulation increases
tyrosine phosphorylation of the Tyr-984
Ala mutant receptor indicates
that additional steric restraints in the receptor are still operational.
Importantly, many other RTKs contain either tyrosine at the equivalent
position of Tyr-984 in the insulin receptor or another hydrophobic residue at
that or a nearby position, which may function similarly to Tyr-984, at least
with respect to C positioning. For example, vascular endothelial growth
factor receptor-2 possesses a tyrosine (Tyr-822) at the equivalent position of
Tyr-984, which in the crystal structure
(31) is disposed similarly to
Tyr-984, hydrogen-bonded to Glu-828, the equivalent of Glu-990 in the insulin
receptor. In the crystal structure of the kinase domain of fibroblast growth
factor receptor-1 (32),
Leu-465, one residue N-terminal to the position of Tyr-984 in the insulin
receptor, is engaged in the
sheet-
C cleft. Finally, in the
crystal structures of Tie2
(33) and EphB2
(34), an isoleucine (Ile-815)
or valine (Val-617), respectively, in the proximal juxtamembrane region
fulfills this structural role.
Therefore, a similar autoinhibitory mechanism may operate in these and
other RTKs, and mutation of the residue positioned in the
sheet-
C cleft would be predicted to result in partial activation. The
degree to which the receptor is activated will depend on the relative strength
of other autoinhibitory mechanisms to which that particular RTK is subject
(35), e.g. activation
loop (IRK and MuSK (36)),
extended juxtamembrane region (EphB2
(34) and c-Kit
(37)), or C-terminal tail
(33). Moreover, the
constitutive dimeric arrangement of receptors in the insulin receptor
subfamily might make receptors in this subfamily particularly sensitive to
mutation of conserved Tyr-984, as discussed above.
The structural characterization of the autoinhibitory role of Tyr-984 in
the basal state kinase affords a novel strategy for the design of small
molecule activators of the insulin receptor. A cell-permeable compound
designed to bind in the sheet-
C cleft and displace Tyr-984
should partially activate the receptor in the absence of insulin and sensitize
the receptor in the presence of insulin. The specific target site for such a
molecule would be the
sheet-
C cleft as configured in the
activated IRK structure (13)
(Fig. 5B).
Two compounds capable of stimulating insulin receptor autophosphorylation
by acting on the cytoplasmic domain have been reported
(38,
39). One of the compounds
(L-783,281) modestly elevates insulin receptor autophosphorylation in the
absence of insulin, whereas the other compound (TLK16998) potentiates receptor
autophosphorylation in the presence of insulin, suggesting two different
mechanisms of action (40).
Interestingly, L-783,281 and a derivative
(41) are of an appropriate
size to bind in the sheet-
C cleft, and L-783,281 was shown to
alter the trypsin susceptibility at Lys-1030
(38), not far spatially from
Tyr-984 (Fig. 5A).
Whether or not the
sheet-
C cleft is the site of action of these
compounds, the existence of such small molecule activators, together with the
data presented here on the autoinhibitory role of Tyr-984, motivates efforts
to design novel insulin receptor activators with potential application to
noninsulin-dependent diabetes mellitus.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK52916 (to
S. R. H.). Financial support for Beamline X12C of the National Synchrotron
Light Source comes principally from the National Institutes of Health and the
Department of Energy. The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Present address: Exelixis, Inc., South San Francisco, CA 94083.
To whom correspondence should be addressed: Skirball Institute of Biomolecular
Medicine, New York University School of Medicine, 540 First Ave., New York, NY
10016. Tel.: 212-263-8938; Fax: 212-263-8951; E-mail:
hubbard{at}saturn.med.nyu.edu.
1 The abbreviations used are: RTK, receptor tyrosine kinase; IRS, insulin
receptor substrate; IGF1, insulin-like growth factor-1; IRR, insulin
receptor-related receptor; IRK, tyrosine kinase domain of the insulin
receptor; HEK, human embryonic kidney.
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ACKNOWLEDGMENTS |
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REFERENCES |
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