From the Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, and the § Unitat de Bioquímica i Biologia Molecular, Departament de Ciències Fisiològiques I, Facultat de Medicina, Universitat de Barcelona, E-08028 Barcelona, Spain
Received for publication, September 5, 2000, and in revised form, March 12, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neuregulins regulate the expression
of acetylcholine receptor genes and induce development of the
neuromuscular junction in muscle. In studying whether
neuregulins regulate glucose uptake in muscle, we analyzed the effect
of a recombinant neuregulin, rheregulin- Skeletal muscle is the main tissue that contributes to glucose
disposal in absorptive conditions. A limiting step in this process is
glucose transport, which is mediated by different glucose transporters;
GLUT1 is responsible for basal transport and GLUT4 is responsible for
insulin- or exercise-stimulated glucose transport through translocation
to the plasma membrane (1). GLUT1 is highly expressed in fetal muscle,
but during perinatal life it is markedly repressed, whereas GLUT4 is
induced (2). This effect is temporally coincident with the process of
innervation of the muscle fiber (3). Therefore, denervated muscle shows
a decrease in GLUT4 and an increase in GLUT1 expression (3-7). A
program of electrical stimulation of the denervated muscle prevents
GLUT4 gene repression (3), which suggests that basal contractile activity dependent on innervation regulates the expression of GLUT4 in
skeletal muscle. Furthermore, GLUT4 is more sensitive than GLUT1 to the
lack of muscle contraction (5, 8), and the extent of induction of GLUT1
depends mainly on the fiber type (6, 7, 9).
Neuregulins are a family of closely related products encoded by a
single gene, neuregulin-1 (10). In the last few years, other related
genes have been identified (neuregulin-2, -3, and -4). They were
isolated initially from ras-transformed mouse
fibroblasts (neu differentiation factor
(NDF)) (11, 12) and human breast cancer
cells (heregulin) (13). Three other factors were isolated from neural
sources: acetylcholine receptor-inducing activity from chicken
brain (14), glial growth factor from bovine brain (15, 16), and sensory
and motor neuron-derived factor (17). More than 15 distinct isoforms
arise by alternative splicing and cell type-specific transcription
initiation sites (reviewed in Refs. 18 and 19). Two major groups can be
distinguished on the basis of whether they are membrane-associated or
soluble isoforms. The first group of neuregulins contain a
transmembrane domain and a cytosolic tail, reside as membrane proteins,
and are released to the extracellular milieu after proteolytic
cleavage. Most of the released forms contain an N-terminal Ig-like
domain that binds to the glycosaminoglycan portion of proteoglycans in
the extracellular matrix. Common to all isoforms, there is a C-terminal
EGF1-like domain defined by six cysteine residues that fold
the domain into compact, protease-resistant Neuregulins have major effects on the growth and development of
epithelial cells (22), and generation of knockout mice has demonstrated that they are essential for the development of cranial nerve, ganglia, and Schwann cell precursors along peripheral nerves in
the trunk (23). In the hearts of mutant embryos for neuregulin, ErbB2
and ErbB4, ventricular trabeculation does not occur, which results in
developmental arrest and embryo death at embryonic day 10.5 (23-25).
Neuregulins also affect the biology of skeletal muscle; they are potent
activators of the expression of acetylcholine receptors (26-28). In
addition, the neuregulins GGF2 and NRG Here we examined the effects of the neuregulin
rheregulin- Cells, Reagents, and Materials--
The L6E9 rat skeletal muscle
cell line was kindly provided by Dr. B. Nadal-Ginard (Harvard
University, Boston, MA). Recombinant heregulin
(rheregulin- Cell Culture--
L6E9 myoblasts were grown in monolayer culture
in Dulbecco's modified Eagle's medium supplemented with 10% (v/v)
fetal bovine serum, 1% (v/v) antibiotics (10,000 units/ml of
penicillin G and 10 mg/ml streptomycin), 2 mM glutamine,
and 25 mM Hepes (pH 7.4). Pre-confluent myoblast (80-90%)
were induced to differentiate by lowering FBS to final concentration of
2% (v/v).
To analyze the acute effect of HRG on glucose transport and
transporters, fully differentiated myotubes were depleted of serum containing 0.2% bovine serum albumin for 4.5 h. 90 min before the
end of this period, HRG was added (except in the time course experiments), and when insulin action was studied, it was added for the
last 30 min of this period. For the chronic HRG treatment, HRG
was added 24 h after the cells were changed to the low serum medium, and studies were carried out after 1, 2, or 3 days of HRG treatment.
2-Deoxy-D-[3H]Glucose
Uptake--
Cells were cultured on 6-well plates. Transport (34) was
initiated by washing the cells twice in a transport solution (20 mM Hepes, 137 mM NaCl, 4.7 mM KCl,
1.2 mM MgSO4, 1.2 mM
KH2PO4, 2.5 mM CaCl2, 2 mM pyruvate, pH 7.4). Cells were then incubated for 10 min
with a transport solution that contained 0.1 mM
2-deoxy-D-glucose and 1 µCi of
2-deoxy-D-[3H]glucose uptake (10 mCi/mmol).
To determine background labeling, we incubated the cells for 10 min
with ice-cold 50 mM glucose in phosphate-buffered saline
buffer (PBS) containing the same specific activity of
2-deoxy-D-[3H]glucose. Uptake was stopped by
the addition of 2 volumes of ice-cold 50 mM glucose in PBS.
Cells were washed twice in the same solution and disrupted with 0.1 M NaOH, 0.1% SDS. Radioactivity was determined by
scintillation counting. Protein was determined by the Bradford method.
Each condition was run in duplicate or triplicate. Glucose transport
was linear during the period assayed (data not shown).
Preparation of Homogenates and Membrane Fractions from L6E9
Myocytes--
Homogenates were obtained from cells cultured on 6-well
plates at 2, 3, or 4 days of differentiation with one plate from each group. Cells were placed on ice, washed twice in ice-cold PBS, and scraped into 2 ml of PBS. Cells were pelleted at 3,000 rpm for 5 min and resuspended in 300 µl of lysis buffer (20 mM
Hepes, 350 mM NaCl, 20% (v/v) glycerol, 1% (v/v) Nonidet
P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, pH 7.9) containing freshly added protease inhibitors (1 mM dithiothreitol, 0.1% (v/v)
phenylmethylsulfonyl fluoride, 0.1% (v/v) aprotinin). The homogenate
was shaken for 30 min at 4 °C and centrifuged at 1,000 rpm for 20 min, and the supernatant (SN) was collected and kept at Fusion Index--
Cells were cultured in 10-cm dishes. After
treatments, cells were washed twice in cold PBS, and 1 mM ZnSO4 in 20% dimethyl sulfoxide was added
at room temperature and incubated for 1 min. Cells were then washed
gently in cold PBS and fixed with 2.5% glutaraldehyde in PBS for 2 min. Cells were then treated for 1 min with 50% ethanol and
rinsed with PBS before staining with filtered 0.04% Giemsa in
PBS, pH 6.8, overnight. Finally, cells were rinsed with tap water.
