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Hypertension-Endocrine Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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XXXXX motif in the tyrosine kinase domain of the insulin
receptor ß-subunit (where represents aromatic amino acids at
positions 1193, 1195, and 1200 of the insulin receptor) (15). In
addition, scaffolding domain peptides derived from caveolin-1 or -3 can
increase tyrosine kinase activity of the insulin receptor (10). Some
patients with syndromes of extreme insulin resistance have mutations of
the insulin receptor in which this caveolin binding motif is disrupted
(e.g. W1193L and W1200S) (16, 17, 18, 19, 20, 21). The W1193L mutant
undergoes accelerated degradation resulting in decreased cell surface
expression of receptors and also has an impairment in
autophosphorylation (18, 19, 20, 21). Although the W1200S mutant seems to be
expressed at higher levels on the cell surface than W1193L,
autophosphorylation of this mutant is severely impaired as well (16, 17). Taken together, both in vitro and in vivo
studies suggest that caveolin may interact with the insulin receptor
and play an important role in insulin signaling. However, the
functional consequences of interactions between caveolin and insulin
receptors are not well understood. In the present study, we used
transient transfection techniques to overexpress wild-type and mutant
forms of caveolin-1 in Cos-7 cells (low levels of endogenous
caveolin-1) and rat adipose cells (high levels of endogenous
caveolin-1) to investigate the role of caveolin-1 in insulin signaling.
In addition, we constructed and characterized several insulin receptor
mutants with a disrupted caveolin-1 binding motif. We demonstrate that
the interaction between insulin receptors and caveolin-1 in intact
cells is dependent upon an intact caveolin-1 scaffolding domain.
Intriguingly, we found that overexpression of caveolin-1 enhanced
insulin signaling in Cos-7 cells while inhibiting insulin action in
adipose cells. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Recombinant caveolin-1 could be
distinguished from the endogenous protein because myc-tagged caveolin-1
migrated more slowly on the gel. We detected abundant endogenous
caveolin-1 in adipose cells while very little of the endogenous protein
was observed in Cos-7 cells. Under our experimental conditions,
transfection efficiencies were approximately 5% for adipose cells (22)
and approximately 30% for Cos-7 cells as determined by expression of
green fluorescent protein (data not shown). Therefore, by comparing the
density of bands representing endogenous and recombinant caveolin-1, we
estimated levels of overexpression of approximately 20-fold in both
Cos-7 cells and adipose cells. A mutant caveolin-1 construct (Cav-Mut)
containing F92A and V94A point mutations in the scaffolding domain of
caveolin-1 was expressed at levels comparable to the wild-type
construct (Cav-WT).
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XXXXX caveolin-1 binding motif present in the kinase domain of
the receptor ß-subunit to evaluate potential interactions between
caveolin-1 and the insulin receptor. In the W1200T mutant, the distal
aromatic amino acid in the binding motif has been changed to threonine,
a residue found in a homologous region of c-src (1). The F1195G
mutation disrupts the binding motif at the central aromatic residue
while the triple mutation (W1193G/F1195G/W1200G) replaces all three
aromatic amino acids with glycine. The K1030A mutant is a
kinase-inactive receptor resulting from disruption of the canonical ATP
binding site.
High level expression of the mature 95-kDa insulin receptor ß-subunit
was observed in lysates derived from Cos-7 cells transfected with
either the wild-type or K1030A receptors (Fig. 2A). However, expression levels for the
caveolin-binding motif mutants were substantially lower. Of the three
binding motif mutants, the highest level of expression was observed
with W1200T. Interestingly, levels of expression for the 210-kDa
proinsulin receptor were similar for all of the insulin receptor
constructs evaluated. Since the extracellular 
-subunit was not
altered in any of our constructs, we performed
[125I]insulin binding studies in parallel with our
immunoblotting experiments to determine the relative numbers of
recombinant receptors present on the cell surface. The results of our
binding studies were consistent with our immunoblotting experiments
(Fig. 2, A and C). That is, the relative number of insulin receptors
present on the cell surface was proportional to the amount of 95-kDa
ß-subunit detected by immunoblotting. When we immunoblotted cell
lysates from experiments shown in Fig. 2A with an antiphosphotyrosine
antibody we observed normal autophosphorylation of the wild-type
insulin receptor, but we could not detect significant
autophosphorylation with any of the mutant insulin receptors (Fig. 2B).
