From the Department of Pharmacology and Therapeutics,
Medical College of Ohio, Toledo, Ohio 43614
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ABSTRACT |
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pp120, a substrate of the insulin receptor
tyrosine kinase, does not undergo ligand-stimulated phosphorylation by
the insulin-like growth factor-1 (IGF-1) receptor. However, replacement
of the C-terminal domain of the IGF-1 receptor -subunit with the
corresponding segment of the insulin receptor restored pp120
phosphorylation by the chimeric receptor. Since pp120 stimulates
receptor-mediated insulin endocytosis when it is phosphorylated, we
examined whether pp120 regulates IGF-1 receptor endocytosis in
transfected NIH 3T3 cells. pp120 failed to alter IGF-1 receptor
endocytosis via either wild-type or chimeric IGF-1 receptors. Thus, the
effect of pp120 on hormone endocytosis is specific to insulin, and the C-terminal domain of the
-subunit of the insulin receptor does not
regulate the effect of pp120 on insulin endocytosis. Mutation of
Tyr960 in the juxtamembrane domain of the insulin
receptor abolished the effect of pp120 to stimulate receptor
endocytosis, without affecting pp120 phosphorylation by the insulin
receptor. These findings suggest that pp120 interacts with two separate
domains of the insulin receptor as follows: a C-terminal domain
required for pp120 phosphorylation and a juxtamembrane domain required for internalization. We propose that the interaction of pp120 with the
juxtamembrane domain is indirect and requires one or more substrates
that bind to Tyr960 in the insulin receptor.
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INTRODUCTION |
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The insulin receptor is essential to mediate the multiple effects
of insulin on target cells (1, 2). Insulin binding to the -subunit
of its receptor activates the tyrosine kinase of the receptor in the
cytoplasmic tail of the
-subunit to phosphorylate the receptor (3)
and other endogenous substrates, including pp1201 (4, 5), insulin
receptor substrate proteins (IRS-1, -2, -3, and -4) (6-10), Shc (11,
12), and others (reviewed in Ref. 10). Phosphorylated substrates engage
in turn the formation of signaling complexes via
phosphotyrosine-containing binding motifs with src
homology-2 (SH2) domains (13) in order to propagate the signals of
insulin in the cell.
pp120 is a plasma membrane glycoprotein expressed in the liver as two spliced variants that differ by the inclusion (full-length) or exclusion (truncated) of 61 out of 71 amino acids of the cytoplasmic domain (14). In contrast to the truncated isoform, full-length pp120 undergoes insulin-stimulated phosphorylation (5). Site-directed mutagenesis revealed that phosphorylation on Ser503 by cAMP-dependent serine kinase occurs in the absence of insulin and that phosphorylation at this site is required for insulin-stimulated tyrosine phosphorylation on Tyr488, the major pp120 phosphorylation site by the insulin receptor kinase (5).
In marked contrast to insulin receptors, insulin-like growth factor-1
(IGF-1) receptors failed to mediate pp120 phosphorylation in response
to IGF-1 (15). This is consistent with the predominant expression of
pp120 and insulin receptors in the liver, an organ with low levels of
IGF-1 receptors (16). Moreover, pp120 phosphorylation by the IGF-1
receptors was restored when the C-terminal domain of the -subunit of
the IGF-1 receptor was replaced by that of the insulin receptor,
suggesting that pp120 phosphorylation by the insulin receptor is
regulated by this domain. These features distinguish pp120 from other
substrates, phosphorylation of which by both the insulin and the IGF-1
receptors is regulated by the juxtamembrane domain of the receptors.
Thus, pp120 may mediate different physiologic functions of the two
receptors, with the insulin receptor regulating metabolism (1) and the
IGF-1 receptor mediating growth and differentiation (17, 18).
