From the Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, December 5, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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Internalization of the insulin receptor (IR) is a
highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of
extracellular matrix (ECM) proteins in the modulation of IR
internalization. Employing Chinese hamster ovary cells that overexpress
IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as
IRS-1, is impaired (e.g. in CHO-T cells overexpressing
IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR
internalization is affected by the context of the ECM proteins to which
the cells adhere. Hence, IR internalization was inhibited 40-60% in
CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin
ligand of the galectin family) when compared with cells adherent onto
fibronectin, collagen, or laminin. Cells adherent to galectin-8
manifested a unique cytoskeletal organization, which involved formation
of cortical actin and generation of F-actin microspikes that contrasted
with the prominent stress-fibers formed when cells adhered to
fibronectin. To better establish a role for actin filament organization
in IR endocytosis, this process was assayed in CHO-T cells (adherent
onto fibronectin), whose actin filaments were disrupted upon treatment
with latrunculin B. Latrunculin B did not affect insulin-induced Tyr
phosphorylation of IR or its ability to phosphorylate its substrates;
still, a 30-50% reduction in the rate of IR internalization was
observed in cells treated with latrunculin B. Treatment of cells with
nocodazole, which disrupts formation of microtubules, did not affect IR
internalization. These results indicate that proper actin, but not
microtubular, organization is a critical requirement for IR
internalization and suggest that integrin-mediated signaling pathways
emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.
Receptor tyrosine kinases (RTKs) rapidly internalize following
ligand binding. Internalized receptors are then sorted to distinct subcellular pathways that lead either to degradation or recycling to
the cell surface (1-3). Similarly, internalization of the insulin
receptor is a multi-step process (4). Following surface redistribution
(5, 6) the receptor-insulin complex progressively concentrates in
clathrin-coated pits that represent the internalization gates
(cf. Ref. 4, for review). The internalized receptor
undergoes sorting, which determines whether it will be subjected to
degradation in lysosomes or whether it will recycle back to the plane
of the membrane (7). Stimulation of the intrinsic Tyr kinase activity of the insulin receptor (IR)1
following insulin binding is a prerequisite for surface redistribution of receptor-insulin complexes; accordingly, mutations of IR that abolish its kinase activity or mutations that replace amino acids that
constitute putative "internalization signal" motifs inhibit IR
internalization (8-13).
The need for IR kinase (IRK) activity to promote receptor
internalization raises the question of whether IR substrates regulate this process. Several proteins were identified as potential IR substrates. Most studied are the Shc proteins, Gab-1, Cbl, and the
family of insulin receptor substrates (IRS-1 to IRS-4) (reviewed in
Refs. 14 and 15). IRS proteins share a highly conserved amino terminus,
which contains a pleckstrin homology (PH) domain that serves to bind
phosphoinositide lipids and localize the IRS proteins to cellular
membranes. A phosphotyrosine-binding (PTB) domain flanks the PH domain
and binds IRS proteins to the NPEY motif within the juxtamembrane (JM)
domain of the insulin receptor (15). The carboxyl terminus of IRS
proteins contains several Tyr residues that, upon phosphorylation, act
as docking sites for the downstream effectors that mediate the
metabolic and growth-promoting functions of insulin (14, 15).
Agents and conditions that modulate cell adhesion also regulate insulin
receptor internalization. For example, carcinoembryonic antigen-related
cell adhesion molecule 1 (CEACAM1) serves as an IR substrate and
up-regulates receptor-mediated insulin endocytosis (16). Still, the
interplay between receptor endocytosis and cell adhesion is poorly
understood. In the present study we undertook to address this question
and investigated the effects of insulin receptor substrates and
extracellular matrix proteins (ECMs) on IR endocytosis. Our results
indicate that, although IR internalization proceeds unaffected even
when Tyr phosphorylation of IRS proteins is impaired, this process
depends upon the context of ECM proteins to which the cells adhere.
Hence, signaling pathways emitted upon cell adhesion to different
matrices and the altered cytoskeletal organizations generated thereof
affect the itinerary of the insulin receptor.