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). In these cells, Cav-WT was
easily detected in insulin receptor immunoprecipitates (Fig. 3, lane
3). In contrast, the interaction of the scaffolding-domain mutant
(Cav-Mut) with the wild-type insulin receptor appeared to be
significantly reduced (Fig. 3, lane 4). Interestingly, significant
coimmunoprecipitation of Cav-WT with the W1200T insulin receptor mutant
was observed even though this receptor has a disrupted XXXXX
caveolin-1 binding motif (Fig. 3, lane 5). Similar to our results with
the wild-type insulin receptor, coimmunoprecipitation of Cav-Mut with
W1200T was significantly impaired (Fig. 3, lane 6). We also observed
significant coimmunoprecipitation of Cav-WT with a kinase-inactive
insulin receptor mutant (with an intact caveolin binding motif) that
was expressed at levels comparable to the wild-type receptor (data not
shown).
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). Since adipose cells express high
levels of endogenous insulin receptors, it was not necessary to
cotransfect the cells with recombinant insulin receptors. Consistent
with our results in Cos-7 cells, Cav-WT coimmunoprecipitated with
insulin receptors much better than Cav-Mut (Fig. 4, lanes 3–6). In
addition, insulin stimulation did not alter the ability of Cav-WT to
coimmunoprecipitate with the insulin receptor (Fig. 4, lanes 3–4). We
observed similar coimmunoprecipitation of endogenous insulin receptors
with endogenous caveolin-1 in freshly isolated untransfected adipose
cells (data not shown). Taken together, our results from both Cos-7
cells and adipose cells are consistent with the hypothesis that
interactions between caveolin-1 and insulin receptors in intact cells
depend upon the caveolin-1 scaffolding domain.
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). Interestingly,
co-overexpression of Cav-WT significantly enhanced insulin-stimulated
phosphorylation of Elk-1 to levels approximately twice those observed
in the control cells. In contrast, overexpression of Cav-Mut did not
significantly affect phosphorylation of Elk-1, suggesting that the
ability of Cav-WT to modulate insulin signaling is dependent upon an
intact scaffolding domain. In some experiments [125
I]insulin binding studies and anti-myc immunoblots were performed in
parallel to confirm comparable overexpression of recombinant insulin
receptors and caveolin constructs in all groups (data not shown). In
contrast to cells overexpressing wild-type insulin receptors, Elk-1
phosphorylation did not significantly increase upon insulin stimulation
of cells overexpressing the K1030A kinase-inactive insulin receptor
mutant (Fig. 5B). Co-overexpression of Cav-WT or Cav-Mut with the
K1030A receptor did not significantly change Elk-1 phosphorylation in
either the absence or the presence of insulin. We obtained similar
results from cells overexpressing the W1200T mutant insulin receptor as
might be expected from a receptor that does not undergo
autophosphorylation (data not shown).
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). Similar to our results with Elk-1,
insulin stimulated a significant increase in ERK-2 phosphorylation in
control cells (Fig. 6A, lanes 1–2). However, in contrast to our
results with Elk-1, overexpression of Cav-WT blocked the ability of
insulin to stimulate ERK2 phosphorylation (Fig. 6A, lanes 3–4).
Nevertheless, this effect of Cav-WT appeared to depend upon an intact
scaffolding domain because insulin-stimulated phosphorylation of ERK-2
in cells overexpressing Cav-Mut was similar to that of the control
cells (Fig. 6A, lanes 5–6).