An important mechanism to regulate plasma insulin levels is
receptor-mediated rapid vesicular endocytosis of insulin (19), followed
by degradation (20). Whereas activation of the receptor kinase is
required for this endocytosis (21-23), the molecular events involved
in this process are not yet well defined. The juxtamembrane domain of
the insulin receptor contains two tyrosine-centered sequences
(Gly-Pro-Leu-Tyr953 and Asn-Pro-Glu-Tyr960) in
tight -turn structures that conform to the internalization signaling
motif (24). However, the role of these sequences in insulin receptor
endocytosis is controversial despite the general agreement that the
juxtamembrane domain plays a significant role (22, 25). Although most
reports agree that the Gly-Pro-Leu-Tyr953 sequence is
required for insulin-stimulated receptor endocytosis, Berhanu et
al. (26) observed that this sequence is not necessary for
insulin-stimulated endocytosis of insulin receptor isoform B (exon
11+). The sequence around Tyr960 matches the
internalization motif of the low density lipoprotein receptor
(Asn-Pro-X-Tyr) (27) and is conserved in the IGF-1 receptor.
This sequence is required for ligand-induced IGF-1 receptor endocytosis
(28), but it is not required for insulin-induced insulin receptor
endocytosis (25). Instead, it is required for substrates binding such
as IRS-1 and Shc (29-31). Since Tyr960 mediates binding of
substrates to the insulin receptor, it is possible that these
substrates play a role in insulin-mediated receptor endocytosis.
However, phosphorylation of IRS-1 did not regulate insulin-receptor
endocytosis in transfected Chinese hamster ovary cells (32). Shc, a
substrate of the insulin and the epidermal growth factor (EGF)
receptors, has been shown to bind to adaptor proteins in the clathrin
coat of the endocytotic vesicles to participate in ligand-stimulated
endocytosis of EGF receptors (33). Even though it may play a similar
role in insulin receptor endocytosis, this hypothesis has not yet been
tested.
We have observed that pp120 co-expression with insulin receptors
enhanced receptor-mediated insulin endocytosis and degradation in
transfected cells and that this effect required pp120 phosphorylation by the insulin receptor tyrosine kinase (34, 35). More recently, we
have observed that pp120 stimulated insulin endocytosis without being
directly associated with the insulin
receptor.2 This effect
required insulin-stimulated phosphorylation of pp120 by the receptor
tyrosine kinase.2 Since pp120 phosphorylation by the
insulin receptor is regulated by the C-terminal domain of the
-subunit of the receptor (15), we have examined in these studies
whether this domain regulates the effect of pp120 on insulin-induced
receptor endocytosis in transfected NIH 3T3 cells. We have observed
that the C terminus of the
-subunit of the insulin receptor was not
directly involved in regulating the stimulatory effect of pp120 on
insulin endocytosis. Instead, phosphorylation on Tyr960 in
the juxtamembrane domain of the receptor was required for the effect of
pp120 on insulin endocytosis. This is consistent with the hypothesis
that the juxtamembrane domain of the receptor is involved in insulin
endocytosis, perhaps by mediating complex formation between pp120 and
the insulin receptor via other signaling molecules.
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EXPERIMENTAL PROCEDURES |
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Materials--
LipofectAMINE reagent, G418 (Geneticin), and
protein A-agarose were purchased from Life Technologies, Inc.
Hygromycin B was purchased from Calbiochem. 125I-Insulin
and 125I-IGF-1 (2000 Ci/nmol, radioimmunoassay grade), the
sheep horseradish peroxidase-labeled anti-rabbit antibody, and the
enhanced chemiluminescence (ECL) reagents were purchased from Amersham
Pharmacia Biotech. Protease inhibitors were purchased from Boehringer
Mannheim. All reagents for polyacrylamide gel electrophoresis were
purchased from Bio-Rad. Human insulin was purchased from Lilly and
insulin-free bovine serum albumin from Intergen Co. (Des Plaines, IL).
Recombinant human IGF-1, monoclonal anti-phosphotyrosine (-Tyr(P))
antibodies, polyclonal anti-IRS-1 (
-IRS-1) antibody, and fetal calf
serum were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). pp120 antibodies used in these studies were previously described (5). Briefly, the monoclonal antibody used to immunoprecipitate pp120
(
-HA4, an identical protein to pp120) was purified from ascites
fluid from HA4 c19 cells purchased from the Developmental Studies
Hybridoma Bank (Department of Biology, University of Iowa, Iowa City).
The polyclonal antibody used to immunoblot pp120 (
-295) was raised
in rabbit against a peptide (amino acids 51-64) in the extracellular
domain of rat liver pp120. Ab-53, a polyclonal antibody raised in
rabbit against a conserved region in the tyrosine kinase domain of the
insulin and IGF-1 receptors, was described previously (36).