Under an optical microscope, several randomly chosen fields were
photographed, and the nuclei per cell were counted.
Animals and Tissue Sampling--
For studies of incubated soleus
muscle, male Wistar rats (250 g) were anesthetized with pentobarbital,
5-7 mg/100 g of body weight, and strips were isolated by a
modification of the method of Crettaz et al. (36).
For denervation studies, the peroneal nerve of anesthetized male rats
(ketamine, 20 mg/kg of body weight) was severed unilaterally. Three
days after denervation, tibialis anterior and extensor digitorum longus muscles were dissected and frozen in liquid nitrogen. All procedures were reviewed and approved by the local ethics committee. Muscles were processed to obtain total membranes as reported previously (37).
Glucose Transport by Strips of Soleus
Muscles--
Isolated strips of soleus muscles were incubated as
reported (38). HRG (3 nM, 120 min) was added after 30 min
of muscle incubation, and 1 h later, insulin (100 nM,
60 min) was added to the medium. Previous to the uptake period, muscles
were washed for 10 min with a glucose-depleted medium. Thereafter,
muscles were incubated in the presence of
2-deoxy-D-[14C]glucose uptake (1 mCi/mmol)
and D-[3H]mannitol (0.5 mCi/mmol), as an
extracellular space marker, for 20 min, the time in which linear
conditions are maintained. Muscles were then frozen and processed as
reported (38).
Electrophoresis and Immunoblotting of
Membranes--
SDS-polyacrylamide gel electrophoresis was performed on
membrane protein. Proteins were transferred to Immobilon in buffer consisting of 20% methanol, 200 mM glycine, 25 mM Tris, pH 8.3. After transfer, the filters were blocked
with 5% nonfat dry milk in Tris-buffered saline solution for 1 h
at room temperature and then incubated overnight at 4 °C with
antibodies directed against GLUT4 (1:800), GLUT1 (1: 1300),
GLUT3 (1:500), ErbB3 (1:500), ErbB2 (1:500), myosin heavy chain (1:20),
Reverse Transcriptase-PCR from Total RNA--
cDNA was
synthesized from 2 µg of total RNA at 20 µl of final reaction
volume the using SuperScript II Rnase H Expression of ErbB2 and ErbB3 Receptors in L6E9 Muscle
Cells--
As a first step, we assayed the level of neuregulin
receptors ErbB2 and ErbB3 in total membrane extracts obtained from L6E9 myoblasts and cells after 2 and 3 days of differentiation induced by
exposure to low serum conditions (Fig.
1). The ErbB2 content was maximal in
myoblasts, and expression was somewhat reduced during differentiation.
In contrast, ErbB3 levels were low in myoblasts but rose during
differentiation (a 2-fold increase on day 3 of differentiation) (Fig.
1). Under these conditions, the abundance of the Heregulin Acutely Stimulates Glucose Transport and Glucose
Transporter Translocation in Muscle--
Fully differentiated L6E9
myotubes were incubated with different concentrations of HRG for 90 min
(Fig. 2A). HRG stimulated glucose transport with half-maximal effects detected at 2 nM HRG and maximal effects (2-2.5-fold stimulation of
glucose transport) observed between 3 and 5 nM HRG, which
is consistent with other reports on the effects of neuregulins (26, 30,
39). Time course studies were also performed at maximal concentrations
of HRG (3 nM) (Fig. 2B). The maximal effect of
HRG on glucose transport was reached between 60 and 90 min of
incubation.
The effect of a maximal concentration of HRG (3 nM for 90 min) on glucose transport was comparable with the effect caused by a supramaximal concentration of insulin (1 µM for 30 min) (Fig. 2C). Furthermore, the combination of HRG and
insulin caused an additive stimulation of glucose transport (Fig.
2C).
To determine whether the effect of HRG on glucose transport required
phosphatidylinositol 3-kinase activity, wortmannin (1 µM)
(40) was added 30 min prior to HRG. Insulin-stimulated glucose transport was inhibited by wortmannin, and under these conditions wortmannin also blocked HRG-induced glucose transport (Fig.
3).
Incubation of L6E9 myotubes for 90 min in the presence of HRG did not
alter the cell content of GLUT4, GLUT1, or GLUT3 (data not shown). In
the next step, L6E9 myotubes were incubated in the absence or presence
of 3 nM HRG for 90 min and then were further subjected to
subcellular fractionation of membranes. This yielded two membrane
fractions: PM, which were highly enriched in plasma membrane
markers such as the
To determine whether HRG affects glucose transport in rat skeletal
muscle, strips of soleus muscles were incubated in the absence or
presence of HRG and/or insulin. HRG induced a 73% increase on glucose
uptake. HRG and insulin showed additive effects on glucose transport
(Fig. 5).
Chronic Incubation with Heregulin Stimulates Glucose Transport and
Regulates Transporter Expression in Muscle Cells--
Next, we studied
the effect of chronic incubation with HRG on glucose transport in L6E9
cells. To this end, preconfluent cells were induced to differentiate by
decreasing the serum content (2% FBS), and after 1 day, 3 nM HRG was added for 24, 48, or 72 h. Cells treated
with HRG showed enhanced myotube formation (Fig. 6A), which was detected after
24 h of HRG addition (fusion index values were enhanced 34, 32, and 18% at 24, 48, or 72 h, respectively, after heregulin
addition) (Fig. 6B). An enhanced rate of cell fusion induced
by neuregulins is in agreement with previous reports in other muscle
cell lines (29, 30). Chronic HRG caused an enhanced expression of
myosin heavy chain (MHC, Fig. 6C), a late myogenic protein. GLUT4 is also induced during late myogenesis, showing high levels of expression after 3 days of differentiation (Fig.
7A). However, long-term
incubation with HRG reduced GLUT4 expression 60% after 72 h (Fig. 7). In contrast, HRG increased the expression of GLUT1 and
GLUT3 with maximal effects at 48 h of treatment (60 and 36%
increases, respectively) (Fig. 7A).
The effect of HRG on GLUT4 expression in L6E9 was specific, and the
abundance of other components of GLUT4 vesicles such as IRAP (41),
SCAMPs (42), VAMP2 (43), or cellubrevin (44) remained unaltered by HRG
(Fig. 7B).
Exposure to HRG for 48 h stimulated glucose transport in L6E9
myotubes to the same extent as insulin, and when both factors where
tested together, the effect was additive (Fig.