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, upper panel). In the
absence of insulin, ERK2 was phosphorylated at a detectable level in
control cells cotransfected with an empty expression vector (pCIS2) and
HA-ERK2. Upon insulin stimulation, we observed an approximately 3-fold
increase in ERK2 phosphorylation (Fig. 7A, lanes 1–2; Fig. 7C).
Co-overexpression of either Cav-WT or Cav-Mut resulted in a significant
approximately 3-fold decrease in basal ERK2 phosphorylation (in the
absence of insulin) (Fig. 7A, lanes 3 and 6; Fig. 7C). No significant
differences in the level of ERK2 phosphorylation were observed between
any of the groups in the presence of insulin (Fig. 7A, lanes 2, 4, and
6; Fig. 7C). Note that cell lysates used in these experiments did not
include the nuclear fraction so that the amount of ERK2 recovered in
anti-HA immunoprecipitates was slightly less in the samples derived
from insulin-stimulated cells (presumably because some of the ERK2
underwent translocation to the nucleus upon insulin stimulation) (Fig. 7A, lower panel). As a control experiment, we verified
comparable expression of the various transgenes in each experimental
group by immunoblotting cell lysates with antibodies against either the
HA epitope (Fig. 7B, upper panel) or the Myc epitope (Fig. 7B, lower panel). Interestingly, when anti-HA
immunoprecipitates were immunblotted with an antibody against myc, we
detected comparable coimmunoprecipitation of Cav-WT or Cav-Mut with
HA-ERK2 that was unaffected by treatment of the cells with insulin
(Fig. 8). These results suggested that
interactions between ERK2 and caveolin-1 do not depend on the
caveolin-1 scaffolding domain (in contrast to interactions with the
insulin receptor). Thus, interactions of caveolin-1 with ERK2 may help
to explain why overexpression of either Cav-WT or Cav-Mut inhibits
basal phosphorylation of ERK2 in adipose cells.
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). We observed an
approximately 2-fold recruitment of GLUT4 to the cell surface in
response to insulin stimulation in control cells cotransfected with
pCIS2 and GLUT-HA. Interestingly, overexpression of either Cav-WT or
Cav-Mut caused a small, but statistically significant, decrease in the
insulin dose-response curves (when compared with the control).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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XXXXX
caveolin-1 binding motifs. The choice of these mutations was based upon
alanine scanning mutagenesis studies of interactions between caveolin-1
peptides and the epidermal growth factor receptor (15). Previous
peptide binding studies demonstrated that the scaffolding domain of
caveolin-1 (residues 82–101) interacts specifically with the caveolin
binding motif present in the ß-subunit of the insulin receptor as
well as a number of other receptor tyrosine kinases and downstream
signaling molecules (8, 10, 15). In addition, the insulin receptor is
enriched in subcellular fractions containing caveolae (23, 24). In
keeping with these previous studies, our coimmunoprecipitation
experiments showed, for the first time, that the full- length insulin
receptor and caveolin-1 can interact in intact cells. While insulin
stimulation did not result in any detectable change in this
interaction, the markedly impaired ability of Cav-Mut to
coimmunoprecipitate with the insulin receptor suggests that
interactions between insulin receptors and caveolin-1 depend upon the
caveolin-1 scaffolding domain. Our results are consistent with a direct
interaction in intact cells between the scaffolding domain of
caveolin-1 and the caveolin binding motif in the insulin receptor. As a complementary approach to studying interactions between the insulin receptor and caveolin-1, we generated several insulin receptor mutants with a disrupted caveolin binding motif (residues 1193–1200; WSFGVVLW). This region of the receptor is likely to be important for signaling because it is located just distal to the three critical tyrosine phosphorylation sites in the activation loop of the kinase domain (residues 1158, 1162, and 1163) (25) and is also well conserved in homologous regions of many other tyrosine kinases (2). We were unable to detect insulin-stimulated autophosphorylation of any of our mutant receptors. Moreover, it was difficult to evaluate interactions of caveolin-1 with the W1193G receptor mutant and the W1193G/F1195G/W1200G triple mutant because mature insulin receptors were poorly expressed on the cell surface for these constructs (although the proreceptor seemed to be present). Thus, the region of the insulin receptor containing the caveolin-1 binding motif may be necessary for the catalytic function of the kinase domain and may also be important for normal posttranslational receptor processing. Interestingly, these results are consistent with previous characterizations of a similar naturally occurring human insulin receptor mutation (W1193L) that has impaired autophosphorylation, undergoes accelerated degradation of the proreceptor (due to binding heat shock protein 90), and has markedly decreased cell surface expression (18, 19, 21).