Materials--
Recombinant human insulin was a gift from
Novo-Nordisk (Copenhagen, Denmark). Protein A-Sepharose CL-4B,
poly-L-lysine, fibronectin, laminin,
L-histidinol, puromycin, and TRITC-labeled phalloidin were
purchased from Sigma. Monoclonal PY-20, annexin II, and polyclonal IR
Cell Lines--
CHO cells that overexpress IR (CHO-T cells) and
CHO-T cells that co-express either wild-type mouse IRS-1 or IRS-1 whose
PH domain has been deleted were generated as we described previously (19). To generate CHO-T cells that co-express the Myc-tagged PH-PTB
domain of wild-type IRS-1, the sequence encoding Myc-PH-PTB was
amplified by PCR using the pcDNAIII-Myc-tagged IRS-1 (20) as
a template. The 5' primer (5'-AAGCTTAAGATATCGATCATATG-3') was located
at nucleotide Immunoprecipitation--
Cells were solubilized at 4 °C in
buffer A (25 mM Tris-HCl, 2 mM sodium
orthovanadate, 0.5 mM EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 80 mM
Adhesion of CHO-T Cells to Different ECM
Proteins--
Six-centimeter plates were precoated with fibronectin
(10 µg/ml), galectin-8 (25 µg/ml), laminin (10 µg/ml), or
collagen (10 µg/ml) for 2 h at 25 °C. CHO-T cells, grown on
tissue culture plates, were detached from the plates with 5 mM EDTA and seeded in serum-free medium on the plates
coated with the different ECM proteins. Cells were allowed to adhere
for 6 h at 37 °C and then subjected to further treatments as indicated.
Assays of Insulin Internalization--
Cells were incubated with
125I-insulin (10 Assays of Receptor Internalization--
CHO-T cells were allowed
to adhere to plates coated with different ECM proteins as described
above. Thereafter, the cells were cooled to 4 °C and labeled for 45 min with sulfo-NHS-LC-biotin (0.5 mg/ml) in buffer D (0.1 mM CaCl2, 1 mM MgCl2,
pH 7.4 in PBS). The labeled cells were washed 3× with buffer E (0.1 mM CaCl2, 1 mM MgCl2,
15 mM glycine in PBS, pH 7.4) and then further incubated with the indicated concentrations of insulin (in buffer B) at 15 °C
for 3 h to allow for insulin binding. To initiate receptor internalization, the cells were transferred to 37 °C for 20 min and
back to 4 °C to stop the internalization process. To remove any
remaining cell-surface receptor, the cells were subjected to
trypsinization (30 min at 4 °C) with 1 mg/ml trypsin in buffer F
(serum-free F12 medium, 20 mM Hepes, pH 7.5) followed by
washing with buffer G (serum-free F-12 medium containing 10 mg/ml BSA, pH 7.5). Cells extracts were prepared in buffer C (50 mM
Tris-HCl, 1 mM EDTA, 150 mM NaCl, 10%
glycerol, 1% Nonidet P-40 and protease inhibitor, 1:1000, pH 7.4) and
biotin labeled-proteins were precipitated with immobilized NeutrAvidin
for 2h at 4 °C while shaking. The beads were washed 2× with buffer
C and 2× with PBS and then boiled in 60µl of Laemmli sample buffer.
Samples were resolved by means of 10% SDS-PAGE and immunoblotted with
IR antibody ( Immunofluorescence Analysis--
CHO-T cells were allowed to
adhere for 6 h at 37 °C to cover glasses precoated with the
indicated ECM proteins. Cells were washed and fixed for 30 min with
paraformaldehyde-Triton X-100 (3:0.5%). Following fixation, the cells
were stained with TRITC-phalloidin, and actin organization was assessed
under a fluorescence microscope.
Deletion of the PH Domain of IRS-1 Impairs Its Tyr Phosphorylation
without Affecting IR Internalization--
Internalization of the
insulin receptor is a multi-step process requiring an active insulin
receptor kinase. However, the involvement of IR substrates in this
process remains unclear. To assess the role of IRS-1 in mediating IR
internalization, we made use of IRS-1, whose PH domain has been deleted
(IRS-1 Overexpression of the Isolated PH-PTB Domain of IRS-1 Inhibits
Insulin-induced Tyr Phosphorylation of IRS-1 but Not IR
Internalization--
IRS proteins contain at their N terminus a PH
domain flanked by a PTB domain. To generate dominant negative
inhibitors to IRS-1, a construct encoding the (Myc tagged) PH-PTB
region was introduced into CHO-T cells. As shown in Fig.
2, insulin-induced Tyr phosphorylation of
IRS was decreased by ~45% in cells stably overexpressing the PH-PTB
domains, but IR internalization (assayed by insulin internalization)
was not altered upon expression of this construct. Hence, the results
presented in Figs. 1 and 2 support the notion that insulin receptor
internalization occurs, by and large, independent of IRS-1
phosphorylation, although we cannot rule out the possibility that the
residual Tyr-phosphorylated IRS-1 could affect IR internalization.
Ligand-induced Insulin Receptor Internalization Is Modulated by
Extracellular Matrix Proteins (ECMs)--
Agents and conditions that
modulate cell adhesion were shown to affect insulin receptor signaling.