8A). Western blot analysis of
plasma membranes showed an increase in GLUT1 and GLUT3 and a decrease
in GLUT4 from HRG-treated cells (Fig. 8B).
Expression of ErbB2 and ErbB3 Receptors in Denervated
Muscle--
The induction of GLUT1 and repression of GLUT4 in L6E9
muscle cells resemble the effects observed upon denervation of the muscle fiber (3), and therefore we examined the expression of
the neuregulin receptors ErbB2 and ErbB3 in denervated muscles. There
was a 2-3-fold increase in the abundance of both ErbB2 and ErbB3 in
total membrane extracts from denervated muscle (Fig. 9A). We also determined
neuregulin expression in control and denervated muscle. To this end,
specific primers were generated to detect all possible neuregulin
isoforms expressed by muscle. Neuregulin mRNAs were amplified by
reverse transcriptase-PCR in a way that ensured nonsaturating
concentrations. Muscle expressed low levels of neuregulins in
either innervated or denervated muscle, and denervation caused
no alteration in the expression level (Fig. 9B), which is in
agreement with a previous report (33).
Neuregulins play a central role in muscle biology. They are
synthesized by myoblast cells and initiate an autocrine signaling pathway that promotes myogenic differentiation (30). In addition, neuregulins regulate the expression of acetylcholine receptors and
utrophin in muscle (26-28, 45, 46). In the muscle, the neuregulin
receptors ErbB2 and ErbB3 are found only in the neuromuscular junction,
and it is thought that neuregulins maintain the protein composition,
and therefore the functional properties, of the neuromuscular junction.
In this study we provide evidence for a metabolic role of neuregulins
in muscle, i.e. neuregulins stimulate glucose transport in
muscle cells by various mechanisms. On the one hand, they promote rapid
translocation of glucose transporters from an intracellular site to the
plasma membrane. On the other hand, neuregulins cause up-regulation of
GLUT1 and GLUT3 glucose transporters, which is concomitant with an
enhanced abundance at the plasma membrane in conditions in which GLUT4
is markedly repressed. These results suggest that in the mature muscle
fiber, neuregulins regulate glucose uptake in or near the neuromuscular
junction through changes in glucose transporter distribution or in
glucose transporter expression.
We have shown in this study that heregulin acutely stimulates glucose
transport in muscle cells and tissue. To our knowledge, this is the
first report of a rapid effect of neuregulins that is independent of
changes in gene expression. The rapid effect of HRG on glucose
transport was explained by the translocation of GLUT4, GLUT1, and GLUT3
glucose transporters, and it was independent of changes in glucose
transporter expression. The translocation of the glucose transporter in
response to HRG was comparable with the effect of insulin. Furthermore,
the stimulation of glucose transport by insulin and HRG was additive,
which suggests the activation of different or complementary mechanisms.
It has been reported that ErbB3 neuregulin receptor activates
phosphatidylinositol 3-kinase activity and that neuregulins activate
Akt/protein kinase B (47-49). In this regard, we found that
the effect of HRG on glucose transport was completely blocked by
wortmannin, suggesting that it requires an intact phosphatidylinositol
3-kinase activity.
During myogenic differentiation, basal glucose uptake decreases as a
consequence of the down-regulation of GLUT1 and GLUT3. Here, we have
shown that heregulin blocks the down-regulation of both GLUT1 and GLUT3
associated with muscle cell differentiation and increases their
abundance at the plasma membrane, which explains the stimulation of
glucose transport in muscle cells subjected to chronic treatment with
heregulin. For GLUT1 expression, we know that gene transcription is a
crucial regulatory step in muscle cells (50); therefore, it is likely
that heregulin changes GLUT1 gene transcription. In connection with the
factors that regulate GLUT1 gene transcription, we have previously
established that Sp1 transactivates GLUT1 gene transcription (50),
whereas Sp3 represses the transcriptional activity of the GLUT1
promoter in L6E9 cells (50, 51). Furthermore, myoblasts have high
levels of Sp1 and Sp3, and during onset of myogenesis there is a
decrease in the Sp1 content, so that the Sp1/Sp3 ratio falls, which is concomitant to GLUT1 repression (51). In this connection it has been
reported that neuregulins phosphorylate Sp1, which is involved
in the activation of the expression of acetylcholine receptor
HRG potentiates both myoblast proliferation (an increase of 44% in
L6E9 cells; data not shown) and myotube formation, the latter being in
keeping with previous observations (30). In this regard, the effect of
heregulin is similar to the effects of insulin-like growth
factors, activating both the proliferation and differentiation
of muscle cells (53). The finding that HRG represses expression of
GLUT4 at the time that myotube formation is induced indicates
that both effects are not dependent on each other. In addition, this
effect is specific for GLUT4 and HRG does not compromise the expression
of other proteins such as IRAP, VAMP2, SCAMPs, or cellubrevin that
colocalize with GLUT4 in intracellular compartments of the muscle.
Muscle denervation causes GLUT1 induction and GLUT4 repression
(3-7), but the mechanisms are largely unknown. Thus, denervation increases muscle cAMP (54), and chronic incubation with permeable cAMP
analogues down-regulates GLUT4 and up-regulates GLUT1 in L6E9 myotubes
(55), so cAMP may participate in the effects of denervation.
Furthermore, muscle denervation enhances Sp1 and Sp3 binding activity
(51), which indicates that denervation-mediated enhancement in GLUT1
gene transcription may be also explained by activation of the Sp1 site
of the proximal GLUT1 promoter. Muscle denervation represses GLUT4 gene
transcription, which requires a DNA fragment encompassing 730 base
pairs from the transcription initiation site (56); however, the
regulatory sites and the transcription factors involved are unknown. We
show that muscle denervation in the rat up-regulates the neuregulin
receptors ErbB2 and ErbB3, which is concomitant with unaltered levels
of neuregulin mRNA. This suggests that the effects of muscle
denervation on glucose transporter expression are mediated by enhanced
neuregulin action in the muscle fiber.
Another important aspect refers to the cellular distribution of
neuregulin receptors in the muscle fiber. In innervated muscle, neuregulin receptors are limited to the neuromuscular junction (31,
32); therefore, the biological effects of neuregulin may be restricted
to this domain of the muscle fiber. Immunocytochemical evidence
indicates that after muscle denervation, ErbB2 abundance diminishes,
whereas ErbB3 does not change at the neuromuscular junction (31). As a
whole, this suggests that newly synthesized ErbB2 and ErbB3, in the
muscle fiber after denervation, are not concentrated at the
neuromuscular junction but are spread over the membrane surface. Thus,
neuregulins may alter muscle physiology in extrajunctional areas after
muscle denervation and may have an impact on glucose uptake in the
muscle fiber.