To avoid potential problems with abnormal folding that may be occurring with the W1193G receptor and the triple mutant, we designed a W1200T mutant that disrupts the caveolin binding motif by mimicking the homologous region of c-src (WSFGILLT in region IX of the kinase domain). Although the WSFGILLT sequence in c-src does not perfectly match the canonical caveolin binding motif (15), the scaffolding domain of caveolin-1 is known to interact directly with c-src (26). It remains possible that this imperfect motif may also bind to the scaffolding domain of caveolin-1 since the region of c-src that binds to caveolin-1 has not been elucidated. For example, the reductase domain of endothelial nitric oxide synthase (eNOSr) also does not contain a known caveolin binding motif, but caveolin-1 binds to eNOSr and the scaffolding domain of caveolin-1 inhibits eNOSr activity (27). We observed significant coimmunoprecipitation of Cav-WT with the W1200T insulin receptor mutant while interaction with Cav-Mut was significantly impaired. Thus, similar to interactions between wild-type insulin receptors and caveolin-1, the scaffolding domain of caveolin-1 is important for binding to the W1200T receptor. Our results also suggest that either the canonical caveolin-1 binding motif is not strictly required for the insulin receptor to bind caveolin-1 or that caveolin-1 is capable of binding to another region of the insulin receptor. Although the W1200T receptor was not expressed as well as the wild-type receptor, it did achieve higher levels of expression than the W1193G mutant or triple-mutant. Similar to our other two receptor mutants, W1200T also had impaired autophosphorylation. This was intriguing because a threonine residue in the homologous position of c-src does not interfere with catalytic activity. Nevertheless, a similar naturally occurring insulin receptor mutation (W1200S) that causes severe insulin resistance in a dominant fashion is also kinase inactive (16, 17).
Functional Consequences of Overexpression of Caveolin-1
We chose to explore the functional role of caveolin-1 in insulin
signaling in both Cos-7 cells that express little endogenous caveolin-1
as well as in rat adipose cell that express high levels of caveolin-1.
Terminally differentiated cells including classical insulin targets
such as skeletal muscle and adipose cells often have abundant caveolae
and high levels of endogenous caveolin (8). In contrast,
undifferentiated or dedifferentiated transformed cells usually have few
or no caveolae and lower levels of caveolin (11, 12, 28). Oncogenic
transformation by activated Neu lowers the level of endogenous
caveolin-1 in fibroblasts (29). Moreover, down-regulation of caveolin-1
by stable transfection of antisense constructs in NIH 3T3 cells results
in increased cellular transformation (30). Conversely, overexpression
of recombinant caveolin-1 in transformed cells results in reversion of
this phenotype (31). This negative correlation between caveolin-1
expression and cellular transformation suggests that caveolin-1 may
have an inhibitory effect on growth factor or oncogenic signaling.
Interestingly, caveolin-1 only binds to wild-type Ras and c-src but not
to activated Ras or v-src, and overexpression of caveolin-1 inhibits
c-src autophosphorylation in transfected 293T cells (26, 32).