For example, activation of Insulin Receptor Signaling and Basal Internalization Rates Are
Regulated by ECM Proteins--
To determine whether the reduced
internalization of cells adherent onto galectin-8 reflects impairments
in IRK activity, insulin-stimulated Tyr phosphorylation of IR and IRS
proteins was assayed using CHO-T cells grown on different matrixes. As
shown in Fig. 4 (top), maximal insulin-induced Tyr phosphorylation of IR, assayed at 100 nM insulin, was decreased in cells adherent to galectin-8
when compared with cells adherent to fibronectin laminin or
poly-L-lysine. The decrease in Tyr phosphorylation of IRS-1
was much less severe. To determine whether the reduced
autophosphorylation of IR could account for its impaired
internalization, we established conditions in which insulin-induced Tyr
phosphorylation of IR and IRS-1 were comparable in cells grown on
either matrix. As shown in Fig. 4 (bottom), this could be
achieved when cells grown on fibronectin or galectin-8 were stimulated
for 3 min with 3 and 100 nM insulin, respectively. Pretreatment of cells grown on galectin-8 but not on fibronectin with
vanadate, an inhibitor of protein tyrosine phosphatases, also resulted
in comparable levels of Tyr phosphorylation of IR and IRS-1 (not
shown).
IR internalization was then re-examined under conditions in which
insulin-induced Tyr phosphorylation of IR and IRS were comparable. As
shown Fig. 5, IR internalization, assayed
following its biotinylation, was still severely impaired in cells
adherent to galectin-8 even though the insulin-induced Tyr
phosphorylations of IR and IRS-1 were comparable. Defects in IR
internalization were evident whether the internalization of IR itself
(Fig. 5) or 125I-insulin (Fig.
6) was monitored. These results indicate
that inhibition of IR internalization, which occurs upon cell adhesion to galectin-8, cannot be attributed solely to differences in
insulin-induced Tyr phosphorylation of IR and IRS-1. Cell adhesion to
galectin-8 did not inhibit only the ligand-stimulated internalization
of IR but also impaired its basal internalization rate. As shown in
Fig. 7, the basal (ligand-independent)
rate of IR internalization was inhibited ~30% in cells adherent to
galectin-8, indicating that cell adhesion to galectin-8 also affects
the steady-state surface expression of insulin receptors even in the
absence of a ligand.
Cytoskeletal Organization Affects IR Internalization--
A key
feature that distinguishes cells adherent to galectin-8 is their unique
cytoskeletal organization. Prominent stress fibers that traverse the
cell body are readily detected in cells adherent onto fibronectin but
are less abundant in cells adherent to galectin-8 (Fig.
8). Instead, the formation of cortical
actin (23) and the generation of an F-actin microspike (24)
characterize adhesion to galectin-8. Second, although vinculin and
paxillin are associated with large focal contacts in cells adherent to fibronectin, the number and size of vinculin-and paxillin-containing focal contacts is reduced in cells attached to galectin-8 (23). The
differences in actin-microfilament organization are not accompanied by
differences in microtubules organization, and a similar microtubular network develops when cell adhere to galectin-8 or fibronectin (24).
To establish a role for actin filaments in IR endocytosis,
insulin-induced IR internalization was assayed in CHO-T cells (adherent onto fibronectin) whose actin filaments were disrupted upon treatment with latrunculin B, a drug that sequesters actin monomers. Latrunculin B did not affect insulin-induced Tyr-phosphorylation of IR or the
ability of IRK to phosphorylate its substrates IRS-1 and annexin II
(Fig. 9A). Accordingly,
Latrunculin B did not impair activation of downstream effectors of IR
such as mitogen-activated protein kinase (MAPK) (not shown). Still a
30-50% reduction in the rate of IR internalization was observed in
cells treated with latrunculin B (Fig. 9B). These results
indicate that proper actin organization is a critical requirement for
IR internalization.
Next, we wished to determine whether disruption of microtubule
filaments, which inhibit insulin-induced GLUT-4 translocation (25),
affects IR internalization. CHO-T cells adherent onto fibronectin were
treated with nocodazole, and IR internalization was measured. As shown
in Fig. 9C, there was no prominent change in IR
internalization in cells treated with nocodazole when compared with
control. However, we could demonstrate a ~25% reduction in insulin-induced Tyr phosphorylation of IRS-1 in cells treated with
nocodazole (Fig. 9D). These results indicate that
microtubule integrity is not required for insulin-stimulated IR
internalization despite its importance for insulin-induced GLUT-4 translocation.