In summary, HRG regulates glucose uptake in muscle cells and in rat
muscle by mechanisms that involve, at least in L6E9 cells, either the
rapid redistribution of glucose transporters or the regulation of
glucose transporter expression. Neuregulins may regulate glucose
disposal in or near the neuromuscular junction in the innervated muscle
fiber. Based on the fact that HRG up-regulates GLUT1 and down-regulates
GLUT4 in muscle cells and that the abundance of neuregulin receptors
increases after denervation, we also postulate that neuregulins may
participate in the adaptations in glucose uptake that take place in the
muscle fiber after denervation.
1-(177-244) (HRG), on L6E9 muscle
cells, which express the neuregulin receptors ErbB2 and ErbB3. L6E9
responded acutely to HRG by a time- and
concentration-dependent stimulation of 2-deoxyglucose uptake. HRG-induced stimulation of glucose transport was additive to
the effect of insulin. The acute stimulation of the glucose transport
induced by HRG was a consequence of the translocation of GLUT4, GLUT1,
and GLUT3 glucose carriers to the cell surface. The effect of HRG on
glucose transport was dependent on phosphatidylinositol 3-kinase
activity. HRG also stimulated glucose transport in the incubated soleus
muscle and was additive to the effect of insulin. Chronic exposure of
L6E9 cells to HRG potentiated myogenic differentiation, and under these
conditions, glucose transport was also stimulated. The activation of
glucose transport after chronic HRG exposure was due to enhanced cell
content of GLUT1 and GLUT3 and to increased abundance of these carriers
at the plasma membrane. However, under these conditions, GLUT4
expression was markedly down-regulated. Muscle denervation is
associated with GLUT1 induction and GLUT4 repression. In this
connection, muscle denervation caused a marked increase in the content
of ErbB2 and ErbB3 receptors, which occurred in the absence of
alterations in neuregulin mRNA levels. This fact suggests
that neuregulins regulate glucose transporter expression in denervated
muscle. We conclude that neuregulins regulate glucose uptake in L6E9
muscle cells by mechanisms involving the recruitment of glucose
transporters to the cell surface and modulation of their expression.
Neuregulins may also participate in the adaptations in glucose
transport that take place in the muscle fiber after denervation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets by forming three
disulfide bonds. The EGF-like domain is sufficient to elicit biological responses. After proteolysis, the EGF-like domain is released and binds
to members of the ErbB family of tyrosine kinase receptors, ErbB3
(HER3) and ErbB4 (HER4). ErbB4 shows ligand-stimulated tyrosine kinase
activity. ErbB3, however, is a tyrosine kinase-deficient receptor
because an aspartate and a glutamate in the kinase domain that are
critical for autophosphorylation are replaced by other residues.
Neuregulin binding to ErbB3 signals through heterodimerization with
ErbB2 (HER2, c-neu), which displays tyrosine kinase activity (20, 21).
1 activate myogenic
differentiation (29, 30). There is also evidence for the operation of a
neuregulin-ErbB3 autocrine signaling pathway during an early stage of
myoblast differentiation (30). In the mature muscle fiber, ErbB2 and
ErbB3 are concentrated at the neuromuscular junction, and therefore it
is thought that neuregulins regulate the protein composition and the
functioning of the neuromuscular junction (26, 27, 31-33).
1-(177-244) on glucose uptake in L6E9 muscle
cells and the mechanism involved. Our results indicate that HRG
stimulates glucose transport and translocates glucose transporters to
the cell surface in muscle cells. Additionally, chronic exposure to HRG
also stimulates glucose transport and causes alteration of the
expression pattern of glucose transporters in muscle cells. Our data
are compatible with a model in which neuregulins regulate glucose
disposal in or near the neuromuscular junction in innervated muscle
fiber. In addition, neuregulin may also participate in the adaptations
in glucose uptake that take place in the muscle fiber after denervation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-(177-244) (HRG)) was obtained from
Genentech, Inc. (South San Francisco, CA). Dulbecco's modified
Eagle's medium, fetal bovine serum (FBS), glutamine, and antibiotics
were purchased from BioWhittaker (Walkersville, MD). Most commonly used
chemicals and wortmannin were from Sigma. Pepstatin, leupeptin,
and aprotinin were from ICN (Costa Mesa, CA). Immobilon polyvinylidene
difluoride was obtained from Millipore Corp. (Bedford, MA). Purified
porcine insulin was a kind gift from Lilly Co. ECL reagents were from Amersham Pharmacia Biotech UK (Little Chalfont, Buckinghamshire, UK).
2-Deoxy-D-[3H]glucose was obtained from
American Radiolabeled Chemicals, Inc. (St. Louis, MO).
2-Deoxy-D-[14C]glucose,
D-[3H]mannitol, and the tissue solubilizer
Protosol were obtained from PerkinElmer Life Sciences. All
chemicals were of the highest purity grade available. Bradford reagent
and all electrophoresis agents and molecular weight markers were
obtained from Bio-Rad (Hercules, CA). Polyclonal antibody OSCRX (raised
against the 15 C-terminal amino acid residues from GLUT4) was
produced in our laboratory by Dr. Conxi Mora. Anti-GLUT1 antibody
was purchased from Diagnostic International (Karlsdorf, Germany).
Anti-rat/mouse GLUT3 antibody was kindly provided by Dr. Gwyn W. Gould
(University of Glasgow, UK). Anti-ErbB3 (C-17) and ErbB2 (Neu) (C-18)
antibodies were purchased from Santa Cruz Biotechnologies, Inc. (Santa
Cruz, CA). Monoclonal antibodies against myosin heavy chain (MF20) and against
1-Na+/K+-ATPase (
6F)
were purchased from Developmental Studies Hybridoma Bank (University of
Iowa, Iowa City, IA). Anti-IRAP and anti-SCAMPs antibodies (3F8) were
kindly provided by Dr. Paul Pilch (Boston University, Boston,
MA). Anti-VAMP2 and anti-cellubrevin antibodies were kindly provided by
Dr. Joan Blasi (Universitat de Barcelona). Tissue culture material was
purchased from Corning Inc.
20 °C. For
total membrane preparation (35), 3-4 10-cm dishes were used for each
experimental group. Cells were washed and scraped into 10 ml of PBS as
described previously and pelleted at 1,000 rpm for 5 min. They were
then resuspended in 3 ml/dish of cold homogenization buffer (250 mM sucrose, 2 mM EGTA, 5 mM sodium
azide, 20 mM Hepes, pH 7.4) containing freshly added
protease inhibitors (200 µM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM pepstatin)
and homogenized in a glass Dounce homogenizer (pestle A, 20 strokes).