Furthermore, numerous studies suggest that interactions between
caveolin and a variety of signaling molecules, including G proteins,
src family kinases, and growth factor receptors, may serve to sequester
inactive signaling molecules and inhibit signaling (for review see Ref.
8). Thus, caveolin-1 seems to generally function in an inhibitory role
in most studies. However, there is some evidence that the role of
caveolins in cellular signaling may be cell type dependent and more
diverse than previously appreciated. For example, the development of
multidrug resistance in some tumor cell lines has been associated with
up-regulation of caveolin-1 and caveolae (33, 34), while suppression of
caveolin-1 in androgen-insensitive metastatic murine prostate cancer
cells leads to a more differentiated phenotype that responds to
androgens by undergoing apoptosis (35).
In Cos-7 cells transiently transfected with insulin receptors, overexpression of Cav-WT resulted in a significant increase in the ability of insulin to phosphorylate Elk-1 (while overexpression of Cav-Mut was without effect). This was dependent upon the presence of a catalytically active insulin receptor since cells expressing the kinase-inactive mutants K1030A or W1200T did not phosphorylate Elk-1 in response to insulin (in either the absence or presence of recombinant caveolin-1). Although our results do not fit with the paradigm of caveolin acting as an inhibitor of signaling, they are in agreement with a recent report demonstrating that overexpression of caveolin-3 may activate the insulin receptor kinase in HEK293T cells (10). That study also showed that scaffolding-domain peptides from caveolin-1 or -3 can bind and activate the insulin receptor kinase in vitro. Taken together with our demonstration that the insulin receptor binds to caveolin-1 in intact cells, it seems likely that our Elk-1 phosphorylation results are explained, at least in part, by interaction of caveolin-1 with the insulin receptor. It is also possible that the scaffolding domain of caveolin-1 mediates interactions with other insulin-signaling proteins to enhance Elk-1 phosphorylation. Indeed, previous studies have demonstrated that signaling molecules related to Elk-1 phosphorylation, such as Ras, Raf, MAPK/ERK kinase, and ERK2, are associated with caveolae (36) and caveolin-1 can bind to Ras (32) and ERK2 (this study). However, these interactions seem less likely to explain our results because the binding of caveolin-1 to these molecules may sequester inactive forms and tend to inhibit signaling (8). Indeed, in Cos-7 cells, overexpression of Cav-WT suppressed insulin-stimulated phosphorylation of ERK-2 while overexpression of Cav-Mut was without effect on ERK2 phosphorylation.
To study effects of overexpression of caveolin-1 on MAPK-dependent pathways in rat adipose cells, we examined phosphorylation of a cotransfected ERK2 in response to insulin stimulation. In contrast to our results in Cos-7 cells, overexpression of either Cav-WT or Cav-Mut decreased the basal level of ERK2 phosphorylation (in the absence of insulin) but was without effect on insulin-stimulated phosphorylation. It is unlikely that these results are explained by interactions of caveolin-1 with the insulin receptor since overexpression of Cav-Mut (which does not bind well to the insulin receptor) yielded similar results. Somewhat surprisingly, we found that both Cav-WT and Cav-Mut coimmunoprecipitated with ERK2 in adipose cells. Furthermore, this interaction was not affected by treating the cells with insulin. When we scanned the amino acid sequence of human ERK2, we could not identify any known caveolin scaffolding-domain binding motifs. Taken together, these findings suggest that an intact scaffolding domain may not be necessary for interactions of caveolin-1 with ERK2 in adipose cells.
When we investigated effects of overexpression of caveolin-1 on a
MAPK-independent function in rat adipose cells, we found that both
Cav-WT and Cav-Mut caused a small decrease in insulin-induced
translocation of GLUT4. Since both the wild-type and mutant caveolin
mediated a similar effect, it is unlikely that this was caused by
interactions of the caveolin-scaffolding domain with the insulin
receptor. Downstream effectors of insulin action that have been
implicated in mediating recruitment of GLUT4 to the cell surface such
as PKC-
(37) are also known to interact with caveolin. However,
these interactions are also unlikely to explain our data because the
scaffolding domain of caveolin-1 inhibits PKC- activity (38), and
our effects are observed with both Cav-WT and Cav-Mut.