The present study provides evidence that the cellular environment
into which the cells adhere directly affects insulin signaling and
insulin responsiveness. In particular, we show that the interaction of
different ECM proteins with cell surface integrins results in a
different cytoskeletal organization that affects the rate of
endocytosis of the insulin receptor. The effects of the ECM proteins on
receptor internalization are independent of their effects on IRK
activity and its ability to phosphorylate downstream effectors such as
the IRS proteins. Hence, insulin signaling and insulin responsiveness
are dually regulated by the adhesive properties of the cells. On one
hand, ligation of ECM proteins by cell surface receptors such as
integrins generates signaling cascades that modulate the activity of
the insulin receptor kinase, whereas, on the other hand, the
alterations in cytoskeletal organization that take place upon cell
adhesion dictate the rate and extent of internalization of the insulin receptor.
Several lines of evidence support this conclusion. First, we could show
that insulin receptor internalization occurs independent of its ability
to phosphorylate the IRS proteins that play a central role in insulin
signaling (15, 26). Impairment of IRS-1 signaling either by the
introduction of IRS-1 whose PH domain has been deleted, making it a
poor substrate for IRK, or overexpression of the isolated PH/PTB domain
of IRS-1, which acts as a partial dominant negative inhibitor of
endogenous IRS-1, did not affect the rate and extent of IR endocytosis.
Although we cannot rule out the possibility that the residual
Tyr-phosphorylated IRS-1 is necessary to promote IR internalization,
our findings strongly suggest that downstream effectors of IRS proteins
such as PI3K have little role in mediating early steps of insulin
receptor endocytosis. These results are consistent with previous
studies, which demonstrated that inhibitors of PI3K do not impair
insulin receptor internalization (27, 28). PI3K has been implicated in
the divergence of PDGF receptors into a degradative pathway without
affecting the initial steps of PDGF receptor endocytosis (29). In that
respect, early events of endocytosis of the insulin and PDGF receptors
appear to proceed by mechanisms that are independent of PI3K.
Although modulators of IRS-1 signaling do not affect IR endocytosis,
the nature of the ECM proteins onto which the cells adhere seems to
have a profound effect on this process. ECM proteins exert a dual
effect on the internalization rate of IR. First, ECM proteins such as
galectin-8, which inhibit IR internalization, fail to support IRK
activity at the same intensity as other ECM proteins, represented by
fibronectin. Because a functional IRK is a prerequisite for proper IR
internalization (8-13), partial inhibition of its activity when cells
adhere to galectin-8 could account for the reduction in IR
internalization. Second, and more important, the differences in
cytoskeletal organization, which take place when cells adhere to
different matrices, seem to play a critical role. Hence, whereas cells
adherent to fibronectin develop an elaborated network of actin bundles
associated with well developed focal contacts, cells attached to
galectin-8 are characterized by the formation of cortical actin,
elaborate F-actin microspikes, and a poorly organized network of actin
microfilaments, which is associated with small focal contacts
distributed mainly at the cell periphery (23, 24). Many of these
adhesion sites contain lower amounts of vinculin or paxillin (23).
The differences in cytoskeletal organization observed when cells adhere
either to galectin-8 or to fibronectin stem from fundamental differences in their structure, function, and mode of interaction with
integrins. Whereas fibronectin is considered to be a conventional ECM
protein that forms protein-protein interactions with integrins, galectin-8 (18, 22-24, 30), a member of the galectin family (31), is a
mammalian lectin that interacts with sugar moieties of integrins (22).
It is a secreted protein, widely expressed, which is made of two
homologous carbohydrate recognition domains (CRDs) linked by a short
(~26 amino acids) peptide. Upon secretion, galectin-8 binds to a
subset of cell surface integrins, which include integrin
The differences in cytoskeletal organization of cells adherent to
galectin-8 or fibronectin involve numerous proteins, each having the
potential to modulate the internalization of the insulin receptor. We
can rule out the effects of cytoskeletal organization on protein Tyr
phosphatases (PTPs), because reduced internalization of IR in cells
adherent to galectin-8 could not be potentiated in the presence of
vanadate, a potent inhibitor of PTP activity. Our results do implicate
actin as one of the key components involved. Actin has been proposed to
play both a positive and negative role in the regulation of various
vesicle trafficking events. In some systems, polymerized actin has been
implicated as a barrier that undergoes depolymerization during vesicle
trafficking. On the other hand, actin is also thought to play a
positive role by forming scaffolds for transport vesicles to move along
during vesicle sorting decisions (33, 34).
Actin has already been implicated as playing a role in insulin action
and as a key component of insulin-induced GLUT4 translocation (35).