Construction of Expression Vectors--
Synthesis and subcloning
into a bovine papilloma virus-based expression vector (Amersham
Pharmacia Biotech) of the cDNA encoding the full-length wild-type
(WT) isoform of the rat pp120 and of human insulin receptors-A isoform
(hIR) were described previously (5). Synthesis and subcloning into
bovine papilloma virus-based expression vector of recombinant cDNAs
encoding wild-type (WT) human IGF-1 receptors (hIGF-1R) were originally
described (37). Construction of the bovine papilloma virus-based
expression vector plasmid construct containing the cDNA that
encodes chimeric human IGF-1 receptors (CHI) in which the entire
C-terminal domain (amino acids 1230-1337) of the -subunit was
replaced by the corresponding tail of the insulin receptor (amino acids
1245-1343) was described previously (38). The cDNA encoding the
Y960F hIR mutant was synthesized by Kaburagi et al. (25),
and resubcloned into a modified pGEM7Z plasmid that contained a
glycerolphosphate kinase 1 promoter and poly(A)+ signals by
Accili et al. (1).
Cell Culture-- NIH 3T3 mouse skin fibroblasts were maintained in Dulbecco's modified Eagle's medium (Biofluids Inc., Rockville, MD) containing 10% fetal calf serum and 2 mM glutamine (Biofluids, Inc). Cells expressing insulin and IGF-1 receptors either alone or in addition to full-length pp120 were maintained in medium supplemented with G418 (600 µg/ml, Life Technologies, Inc.) at 37 °C in 5% CO2. Cells co-expressing Y960F insulin receptors and pp120 were routinely maintained in medium supplemented with G418 (600 µg/ml) and hygromycin B (200 µg/ml).
Transfection--
Stable transfection of NIH 3T3 cells with
cDNAs encoding hIGF-1 receptors (WT and CHI) alone or in addition
to full-length pp120 were described previously (15). Stable
transfection of NIH 3T3 cells with cDNAs encoding WT hIR in
addition to full-length pp120 were also previously described (5).
Stable transfection of NIH 3T3 cells with cDNAs encoding Y960F hIR
was achieved by the LipofectAMINE method (Life Technologies, Inc.) in
the presence of 1.5 µg of the pRSV-Neor
neomycin-resistant gene as we have previously described (34). Stable
transfection of NIH 3T3 cells expressing Y960F hIR with full-length
pp120 was also achieved by the LipofectAMINE method in the presence of
1.5 µg of pREP4-Hygror hygromycin-resistant gene.
Individual clones were picked and expanded, and confluent cells were
lysed in 1% Triton X-100 for analysis on 7.5% SDS-polyacrylamide
(SDS-PAGE) gels and screening for pp120 expression by immunoblotting
with a pp120 polypeptide antibody (-295). As previously indicated,
IGF-1 and insulin binding assays were performed on ~80% confluent
cells grown in 6-well plates to screen for insulin and IGF-1 receptor
expression (15, 35). Clones used in these studies typically expressed
~2.5-3.5 × 105 hIR and 1.0-2.0 × 106 hIGF-1R per cell.
Ligand Binding and Internalization-- As described previously (35), confluent monolayer of cells were maintained in 6-well plates in triplicate and allowed to grow to ~80% confluency. Cells were incubated overnight at 4 °C in binding buffer (100 mM Hepes, pH 7.4, 120 mM NaCl, 1.2 mM MgSO4, 1 mM EDTA, 15 mM CH3COONa, 10 mM glucose, and 1% BSA) containing 20 pM (50,000 cpm/ml) 125I-insulin or 125I-IGF-1 and incubated with prewarmed binding buffer at 37 °C for 0-90 min following removal of unbound ligand with ice-cold PBS, pH 7.4. At the end of each incubation period, cells were washed 3 × with ice-cold PBS, pH 7.4, and incubated in 1 ml of 0.1% BSA-supplemented PBS, pH 3.5, for 10 min. The acid wash was then collected to count acid-sensitive radioactivity that corresponds to noninternalized ligand. Cells were then washed 3 × with ice-cold PBS, pH 7.4, solubilized in 1.0 ml of ice-cold 0.4 N NaOH, 0.1% BSA for 30 min, and collected to count acid-resistant radioactivity that corresponds to internalized ligand. Specifically bound ligand was calculated as the sum of acid-sensitive plus acid-resistant ligand. Internalized insulin was calculated as percent acid-resistant per specifically bound ligand. Experiments were performed in triplicate and repeated three times on at least two different clones of each cell type. Since NIH 3T3 cells predominantly express IGF-1 binding protein-6 and some IGF-1 binding protein-1 (39) which do not associate with the cell surface and/or extracellular matrix of cultured cells, we did not expect IGF-1 binding proteins to interfere with the IGF-1 binding experiments described above.