Homogenate was centrifuged at 2000 rpm for 5 min, and the supernatant
(SN1) was kept. The pellet was resuspended in 1.5 ml of
homogenization buffer/dish, re-homogenized, and centrifuged in the same
way. The new pellet was discarded, and the supernatant
(SN2) was pooled with SN1 and centrifuged at
190,000 × g for 1 h 10 min. The pellet (total
membranes) was collected and resuspended in 250 µl of 20 mM Hepes, pH 7.4. When membrane fractionation was required
(35), cells were cultured on 2-4 15-cm dishes for each experimental
group and treated initially as described above. The pooled
SN1 and SN2 were centrifuged at 24,000 × g for 1 h. The pellet, a partially purified plasma
membrane (PM) fraction, was resuspended in 500 µl of 20 mM Hepes, pH 7.4, and the SN was further centrifuged at
190,000 × g for 1 h. The new pellet, a low
density fraction (LDM) of intracellular origin, was resuspended in 200 µl of Hepes solution.
1-subunit of the Na+/K+ ATPase
(1:100), IRAP (1/1000), SCAMPs (1/3000), VAMP2 (1/1000), and
cellubrevin (1/500), all diluted in 1% (w/v) bovine serum albumin,
0.067% (w/v) sodium azide in Tris-buffered saline, 0.09% (v/v) Tween
20. The immune complex was detected using an ECL
chemiluminescence system. The autoradiograms were quantified using
scanning densitometry. Immunoblots were performed in conditions in
which autoradiographic detection was in the linear response range.
Reverse
Transcriptase System (Life Technologies, Inc.). (dT)15 was
used as the primer at 0.4 µM. Genomic contamination was
monitored by enzyme-free controls. The resulting cDNA was diluted
1/10, and 1 µl of this dilution was amplified by PCR in an MJ
Research PT-100 thermocycler at 25 µl final reaction volume. PCR
primers 5'-TCAGAGCTTCGAATTAACAAAGC-'3 and
5'-GTGGTCATGGCTGATAGATAC CT-'3 (Life Technologies, Inc.), corresponding
to 627-649 and 1608-1630 base pairs, respectively, of rat NDF
(sequence with total identity to all neuregulin isoforms) were
added at 0.4 µM and dNTPs at 0.2 mM. 1.25 units of Taq Expand High Fidelity and its corresponding buffer with Mg2+ were used (Roche Molecular Biochemicals).
PCR was performed as follows: an initial step of 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 52 °C, and 2 min at
72 °C; and a final step of 5 min at 72 °C. Nonsaturating
conditions were ensured by previous assays with the same cDNAs
samples subjected to different number of PCR cycles (20 to 35) and in
which the maintenance of linearity was determined (data not shown). For
electrophoretic analysis, 5 µl of the final reaction volume was
loaded in 1.5% agarose gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
subunit of the Na+/K+-ATPase remained
unaltered, indicating that the pattern of changes detected for
neuregulin receptors was specific. Our results indicate the presence of
neuregulin receptors in L6E9 muscle cells, which is consistent with
reports in different muscle cell lines (25, 32).
View larger version (20K):
[in a new window]
Fig. 1.
ErbB2 and ErbB3 expression in L6E9
cells. Total membranes from L6E9 cells at different stages of
differentiation were obtained and processed for Western blot assay. 25 µg of protein was loaded to each lane. The abundance of
ErbB2 and ErbB3 was determined by using specific polyclonal antibodies.
The specificity of the recognition in Western blot was assayed by
incubation of the antibodies in the presence or absence of antigenic
peptides (data not shown). Cells at day 0, pre-confluent myoblasts
(Mb) (80-90% of confluence); day 2, young myotubes
(Mt); day 3, mature myotubes. Autoradiograms representative
of three different experiments are shown.
View larger version (17K):
[in a new window]
Fig. 2.
Acute effects of HRG on 2-deoxyglucose uptake
in L6E9 myotubes. Cells were seeded on 6-well plates for
2-deoxyglucose uptake studies. Fully differentiated L6E9 myotubes were
serum-depleted for 4.5 h prior to 2-deoxyglucose uptake assays.
A, cells were treated during the last 90 min with different
HRG concentrations ranging from 0 to 10 nM. B,
cells were assayed at 3 nM HRG during different times
ranging from 15 to 120 min. 2-Deoxyglucose uptake values are expressed
per µg of protein after subtraction of background values. Results are
the mean ± S.E. of 3-12 observations per group and are
expressed as relative values (treated above nontreated cells
transport). Basal transport rates were (dose-response studies)
1.55 ± 0.21 and (time course studies) 1.13 ± 0.11 pmol of
2-deoxyglucose/µg protein × 10 min. C, cells were
treated with 3 nM HRG (H; during the last 90 min
of incubation), with 1 µM insulin (I; during
the last 30 min of incubation), or with both insulin and HRG
(I + H). 2-Deoxyglucose uptake values were
expressed per µg of protein after subtraction of background values.
Results are the mean ± S.E. of 3-4 observations/group. *,
indicates significant differences with basal group at p < 0.01; , indicates significant differences with the insulin- or
HRG-treated groups at p < 0.01 (Student's
t test). Basal transport rate was 0.97 ± 0.10 pmol of
2-deoxyglucose/µg protein × 10 min. B, basal.
View larger version (17K):
[in a new window]
Fig. 3.
Effects of wortmannin on
heregulin-stimulated glucose uptake in L6E9
myotubes. Differentiated myotubes were serum-depleted for 4.5 h before to the assay of 2-deoxyglucose uptake. Cells were
treated (filled bars) or not (open bars) with
wortmannin (1 µM) for 120 min in basal conditions
(B), in HRG-treated cells (H, 3 nM,
90 min), or in insulin-treated cells (I, 1 µM,
30 min). Results are the mean ± S.E. of 7-11 different
experiments. *, indicates significant differences with basal group at
p < 0.01. prot, protein.
1 subunit of the
Na+/K+-ATPase, and LDM, which are of
intracellular origin. A typical experiment starting with four 15-cm
dishes yielded ~3-5 mg of membrane proteins in the PM fraction and
0.5-1.5 mg of membrane proteins in the LDM fraction. Incubation of
cells for 90 min in the presence of HRG caused a 43% increase in the
abundance of GLUT4 in PM fractions and a 50% decrease in GLUT4 in LDMs
(Fig. 4, A and B),
consistent with HRG-induced GLUT4 translocation. HRG also caused a 45 and a 40% increase in the abundance of GLUT1 and GLUT3 proteins,
respectively, in PM, which suggests that HRG also redistributed GLUT1
and GLUT3 to the cell surface (Fig. 4, A and B).