Actin disassembly abolishes insulin-induced phosphorylation and
activation of extracellular signal regulated kinase (ERK) and p38
mitogen-activated protein kinase (MAPK) but does not prevent activation
of PI3K and its downstream effectors (36). It has also been shown that
the actin cytoskeleton is required for transferrin endocytosis in A431
cells (37). However, the importance of actin filaments for IR
endocytosis was not studied previously. The present findings indicate
that disruption of actin filaments by latrunculin B impairs IR
endocytosis. Moreover, natural changes in actin organization, which
occur when cells adhere to different matrices, affect IR endocytosis.
It appears that specific polymerized actin structures in the form of
filamentous actin tracks are required to maintain proper IR
internalization. Accordingly, IR internalization is impaired when cells
adhere to matrices such as galectin-8 wherein actin is organized in
cortical elements, exhibiting poorly developed filamentous structures.
Cell adhesion to galectin-8 does not inhibit only the ligand-stimulated
internalization of IR but also impairs its basal internalization rate,
indicating that cell adhesion to galectin-8 might also affect the
steady-state surface expression of insulin receptors even in the
absence of a ligand. Of note, cell adhesion to galectin-8 leads to
remodeling of actin organization rather than actin depolymerization.
This conclusion is supported by the fact that focal adhesion kinase
(FAK) is activated to the same extent when cells adhere to galectin-8
or fibronectin (23), whereas actin disassembly abolishes Tyr
phosphorylation of FAK (35).
The impact of insulin itself on cytoskeletal organization is well
documented. Several studies have shown that insulin induces cell
membrane ruffling, stress fiber breakdown, and microspike formation as
well as dynamical cortical actin remodeling (38-40). Certain aspects
of insulin-induced cytoskeletal remodeling, such as the induction of
membrane ruffling, are impaired by inhibitors of PI3K, indicating that
such elements might not be directly involved in insulin-induced IR
endocytosis. In contrast, other features associated with actin
organization that are insensitive to PI3K inhibitors, such as the
formation of cortical actin, which are induced either upon ligation of
integrins by galectin-8 or upon insulin stimulation, could be related
to the inhibition of IR endocytosis. The organization of microtubules
seems to be less critical for proper IR endocytosis, because
impairment of microtubular organization upon the addition of nocodazole
fails to affect IR endocytosis. In that respect, insulin-induced
endocytosis of the insulin receptor differs from insulin-induced
endocytosis of the glucose transporter GLUT4 that depends upon intact
microtubular network and the presence of microtubule motors (25).
Regardless of the nature of the cytoskeletal elements involved in
modulating IR endocytosis, our results clearly indicate that the
constituents of the extracellular matrix onto which the cells adhere
modulate both IRK activity and the signaling pathways emitted thereof,
as well as the itinerary, surface levels, and cellular content of IR.
This conclusion has somewhat broader implications, because it suggests
that the agents and conditions that modulate the structural components
constituting the extracellular matrix might induce insulin resistance
and diabetes by altering the surface availability of the insulin
receptor. Indeed, carcinoembryonic antigen-related cell adhesion
molecule 1 serves as an IR substrate and up-regulates receptor-mediated
insulin endocytosis and degradation to enhance insulin clearance in
liver (41). Recent studies have also reported the presence of high
serum concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1,
and the mammalian lectin sE-selectin) in patients with type 2 diabetes
(42, 43). Moreover, the levels of sE-selectin correlate positively with
the degree of hyperglycemia (44). The elevated levels of sE-selectin
are of particular relevance, because they suggest that mammalian
lectins such as selectins or galectins might affect insulin action
under physiological or pathological conditions by modulation of IR
endocytosis. Further studies are required to put this intriguing
hypothesis to the test.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit antibodies were obtained from BD Transduction Laboratories. Polyclonal IR antibodies (
subunit) were purchased from Santa Cruz
Biotechnology. LipofectAMINE was obtained from Invitrogen. The
polyclonal IRS-1 antibody was prepared as described (17). Sulfo-NHS-LC-biotin and immobilized NeutrAvidin were purchased from
Pierce. Recombinant galectin-8 was generated as described (18).
56. The 3' primer (5'-TTAGTTGAGTGGGGGGTGCAGCCT-3') was
located at nucleotide 1095 of the mouse IRS-1 gene. The PCR product was ligated into a pGEM-T plasmid (Promega), according to the
manufacturer's instructions. The Myc-tagged PH-PTB construct was
excised from pGEM-T with EcoRV and NotI,
gel-purified, and ligated into pcDNAIII/Amp (Invitrogen) to
generate pcDNAIII-Myc-PH-PTB. CHO-T cells were co-transfected with
pBabe-Puromycin and the pcDNAIII-Myc-PH-PTB plasmid (at a 1:10
ratio) using LipofectAMINE as described previously (20). Following
transfection, cells were incubated in non-selective medium and, 48 h thereafter, puromycin (10 µg/ml) was added for selection of stable
colonies. Puromycin-resistant clones that overexpressed Myc-PH-PTB were
isolated and further propagated.