Statistical Analysis-- Curves were compared by a multivariate analysis of variance, and individual points were compared by paired t tests. p values of less than 0.05 were considered statistically significant.
Biotin Labeling of Surface Membrane Proteins-- Following incubation in the absence or presence of 100 nM insulin for 20 min at 37 °C, cells were incubated for 30 min at 4 °C with biotin (1 mg/ml) in phosphate-buffered saline (PBS), pH 7.4, supplemented with 0.1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA as we have described previously (34). Cells were then incubated at 4 °C for 1 h with buffer alone or with Pronase (2.5 mg/ml). Following lysis in 1% Triton X-100 in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 10 µg/ml of each of the following protease inhibitors: antipain dihydrochloride, pepstatin A, leupeptin, aprotinin, and bacitracin) and immunoprecipitation with pp120/HA4 monoclonal antibody (5), proteins were electrophoresed through 7.5% SDS-PAGE, transferred to nitrocellulose membranes (Schleicher & Schuell), and immunoblotted with horseradish peroxidase (HRP)-labeled streptavidin followed by detection with enhanced chemiluminescence (ECL) as we previously described (34). The difference in the amount of biotin-labeled pp120 before and after insulin treatment was calculated as percent biotin-labeled pp120 in the absence of insulin and used as measure for the amount of pp120 internalized in response to insulin. Experiments were repeated three times for each cell type to allow statistical analysis.
Phosphorylation of pp120 in Intact Cells--
NIH 3T3 cells
co-expressing full-length WT pp120 and insulin receptors (WT and Y960F
mutant) were expanded to confluence in 100-mm plates. Following
overnight incubation in serum-free Dulbecco's modified Eagle's medium
containing 0.1% insulin-free BSA and 25 mM Hepes, pH 7.4, cells were treated with either buffer alone or insulin
(107 M) for 5 min prior to lysis in 1%
Triton X-100 in the presence of phosphatase (EDTA, 4 mM;
NaF, 100 mM; sodium pyrophosphate, 10 mM;
sodium phosphate, 10 mM; ATP, 2 mM; sodium
orthovanadate, 20 mM; N-ethylmalemide, 5 mM; and Hepes, 40 mM, pH 7.6), and protease inhibitors. Cell lysates were subjected to immunoprecipitation with a
polyclonal antibody against IRS-1 (
-IRS-1) or a polyclonal antibody
against the insulin receptor (
-IR). For pp120 immunoprecipitation, glycoproteins in the cell lysates were partially purified on wheat germ
agglutinin affinity chromatography (5) prior to being subjected to
immunoprecipitation with a polyclonal insulin receptor antibody and a
monoclonal pp120/HA4 antibody since these two proteins do not
co-immunoprecipitate.2 Following analysis on 7.5%
SDS-PAGE, proteins were transferred on nitrocellulose membranes and
immunoblotted with HRP-coupled
-Tyr(P) antibody to detect
phosphorylated proteins by the ECL detection system (5). Contrary to
previous experiments (40), the
-Tyr(P) antibody detected pp120 under
these conditions. These experiments were carried out with two
independent clones for each construct derived from the same
transfection.
Quantitation of Proteins-- Autoradiograms were initially scanned on an imaging densitometer (Bio-Rad model GS-670), and the proteins were quantitated on the Image NIH version 1.59 Macintosh software program.