The effects of HRG were specific, and the abundance of the
1 subunit of Na+/K+-ATPase was
unaltered after incubation with HRG (Fig. 4A). HRG and
insulin (1 µM for 30 min) had a similar effect on all
glucose transporters (Fig. 4, B and C). The
combination of HRG and insulin (Fig. 4, D and E)
caused an additional stimulation of GLUT4, GLUT1, and GLUT3
translocation in muscle cells (87, 86, and 57% increase at PM,
respectively). In all, our data indicate that HRG causes translocation
of glucose transporters to the cell surface and that HRG is at least as
potent as insulin in activating glucose transport and glucose
transporters translocation in L6E9 myotubes.
View larger version (37K):
[in a new window]
Fig. 4.
Effect of heregulin and insulin on
redistribution of glucose transporters in L6E9 myotubes. L6E9
myotubes were serum-depleted for 4.5 h and thereafter treated or
not during the last 90 min with 3 nM HRG (A and
B), during the last 30 min with 1 µM insulin
(C), or with a combination of both effectors (D
and E). PM and LDM fractions were obtained and assayed by
Western blot to determine the abundance of GLUT1, GLUT3, and GLUT4
glucose transporters. Panel A shows autoradiograms from a
representative experiment (25 µg of protein/lane). Panel B
shows the values of the densitometry corresponding to the abundance of
glucose transporters in plasma membrane fractions after incubation with
HRG; the results are the mean ± S.E. of 3 observations per group.
Panel C shows the values corresponding to the abundance of
glucose transporters in plasma membrane fractions after incubation with
insulin; the results are the mean ± S.E. of 4-5 observations per
group. Panel D shows autoradiograms of a representative
experiment (20 µg of protein/lane). Panel E shows the
values of the densitometry corresponding to the abundance of glucose
transporters in plasma membrane fractions after incubation with HRG and
insulin; results are the mean ± S.E. of 6 observations per group.
*, indicates significant differences compared with the basal group at
p < 0.05 (Student's t test). , indicates
significant differences compared with the insulin + HRG group at
p < 0.05 (Student's t test).
View larger version (32K):
[in a new window]
Fig. 5.
Effect of HRG on glucose uptake in the
incubated soleus muscle. Muscle strips were incubated in the
presence of insulin (I, 100 nM, 60 min), HRG
(H, 3 nM, 120 min), or both (I + H). Following 2-deoxyglucose uptake, muscles were digested,
and radioactivity was measured in a -counter. Results are the
mean ± S.E. of 3-5 separate experiments. *, indicates
significant differences with basal group at p < 0.05;
, indicates significant differences between the insulin and HRG
groups at p < 0.05 (Student's t
test).
View larger version (54K):
[in a new window]
Fig. 6.
Effect on HRG on myotube
formation. L6E9 cells proliferated for 2 days at 10% FBS. Then
the cells were cultured at 2% FBS to allow them to differentiate.
After 24 h of differentiation, cells were incubated in the
presence or absence of 3 nM HRG for different times.
A, control cells and cells treated with HRG for 48 h
are shown. The bar indicates 28 µm. B, fusion
index studies were done in glutaraldehyde-fixed cultures where nuclei
were stained with Giemsa. The fusion index was calculated as the number
of nuclei/cell in a determined area. At least 10 different fields were
randomly analyzed from each plate. Open symbols, nontreated
cells; filled symbols, HRG-treated cells. Results are the
mean ± S.E. of 3 independent experiments; they are shown as
values relative to control cells on differentiation day 2. C, homogenates from control (open circles) and 3 nM HRG-treated (filled circles) cells were
obtained and processed by Western blot to determine myosin heavy chain
(MHC) expression. Three different experiments were run.
Results are shown as absolute increments with respect to day 2 of
differentiation. *, differences between control and HRG-treated groups
were statistically significant at p < 0.05 (Student's
t test).
View larger version (34K):
[in a new window]
Fig. 7.
Effects of chronic HRG on the expression of
glucose transporters, IRAP, SCAMPs, VAMP2, and cellubrevin. L6E9
cells proliferated for 2 days at 10% FBS and then were cultured at 2%
FBS to allow them to differentiate. After 24 h of differentiation,
cells were incubated in the presence or absence of 3 nM HRG
for different times. Total membranes were analyzed to determine GLUT1,
GLUT3, and GLUT4 total content (A) or abundance of IRAP,
SCAMPs, VAMP2, and cellubrevin (B). As a control we used
cells at the myoblastic stage (10% FBS, 80-90% confluence, day 0).
The 1 subunit of Na+/K+-ATPase
was used as a control of protein loading. Representative
autoradiograms are shown (10 µg of protein/lane for GLUT1, GLUT3, and
GLUT4 and 25-30 µg of protein/lane for IRAP, SCAMPs, cellubrevin,
and VAMP2). At least three different experiments were performed.
View larger version (30K):
[in a new window]
Fig. 8.
Glucose transport in heregulin-treated L6E9
muscle cells and abundance of glucose transporters at the plasma
membrane. L6E9 myotubes were treated or not for 48 h with 3 nM HRG. A, at term, cells were depleted of serum
for 4.5 h, and some were treated with 1 µM insulin
during the last 30 min. 2-Deoxyglucose uptake values were expressed per
µg of protein after subtraction of background values. Results are the
mean ± S.E. of 4 different experiments. Basal transport values
were 0.73 ± 0.14 pmol of 2-deoxyglucose/µg protein × 10 min. *, indicates the existence of significant differences compared
with the basal group at p < 0.05; , indicates
significant differences compared with insulin or HRG groups at
p < 0.05 (Student's t test). B,
basal; I, insulin; H, heregulin. B, at
term, plasma membrane fractions were obtained and assayed by Western
blot to determine the abundance of GLUT1, GLUT3, and GLUT4 glucose
transporters. Autoradiograms from a representative experiment are shown
(25 µg of protein/lane). Autoradiograms were subjected to
densitometry; results are the mean ± S.E. of 3-5 independent
experiments. *, indicates significant differences compared with the
basal group at p < 0.01 (Student's t
test).
View larger version (32K):
[in a new window]
Fig. 9.
Expression of ErbB2 and ErbB3 proteins and
neuregulin mRNA in denervated muscle. Total membranes were
obtained from 3-day denervated (Dn), contralateral
rat tibialis anterior muscles (C), or sham-operated muscles
(Sh). Membrane proteins were laid on gels. After blotting,
GLUT4, GLUT1, ErbB2, and ErbB3 were detected by incubation with
specific polyclonal antibodies. Representative autoradiograms are shown
(25 µg of protein/lane). Total RNA was obtained from 3-day denervated
and contralateral extensor digitorum longus muscle. Equal volumes of
total RNA were subjected to reverse transcriptase-PCR with primers
specific for the amplification of neuregulin mRNA. Original RNA
aliquots were subjected to electrophoresis as a loading control (not
shown). A representative image of six different samples is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (52). Thus, HRG might up-regulate GLUT1 expression through changes in Sp1, increasing either its total cellular content or
its active form.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Robin Rycroft for editorial support.