-glycerophosphate, 25 mM NaCl, 1% Triton X-100 and
protease inhibitor mixture, 1:1000, pH 7.4). Cells were centrifuged at
12,000 × g for 15 min at 4 °C, and the supernatants were collected. Aliquots (0.5-1.0 mg) were incubated for 2 h with polyclonal IRS-1 antibodies coupled to 20 µl of protein A-Sepharose beads. Immunocomplexes were washed twice with buffer A and twice with
PBS. Immunocomplexes were resolved by means of SDS-PAGE and immunoblotted with the indicated antibodies.
10 M) for 16 h at 4 °C in buffer B (serum-free F-12 medium, 50 mM
Hepes, 1 mg/ml bovine serum albumin (radioimmunoassay grade), pH
7.5). The cells were washed 3× with PBS, buffer B was re-added, and
the cells were transferred to 37 °C for the indicated times.
Thereafter, cells were washed 2× with buffer B (titrated to pH 4.0).
Following acid wash, the cells were solubilized in PBS containing 1%
Triton X-100 and 1% SDS, and the amount of intracellular
125I was counted in a gamma counter.
subunit).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PH). We have shown previously that such deletion
impairs the ability of IRS-1 to properly juxtapose to the insulin
receptor, making it a poor substrate for the IRK (19). Indeed, as shown
in Fig. 1A, insulin-induced
Tyr-phosphorylation of IRS-1 was decreased ~60% in CHO cells stably
overexpressing the insulin receptor and IRS-1
PH (CHO-TD)
when compared with CHO cells expressing the insulin receptor and
wild-type IRS-1 (CHO-TS). This occurred despite the fact that the
cellular content of IRS-1
PH was higher than that of
wild-type IRS-1. Tyr phosphorylation of IRS-1
PH was
lower by about 30% even when compared with the phosphorylation of the
endogenous IRS-1 protein in CHO-T cells stably overexpressing just the
insulin receptor. Still, as shown in Fig. 1B, insulin internalization, which represents IR internalization, preceded at
comparable rates in CHO-TS and CHO-TD cells, indicating that insulin
receptor internalization occurs independent of IRS-1
phosphorylation.
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Fig. 1.
Effect of IRS-1, whose PH domain has been
deleted (IRS-1 PH), on
insulin-induced Tyr phosphorylation of IRS-1 and IR internalization.
A, CHO cells overexpressing IR (CHO-T cells), CHO-T cells
overexpressing the wild-type IRS-1 (CHO-TS), and CHO-T cells
overexpressing IRS-1
PH (CHO-TD) were incubated with or
without 100 nM insulin for 1 min at 37 °C. Extracts were
prepared, resolved by means of 10% SDS-PAGE, and immunoblotted
(IB) with anti-Tyr(P) (pTyr) and
anti-IRS-1 antibodies (section I). Aliquots (0.5-1 mg) were
immunoprecipitated (IP) with an IRS-1 antibody coupled to
protein-A beads. The beads were then boiled in 50 µl of Laemmli
sample buffer. Samples were resolved by means of 10% SDS-PAGE and
immunoblotted with anti IRS-1 and anti-Tyr(P) antibodies as indicated
(section II). B, CHO-TS and CHO-TD cells were
incubated with 125I-insulin (10
10
M) for 16 h at 4 °C. 125I-insulin
internalization was then assayed following transfer of the cells to
37 °C as described under "Experimental Procedures." Results are
mean ± S.D. of duplicates measurements.
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Fig. 2.
Effect of overexpression of the PH-PTB domain
of IRS-1 on insulin-induced Tyr phosphorylation of IRS-1 and IR
internalization. A, naïve CHO-T cells or cells
overexpressing a Myc-tagged PH-PTB domain of IRS-1
(CHO-TPH-PTB) were incubated for 16 h in serum-free
F-12 prior to stimulation with or without 100 nM insulin
for the indicated times at 37 °C. Cell extracts were prepared, and
samples (100 µg) were resolved by means of 10% SDS-PAGE and
immunoblotted (IB) with anti-Tyr(P) (pTyr)
(section I) or anti-Myc (section
II) antibodies. B, CHO-T and CHO-TPH-PTB
were incubated with 125I-insulin (10 10
M) for 16 h at 4 °C. 125I-insulin
internalization was then assayed following transfer of the cells to
37 °C as described under "Experimental Procedures." Results are
mean ± S.D. of duplicates measurements.