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RESULTS |
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Effect of pp120 on IGF-1 Internalization
(Endocytosis)--
Co-expression of full-length pp120 with insulin
receptors increased insulin endocytosis in NIH 3T3 cells compared with
cells expressing insulin receptors alone (34, 35). To examine whether pp120 similarly regulates IGF-1 internalization, we measured
internalized 125I-IGF-1 in NIH 3T3 cells expressing
comparable amounts of WT IGF-1 receptors per cell with or without
pp120. As Fig. 1A reveals, pp120 expression did not alter the amount of internalized
125I-IGF-1 in cells co-expressing WT hIGF-1 receptors by
comparison to cells expressing WT hIGF-1 receptors alone (Fig.
1A, WT hIGF-1R/pp120 (1) versus WT hIGF-1R).
Since replacement of the C-terminal domain of the -subunit of the
IGF-1 receptor with the corresponding segment of the insulin receptor
restored pp120 phosphorylation by the chimeric receptor, we examined
whether replacement of this domain restored the effect of pp120 on
IGF-1 internalization. When stable clones expressing comparable amounts
of chimeric receptors per cell with or without pp120 were examined, no
significant effect of pp120 on internalized 125I-IGF-1 in
cells co-expressing chimeric receptors was observed relative to cells
expressing chimeric receptors alone (Fig. 1B, CHI
hIGF-1R/pp120 (59) versus CHI hIGF-1R). Failure of pp120 to alter receptor-mediated IGF-1 endocytosis suggests that the effect of
pp120 is specific to the insulin-insulin receptor complex. Moreover,
failure to restore the effect of pp120 on IGF-1 endocytosis by
replacing the C-terminal domain of the
-subunit of the IGF-1 receptor with that of the insulin receptor suggests that this domain
does not regulate the effect of pp120 on receptor-mediated insulin
endocytosis.
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Surface Expression of pp120 in Response to IGF-1--
We have
recently observed that the effect of pp120 on receptor-mediated insulin
endocytosis depends on its ability to be endocytosed along with the
insulin receptor.2 To test whether pp120 participated in
IGF-1 receptor endocytosis, the effect of IGF-1 on surface expression
of pp120 was examined in cells co-expressing IGF-1 receptors (WT and
CHI). To this end, cells were treated with either insulin or IGF-1
prior to biotin labeling followed by Pronase treatment. Cells were
lysed, and the proteins were immunoprecipitated with pp120/HA4
monoclonal antibody, electrophoresed, and immunoblotted with
HRP-labeled streptavidin. The difference in the amount of
biotin-labeled pp120 at the cell surface before and after ligand
treatment was calculated as percent biotin-labeled pp120 in the absence
of ligand and used as measure for the amount of pp120 internalized in
response to ligand (Fig. 2,
graph). Consistent with our previous
findings,2 the amount of
biotin incorporated in the extracellular domain of full-length pp120 in
cells co-expressing WT insulin receptors was substantially decreased by
65.84 ± 2.85% in response to insulin (Fig. 2, WT hIR/pp120 (10),
lane 3 versus 1). In contrast, IGF-1 treatment failed to
decrease the amount of biotin-labeled pp120 in cells co-transfected
with WT IGF-1 receptors (Fig. 2, WT hIGF-1R/pp120 (1), lane 3 versus 1, 6.65 ± 1.69%). IGF-1 treatment did not lead to
a significant decrease in surface expression of pp120 in cells
co-transfected with chimeric IGF-1 receptors, as evidenced by the
modest 15.16 ± 1.88% decrease in biotin-labeled pp120 in these
cells (Fig. 2, CHI hIGF-1R/pp120 (59), lane 3 versus 1). This suggests that in contrast to insulin receptors, pp120 does not
take part in the endocytosis of IGF-1 receptors.
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The Effect of pp120 on Receptor-mediated Insulin Endocytosis Is
Regulated by the Juxtamembrane Domain of the Insulin
Receptor--
Since the C-terminal domain of the -subunit of the
insulin receptor did not directly regulate the effect of pp120 on
receptor-mediated insulin endocytosis (Fig. 1), the hypothesis that the
juxtamembrane domain mediates the stimulatory effect of pp120 on
insulin endocytosis was tested. Phosphorylation on Tyr960
in the juxtamembrane is not required for insulin-induced insulin receptor endocytosis (25). Therefore, mutating Tyr960 in
the insulin receptor was not expected to alter its endocytosis in
response to insulin binding. We investigated whether replacing Tyr960 by a non-phosphorylatable phenylalanine would
interfere with the ability of pp120 to increase receptor endocytosis.