![]() |
FOOTNOTES |
---|
* This work was supported by research grants from the Dirección General de Investigación Científica y Técnica (PM98/0197), the Generalitat de Catalunya (1999SGR00039), the Fondo de Investigaciones Sanitarias (00/2101), and the Fundació Marató de TV3 (991110).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.
Recipient of a pre-doctoral fellowship from the Universitat de
Barcelona, Spain.
¶ To whom correspondence may be addressed: Dept. Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal, 645, E-08028 Barcelona, Spain. Tel.: 34-93-402-1519; Fax: 34-93-402-1559; E-mail: azorzano@porthos.bio.ub.es or aguma@ porthos.bio.ub.es.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M008100200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EGF, epidermal
growth factor;
HRG, rheregulin-1-(177-244);
IRAP, insulin-responsive aminopeptidase;
SCAMPs, secretory
component-associated membrane proteins;
VAMPs, vesicle-associated
membrane proteins;
FBS, fetal bovine serum;
PBS, phosphate-buffered
saline;
SN, supernatant;
PM, plasma membrane;
LDM, low density vesicle
membrane;
PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Douen, A. G.,
Ramlal, T.,
Rastogi, S.,
Bilan, P. J.,
Cartee, G. D.,
Vranic, M.,
Holloszy, J. O.,
and Klip, A.
(1990)
J. Biol. Chem.
265,
13427-13430 |
2. | Santalucía, T., Camps, M., Castelló, A., Muñoz, P., Nuel, A., Testar, X., Palacín, and M., Zorzano, A. (1992) Endocrinology 130, 837-846[Abstract] |
3. |
Castelló, A.,
Cadefau, J.,
Cussó, R.,
Testar, X.,
Hesketh, J. E.,
Palacín, M.,
and Zorzano, A.
(1993)
J. Biol. Chem.
268,
14998-15003 |
4. | Block, N. E., Menick, D. R., Robinson, K. A., and Buse, M. G. (1991) J. Clin. Invest. 88, 1546-1552[Medline] [Order article via Infotrieve] |
5. |
Henriksen, E. J.,
Rodnick, K. J.,
Mondon, C. E.,
James, D. E.,
and Holloszy, J. O.
(1991)
J. Appl. Physiol.
70,
2322-2327 |
6. |
Megeney, L. A.,
Neufer, P. D.,
Dohm, G. L.,
Tan, M. H.,
Blewett, C. A.,
Elder, G. C. B.,
and Bonen, A.
(1993)
Am. J. Physiol.
264,
E583-E593 |
7. | Coderre, L., Monfar, M. M., Chen, K. S., Heydrick, S. J., Kurowski, T. G., Ruderman, N. B., and Pilch, P. F. (1992) Endocrinology 131, 1821-1825[Abstract] |
8. | Didyk, R. B., Anton, E. E., Robinson, K. A., Menick, D. R., and Buse, M. G. (1994) Metabolism 43, 1389-1394[Medline] [Order article via Infotrieve] |
9. |
Elmendorf, J. S.,
Damrau-Abney, A.,
Smith, T. R.,
David, T. S,
and Turinsky, J.
(1997)
Am. J. Physiol.
272,
E661-E670 |
10. |
Meyer, D.,
Yamaai, T.,
Garratt, A.,
Riethmacher-Sonnenberg, E.,
Kane, D.,
Theill, L. E.,
and Birchmeier, C.
(1997)
Development
124,
3575-3586 |
11. | Peles, E., Bacus, S. S., Koski, R. A., Lu, H. S., Wen, D., Ogden, S. G., Levy, R. B., and Yarden, Y. (1992) Cell 69, 205-216[Medline] [Order article via Infotrieve] |
12. | Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y., Trail, G., Hu, S., Silbiger, S. M., Levy, R. B., Koski, R. A., Lu, H. S., and Yarden, Y. (1992) Cell 69, 559-572[Medline] [Order article via Infotrieve] |
13. | Holmes, W. E., Sliwkowski, M. X., Akita, R. W., Henzel, W. J., Lee, J., Park, J. W., Yansura, D., Abadi, N., Raab, H., Lewis, G. D., Shepard, H. M., Kuang, W.-J., Wood, W. I., Goeddel, D. V., and Vandlen, R. L. (1992) Science 256, 1205-1210[Medline] [Order article via Infotrieve] |
14. | Falls, D. L., Rosen, K. M., Corfas, G., Lane, W. S., and Fischbach, G. D. (1993) Cell 72, 801-815[Medline] [Order article via Infotrieve] |
15. |
Goodearl, A. D. J.,
Davis, J. B.,
Mistry, K.,
Minghetti, L.,
Otsu, M.,
Waterfield, M. D.,
and Stroobant, P.
(1993)
J. Biol. Chem.
268,
18095-18102 |
16. | Marchionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-McDonogh, O., Kirk, C., Hendriks, M., Danehy, F., Misumi, D., Sudhalter, J., Kobayashi, K., Wroblewski, D., Lynch, C., Baldassare, M., Hiles, I., Davis, J. B., Hsuan, J. J., Totty, N. F., Otsu, M., McBurney, R. N., Waterfield, M. D., Stroobant, P., and Gwynne, D. (1993) Nature 362, 312-318[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Ho, W.-H.,
Armanini, M. P.,
Nuijens, A.,
Phillips, H. S.,
and Osheroff, P. L.
(1995)
J. Biol. Chem.
270,
14523-14532 |
18. | Lemke, G. (1996) Mol. Cell. Neurosci 7, 247-262[CrossRef][Medline] [Order article via Infotrieve] |
19. | Fischbach, G. D., and Rosen, K. M. (1997) Annu. Rev. Neurosci. 20, 429-458[CrossRef][Medline] [Order article via Infotrieve] |
20. | Carraway, K. L., and Cantley, L. C. (1994) Cell 78, 5-8[Medline] [Order article via Infotrieve] |
21. |
Sliwkowski, M. X.,
Schaefer, G.,
Akita, R. W.,
Lofgren, J. A.,
Fitzpatrick, V. D.,
Nuijens, A.,
Fendly, B. M.,
Cerione, R. A.,
Vandlen, R. L.,
and Carraway, K. L.