5
1 integrins
upon adherence of CHO-T cells onto fibronectin markedly potentiates the
Tyr phosphorylation of IR and IRS-1 (21). Conversely, when adherent
cells are maintained in suspension, the extent of insulin-stimulated IR
autophosphorylation is largely diminished (21). To determine whether
ECM proteins could regulate insulin receptor internalization, we made
use of ECM proteins having different characteristics. In general, two
kinds of ECM proteins were utilized, i.e. those that
interact with integrins through protein-protein interactions,
represented by fibronectin, and those that interact with integrins
through protein-sugar interactions, represented by the mammalian lectin
galectin-8, a
-galactoside-binding protein (22, 23). As shown in
Fig. 3, cells adherent onto fibronectin,
laminin, and collagen showed a similar extent of IR internalization.
However, IR internalization was inhibited ~65% in cells adherent
onto galectin-8. These results indicate that extracellular matrix
proteins modulate insulin receptor internalization. They further
suggest that ligation of integrins via protein-protein interactions
better supports IR endocytosis.
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Fig. 3.
Effects of cell adhesion molecules on insulin
receptor internalization. Six-centimeter plates were precoated
with fibronectin (10 µg/ml), galectin-8 (25 µg/ml), laminin (10 µg/ml), or collagen (10 µg/ml) for 2 h at 25 °C.
Thereafter, CHO-T cells in serum-free medium were seeded on the coated
plates and allowed to adhere to the plates for 6 h at 37 °C.
The cells were then transferred to 4 °C and incubated with
125I-insulin (10 10 M) for 16 h at 4 °C. 125I-insulin internalization was assayed at
zero (
) or 5 min (
) following transfer of the cells back to
37 °C as described under "Experimental Procedures." Results are
mean ± S.D. of duplicates measurements.
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Fig. 4.
Effects of cell adhesion molecules on
insulin-stimulated Tyr phosphorylation of IR and IRS-1. A,
six-centimeter plates were precoated with fibronectin (10 µg/ml),
galectin-8 (25 µg/ml), laminin (10 µg/ml), or
poly-L-lysine (100 µg/ml). CHO-T cells, in serum-free
medium, were seeded on the coated plates for 6 h at 37 °C.
After additional incubation at 4 °C for 16 h, cells were
stimulated with 100 nM insulin for 5 min. Cytosolic
extracts were prepared and boiled in Laemmli sample buffer. Samples
(100 µg) were resolved by means of 10% SDS-PAGE and immunoblotted
(IB) with anti Tyr(P) (pTyr) or IR antibodies.
B, six-centimeter plates were precoated with fibronectin or
galectin-8, and CHO-T cells in serum-free medium were seeded on the
precoated plates as described above. After incubation at 37 °C for
10 h, the cells were stimulated with the indicated insulin
concentrations for 3 min. Extracts were prepared, and samples (100 µg) were resolved by means of 10% SDS-PAGE and immunoblotted
(IB) with Tyr(P) (pTyr) antibodies.
View larger version (28K):
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Fig. 5.
Effect of galectin-8 and fibronectin on IR
internalization under conditions where insulin induces comparable level
of Tyr phosphorylation of IR and IRS-1. Six-centimeter plates were
precoated with fibronectin (10 µg/ml) or galectin-8 (25 µg/ml), and
CHO-T cells in serum-free medium were seeded on the precoated
plates. After incubation at 37 °C for 6 h, the cells
were further incubated at 4 °C for 16h and then surface labeled with
sulfo-NHS-LC-biotin and IR internalization was assayed as described
under "Experimental Procedures." Samples were resolved by means of
10% SDS-PAGE and immunoblotted (IB) with IR antibody ( subunit). Results are mean ± S.D. of duplicates measurements.
P, precipitation.
View larger version (14K):
[in a new window]
Fig. 6.
Effect of vanadate on insulin internalization
in cells adherent to galectin-8. Six-centimeter plates were
precoated with fibronectin (10 µg/ml) or galectin-8 (25 µg/ml), and
CHO-T cells in serum-free medium were seeded on the precoated plates.
The cells were incubated with 125I-insulin
(10 10 M) for 16 h at 4 °C in the
absence or presence of 50 µM sodium orthovanadate, as
indicated. 125I-insulin internalization was assayed as
described under "Experimental Procedures." Results are mean ± S.D. of duplicates measurements
View larger version (34K):
[in a new window]
Fig. 7.
Effects of cell adhesion molecules on basal-
and insulin-induced IR internalization. Six-centimeter plates were
precoated with fibronectin (10 µg/ml) or galectin-8 (25 µg/ml), and
CHO-T cells in serum-free medium were seeded on the precoated plates.