To this end, internalized 125I-insulin was measured in NIH
3T3 cells expressing comparable amounts of Y960F insulin receptors per
cell with or without pp120. Cells expressing WT insulin receptors with
or without pp120 were included as controls. Consistent with our
previous findings (34, 35), full-length pp120 increased internalized
125I-insulin in NIH 3T3 cells co-expressing WT insulin
receptors by comparison to cells expressing WT insulin receptors alone
(Fig. 3A, WT
hIR/pp120(10) versus WT hIR(3006)). In contrast, co-expressing pp120 with Y960F insulin receptors did not increase the amount of
internalized 125I-insulin by comparison to cells expressing
Y960F insulin receptors alone (Fig. 3B, Y960F
hIR/pp120(73-16) versus Y960F hIR(53)). Since the amount of
internalized insulin is only slightly higher in cells expressing Y960F
by comparison to cells expressing wild-type receptors, it is unlikely
that the lack of effect of pp120 on insulin internalization by the
Y960F receptor mutant is attributed to saturation of insulin
internalization in cells expressing this mutant. This suggests that
phosphorylation of the insulin receptor on Tyr960 is
required for pp120 regulation of receptor-mediated insulin endocytosis.
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Internalization of pp120 in Response to Insulin-- We tested whether endocytosis of pp120 depends on phosphorylation of Tyr960 in the juxtamembrane domain of the receptor. To this end, the effect of insulin on surface expression of pp120 was examined in cells co-expressing Y960F insulin receptors and pp120 as described above. Consistent with our previous findings,2 the amount of biotin incorporated in the extracellular domain of full-length pp120 was substantially decreased upon treating cells co-expressing WT insulin receptors with insulin (Fig. 4, WT hIR/pp120(10), lane 3 versus 1). In contrast, insulin treatment failed to decrease the amount of biotin-labeled pp120 in cells co-transfected with Y960F insulin receptors (Fig. 4, Y960F hIR/pp120(73-31), lane 3 versus 1). This suggests that substituting phenylalanine for Tyr960 abolished the ability of insulin to induce a decrease in the surface expression of pp120. Thus, pp120 endocytosis is regulated by phosphorylation on Tyr960 in the juxtamembrane domain of the receptor.
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Phosphorylation of pp120 by Y960F Insulin Receptors in Intact
Cells--
We then investigated whether pp120 phosphorylation depends
on phosphorylation of Tyr960 in the juxtamembrane domain of
the receptor. To this end, cells co-expressing full-length pp120 and
insulin receptors (WT and Y960F) were treated with insulin,
solubilized, and purified on wheat germ agglutinin-agarose affinity
chromatography. The glycoprotein fraction was then subjected to
immunoprecipitation with pp120 and insulin receptor antibodies,
followed by electrophoresis and immunoblotting with an
anti-phosphotyrosine antibody (Fig.
5A). The immunoblot was
reprobed with a pp120 polyclonal antibody to account for the amount of
immunoprecipitated pp120 (Fig. 5B). Corrected for the amount
of insulin receptors and pp120, pp120 phosphorylation by WT insulin
receptors was comparable to that by Y960F insulin receptors. This
suggests that pp120 phosphorylation does not depend on the
phosphorylation of Tyr960 in the juxtamembrane domain of
the insulin receptor. This finding was not surprising since it had been
observed that pp120 phosphorylation was regulated by the
C-terminal domain of the -subunit of the insulin receptor (15).
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DISCUSSION |
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pp120, a substrate of the insulin receptor tyrosine kinase in the hepatocyte, stimulates receptor-mediated insulin endocytosis in transfected cells. Consistent with our previous observations that pp120 failed to regulate receptor-mediated endocytosis of platelet-derived growth factors (34), we have observed in the current studies that the effect of pp120 is specific to the insulin receptor insofar as pp120 failed to regulate IGF-1 receptor endocytosis in response to IGF-1. Since IGF-1 is not cleared in the liver as insulin is, the specific effect of pp120 on insulin endocytosis and degradation proposes a specific physiologic role for pp120 in regulating the cell sensitivity to insulin.