(1994)
J. Biol. Chem.
269,
14661-14665 |
22. | Peles, E., and Yarden, Y. (1993) Bioessays 15, 815-824[Medline] [Order article via Infotrieve] |
23. | Meyer, D., and Birchmeier, C. (1995) Nature 378, 386-390[CrossRef][Medline] [Order article via Infotrieve] |
24. | Lee, K.-F., Simon, H., Chen, H., Bates, B., Hung, M.-C., and Hauser, C. (1995) Nature 378, 394-398[CrossRef][Medline] [Order article via Infotrieve] |
25. | Gassman, M., Casagranda, F., Orioli, D., Simon, H., Lai, C., Klein, R., and Lemke, G. (1995) Nature 378, 390-393[CrossRef][Medline] [Order article via Infotrieve] |
26. | Altiok, N., Bessereau, J.-L., and Changeux, J.-P. (1995) EMBO J. 14, 4258-4266[Abstract] |
27. | Jo, S. A., Zhu, X., Marchionni, M. A., and Burden, S. J. (1995) Nature 373, 158-161[CrossRef][Medline] [Order article via Infotrieve] |
28. | Chu, G. C., Moscoso, L. M., Sliwkowski, M. X., and Merlie, J. P. (1995) Neuron 14, 329-339[Medline] [Order article via Infotrieve] |
29. |
Florini, J. R.,
Samuel, D. S.,
Ewton, D. Z.,
Kirk, C.,
and Sklar, R. M.
(1996)
J. Biol. Chem.
271,
12699-12702 |
30. |
Kim, D.,
Chi, S.,
Lee, K. H.,
Rhee, S.,
Kwon, Y. K.,
Chung, C. H.,
Kwon, H.,
and Kang, M.-S.
(1999)
J. Biol. Chem.
274,
15395-15400 |
31. | Zhu, X., Lai, C., Thomas, S., and Burden, S. J. (1995) EMBO J. 14, 5842-5848[Abstract] |
32. | Moscoso, L. M., Chu, G. C., Gautman, M., Noakes, P. G., Merlie, J. P., and Sanes, J. R. (1995) Dev. Biol. 172, 158-169[CrossRef][Medline] [Order article via Infotrieve] |
33. | Rimer, M., Cohen, I., Lomo, T., Burden, S. J., and McMahan, U. J. (1998) Mol. Cell. Neurosci. 12, 1-15[CrossRef][Medline] [Order article via Infotrieve] |
34. | Kaliman, P., Viñals, F., Testar, X., Palacín, M., and Zorzano, A. (1995) Biochem. J. 312, 471-477[Medline] [Order article via Infotrieve] |
35. |
Mitsumoto, Y.,
and Klip, A.
(1992)
J. Biol. Chem.
267,
4957-4962 |
36. | Crettaz, M., Prentki, M., Zaninetti, D., and Jeanrenaud, B. (1980) Biochem. J. 186, 525-534[Medline] [Order article via Infotrieve] |
37. |
Gumà, A.,
Zierath, J. R.,
Wallberg-Henriksson, H.,
and Klip, A.
(1995)
Am. J. Physiol.
268,
E613-E622 |
38. | Gumà, A., Testar, X., Palacín, M., and Zorzano, A. (1988) Biochem. J. 253, 625-629[Medline] [Order article via Infotrieve] |
39. |
Si, J.,
Miller, D. S.,
and Mei, L.
(1997)
J. Biol. Chem.
272,
10367-10371 |
40. | Tsakiridis, T., McDowell, H., Walker, T., Downes, C. P., Hunda, H. S., Vranic, M., and Klip, A. (1995) Endocrinology 136, 4315-4322[Abstract] |
41. |
Sumitami, S.,
Ramlal, T.,
Somwar, R.,
Séller, S. R.,
and Klip, A.
(1997)
Endocrinology
138,
1029-1034 |
42. | Kandror, K. V., Coderre, L., Pushkin, A. V., and Pilch, P. F. (1995) Biochem. J. 307, 383-390[Medline] [Order article via Infotrieve] |
43. |
Randhawa, V. K.,
Bilan, P. J.,
Khayat, Z. A.,
Daneman, N.,
Liu, Z.,
Ramlal, T.,
Volchuk, A.,
Peng, X.-R.,
Coppola, T.,
Regazzi, R.,
Trimble, W. S.,
and Klip, A.
(2000)
Mol. Biol. Cell
11,
2403-2417 |
44. |
Sevilla, L.,
Tomàs, E.,
Muñoz, P.,
Gumà, A.,
Fischer, Y.,
Thomas, J.,
Ruiz. Montasell, B.,
Testar, X.,
Palacín, M.,
Blasi, J.,
and Zorzano, A.
(1997)
Endocrinology
138,
3006-3015 |
45. |
Gramolini, A. O.,
Angus, L. M.,
Schaeffer, L.,
Burton, E. A.,
Tinsley, J. M.,
Davies, K. E.,
Changeux, J.-P.,
and Jasmin, B. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3223-3227 |
46. |
Khurana, T. S.,
Rosmarin, A. G.,
Shang, J.,
Krag, O. B.,
Das, S.,
and Gammeltoft, S.
(1999)
Mol. Biol. Cell
10,
2075-2086 |
47. |
Carraway, K. L.,
Soltoff, S. P.,
Diamonti, A. J.,
and Cantley, L. C.
(1995)
J. Biol. Chem.
270,
7111-7116 |
48. |
Altiok, S.,
Batt, D.,
Altiok, N.,
Papautsky, A.,
Downward, J.,
Roberts, T. M.,
and Avraham, H.
(1999)
J. Biol. Chem.
274,
32274-32278 |
49. | Liu, W., Li, J., and Roth, R. A. (1999) Biochem. Biophys. Res. Commun. 261, 897-903[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Viñals, F.,
Fandos, C.,
Santalucia, T.,
Ferré, J.,
Testar, X.,
Palacín, M.,
and Zorzano, A.
(1997)
J. Biol. Chem.
272,
12913-12921 |
51. | Fandos, C., Sánchez-Feutrie, M., Santalucia, T., Viñals, F., Cadefau, J., Gumà, A., Cussó, R., Kaliman, P., Canicio, J., Palacín, M., and Zorzano, A. (1999) J. Mol. Biol. 294, 103-119[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Alroy, I.,
Soussan, L.,
Seger, R.,
and Yarden, Y.
(1999)
Mol. Cell. Biol.
19,
1961-1972 |
53. | Florini, J. R., Ewton, D. Z., and Magri, K. A. (1991) Annu. Rev. Physiol. 53, 201-216[CrossRef][Medline] [Order article via Infotrieve] |
54. | Carlsen, R. C. (1975) J. Physiol. 247, 343-361[Abstract] |
55. |
Viñals, F.,
Ferré, J.,
Fandos, C.,
Santalucia, T.,
Testar, X.,
Palacín, M.,
and Zorzano, A.
(1997)
Endocrinology
138,
2521-2529 |
56. |
Jones, J. P.,
Tapscott, E. B.,
Olson, A. L.,
Pessin, J. E.,
and Dohm, G. L.
(1998)
J. Appl. Physiol.
84,
1661-1666 |