After incubation at 37 °C for 6 h, cells were further incubated
at 4 °C for 16 h and then surface-labeled with
sulfo-NHS-LC-biotin. IR internalization was assayed as described under
"Experimental Procedures." Samples were resolved by means of 10%
SDS-PAGE and immunoblotted (IB) with IR antibody ( subunit). Total extracts were immunoblotted with annexin II.
P, precipitation.
View larger version (115K):
[in a new window]
Fig. 8.
Microfilaments organization of CHO-T cells
grown on fibronectin versus galectin-8. CHO-T cells
were seeded on cover glasses precoated with galectin-8 (25 µg/ml) or
fibronectin (10 µg/ml). Following 16 h of incubation at 37 °C
in serum-free medium, the cells were fixed and incubated with
TRITC-labeled phalloidin for actin staining.
View larger version (31K):
[in a new window]
Fig. 9.
Effects of latrunculin B and Nocodazole on
insulin-induced Tyr phosphorylation of IR and IR internalization.
CHO-T cells were seeded on 6-cm plates precoated with 10 µg/ml
fibronectin. A, following 16 h at 37 °C, the cells
were treated for 30 min with 2 µM latrunculin-B and were
further incubated with insulin for the indicated times. Samples were
resolved by means of 10% SDS-PAGE and immunoblotted (IB)
with Tyr(P) (PY) antibodies. B and C,
following 6 h of incubation at 37 °C, cells were incubated with
125I-insulin (10 10 M) for 16 h at 4 °C and buffer; 5 µM latrunculin-B
(B) or 2 µg/ml nocodazole (C) were added for
the last 3 or 4 h, respectively. Cells were transferred back to
37 °C, and 125I-insulin internalization was assayed at
0, 5, and 15 min thereafter as described under "Experimental
Procedures." D, following 6 h incubation at 37 °C,
cells were further incubated for 16 h at 4 °C. 2 µg/ml
nocodazole was added for the last 4 h, and the cells were
stimulated with insulin as indicated at 37 °C. Cytosolic extracts
were prepared, and samples (100 µg) were resolved by means of 10%
SDS-PAGE and immunoblotted (IB) with anti-Tyr(P)
(PY) antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 or
6
1
but not
4
1 (22). Immobilized galectin-8
is equipotent to fibronectin in promoting cell adhesion and spreading,
effects that involve interactions of galectin-8 with sugar-moieties of
integrins (23). Accordingly, cell adhesion to galectin-8 is potentiated
in the presence of Mn2+, whereas adhesion is interrupted in
the presence of soluble galectin-8, integrin
1
inhibitory antibodies, EDTA, or thiodigalactoside, but not by RGD
peptides (23). Hence, formation of protein-protein complexes upon
binding of integrins to fibronectin versus the formation of
protein-sugar complexes between galectin-8 and integrins offers a
molecular aspect for the differences in cytoskeletal organization and
signaling induced by these two matrices. Indeed, the less developed
pattern of actin filaments and focal contacts observed in cells seeded
on galectin-8 resembles the appearance of cells whose integrins were
aggregated in the absence of a ligand (e.g. RGD peptide)
(32), suggesting that galectin-8 presumably fails to occupy the
protein-ligand binding site of integrins, although it effectively
induces aggregation of these receptors (23).
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FOOTNOTES |
---|
* This work was supported by research grants from The Howard M. Siegler Foundation, the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), and the United States-Israel Binational Science Foundation.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.
Present address: Laboratory of Molecular Cell Biology, The
Rockefeller University, 1230 York Ave., New York, NY 10021.
§ Incumbent of the Marte R. Gomez Professorial Chair and to whom correspondence should be addressed. Tel.: 972-8-9342-380; Fax: 972-8-9344-125; E-mail: yehiel.zick@weizmann.ac.il.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M212385200
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ABBREVIATIONS |
---|
The abbreviations used are:
IR, insulin
receptor;
IRK, IR kinase;
IRS, IR substrate;
PH, pleckstrin homology;
PTB, phosphotyrosine binding;
ECM, extracellular matrix protein;
TRITC, tetramethylrhodamine isothiocyanate;
CHO, Chinese hamster ovary;
CHO-T, CHO cells stably overexpressing IR;
CHO-TD, CHO cells stably
overexpressing IR and IRS-1PH;
CHO-TS, CHO cells stably
expressing IR and wild-type IRS-1;
sulfo-NHS-LC-biotin, sulfosuccinimidyl-6-(biotinamido) hexanoate;
PBS, phosphate-buffered saline;
PI3K, phosphatidylinositol 3-kinase;
PTP, protein tyrosine phosphatase.
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