Since IGF-1 receptor does not phosphorylate pp120 in response to IGF-1
(15), failure of pp120 to stimulate receptor-mediated IGF-1 endocytosis
supports our previous observations that the effect of pp120 on insulin
endocytosis requires pp120 phosphorylation by the insulin receptor
tyrosine kinase (34). However, restoration of pp120 phosphorylation by
IGF-1 receptors upon replacing the C-terminal domain of the -subunit
of the receptor with that of the corresponding segment of the insulin
receptor did not confer on pp120 the ability to stimulate endocytosis
of IGF-1 via this chimeric receptor. Thus, pp120 phosphorylation by the
receptor is required but not sufficient to mediate its effect on
hormone endocytosis. This is consistent with our recent report that
pp120 differentially increased insulin endocytosis via the high rather than the low affinity insulin receptor isoform despite being equally phosphorylated by both isoforms (35). Thus, an additional molecular mechanism must underlie the effect of pp120 on insulin endocytosis.
In this report, we have observed that Tyr960 in the
juxtamembrane of the insulin receptor regulated the effect of pp120 on
insulin endocytosis but not its phosphorylation by the insulin
receptor. Since association between pp120 and the insulin receptor
appears to be mediated by other signaling molecules,2 we
propose a model in which insulin receptors phosphorylate pp120 through
the C-terminal domain of the -subunit. The phosphorylated pp120, in
turn, engages one or more molecules that associate with the insulin
receptor via Tyr960 in the juxtamembrane domain of the
insulin receptor. This complex is required for insulin-mediated
receptor endocytosis. The role of pp120 is probably to stabilize this
complex and possibly to mediate its association with structural
components of the clathrin-coated pits, such as adaptor proteins-2,
since it contains tyrosine-centered sequences
(Tyr488-Ser-Val-Leu and Tyr513-Ser-Val-Val)
known to target proteins to adaptor protein-2 (24). Insulin receptor
substrates that bind to this Tyr960 residue include IRS-1,
Shc, GTPase-activating protein-1 (41), and Grb2-associated binding
proteins-1 (42). A role for IRS-1 in receptor-mediated insulin
endocytosis has been ruled out (32), and a role for GTPase-activating
protein-1 and Grb2-associated binding protein-1 in ligand-induced
endocytosis of receptors has not yet been identified. Even though a
role for Shc in insulin-induced endocytosis of insulin receptors is not
yet known, its role in ligand-induced endocytosis of EGF receptors has
been documented (33). More studies are required to identify which of
these molecules, if any, is involved with pp120 in the formation of the
protein complex required for insulin receptor endocytosis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Takashi Kadowaki (University of Tokyo, Japan) and Domenico Accili (NICHD, National Institutes of Health) for providing the cDNA encoding the Y960F insulin receptor mutant. We also thank Dr. Domenico Accili for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by an American Diabetes Association Research award and by Grant 94622 from the National Science Foundation (to S. M. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Medical College of Ohio, 3035 Arlington Ave., HSci Bldg., Rm. 270, Toledo, OH 43614. Tel.: 419-383-4059; Fax: 419-383-2871; E-mail: snajjar{at}opus.mco.edu.
¶ Both authors contributed equally to the project.
1
The abbreviations used are: pp120,
pp120/HA4/C-CAM; IGF-1, human insulin-like growth factor-1; IGF-1R,
human insulin-like growth factor-1 receptor; IR, human insulin
receptor; WT, wild-type receptor; CHI, chimeric IGF-1 receptor in which
the C-terminal domain was replaced by that of the insulin receptor;
EGF, epidermal growth factor; IRS-1, insulin receptor substrate-1; WT
pp120, the full-length isoform of pp120; NIH 3T3, NIH 3T3 mouse skin fibroblasts; and -Tyr(P), anti-phosphotyrosine monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; h, human; PBS,
phosphate-buffered saline; BSA, bovine serum albumin; HRP, horseradish
peroxidase.
2 V. C. Choice, M. J. Howard, M. N. Poy, M. H. Hankin, and S. M. Najjar, submitted for publication.
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REFERENCES |
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