It is interesting that in adipose cells, overexpression of either Cav-WT or Cav-Mut had inhibitory effects on both MAPK-dependent and -independent pathways while in Cos-7 cells, Cav-WT enhanced some insulin-stimulated MAPK-dependent pathways. This differential modulation of signaling may be due to differences in endogenous levels of caveolin-1. For example, since caveolin peptides have been shown to enhance insulin receptor tyrosine kinase activity (10), overexpression of caveolin in cells that have low endogenous levels of caveolin might be predicted to enhance insulin signaling as we observed. However, if the cell already has high endogenous levels of caveolin, its interaction with the insulin receptor may be saturated, and overexpression of caveolin may interfere with other downstream effectors. In addition, it is possible that other proteins that are important for the ability of caveolin to modulate signaling may be expressed in a cell type-dependent fashion. For example, insulin stimulation results in tyrosine phosphorylation of caveolin-1 by fyn only in 3T3-L1 adipocytes but not in undifferentiated preadipocytes (13, 39). A kinase that is induced upon adipocyte differentiation has been implicated in phosphorylation of c-cbl, which can then activate fyn leading to phosphorylation of caveolin (13).
Conclusions
Interactions between caveolin-1 and insulin receptors in intact
cells are dependent upon the caveolin-1 scaffolding domain. Point
mutations disrupting the caveolin-1 binding motif in the insulin
receptor result in markedly decreased cell surface receptor expression
and a defect in receptor autophosphorylation consistent with a
functionally important role for this region of the insulin receptor.
Indeed, a number of patients with syndromes of extreme insulin
resistance have mutations in this very region. Finally, our data
suggest that caveolin may have both inhibitory and stimulatory roles in
insulin signaling that depend upon the cellular context.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cav-WT: HindIII/BamH I fragment (
600-bp)
containing cDNA for myc-tagged canine caveolin-1 [obtained from M.
Lisanti (7, 42)] was blunt-ended and ligated in the sense orientation
into the HpaI site of pCIS2.
Cav-Mut: F92A and V94A point mutations in the scaffolding domain of
caveolin-1 were introduced into Cav-WT using the mutagenic
oligonucleotide 5'-CTT CAC CAC CGC CAC
TGC GAC AAA ATA C-3' and the Morph mutagenesis
kit (5Prime
3Prime; Boulder, CO). This mutagenesis also causes a loss
of a Tsp 45 I restriction site.
hIR-WT: pCIS2 vector containing the cDNA for the human insulin receptor (22).
hIR-K1030A: kinase-inactive point mutant of hIR-WT (obtained from S.I. Taylor)
hIR-W1200T: W1200T point mutant of hIR-WT generated using the mutagenic oligonucleotide 5'-GGC GTG GTC CTT ACC GAA ATC ACT AGC TTG GC-3'. This oligonucleotide also introduced a silent mutation that created an extra restriction site for Bfa I.
hIR triple-mutant: mutant of hIR-WT containing W1193G, F1195G, and
W1200G mutations to disrupt XXXXX caveolin binding motif. The
mutagenic oligonucleotide 5'-CAC TTC TTC TGA CAT
GGG GTC CGG TGG
CGT GGT CCT TGG GGA AAT CAC-3' used also causes
loss of a Bsplu 11 restriction site.
hIR-F1195G: mutant derived from hIR-triple mutant using mutagenic oligonucleotide 5'-CTG ACA TGT GGT CCG GTG GCG TGG TCC TTT GGG AAA TCA CCA GC-3' which changes back F1193 and W1200 and also restores the restriction site for Bsplu 11.
PCIS2-GLUT4 HA: the expression vector for GLUT4-HA was constructed as described previously (43).
HA-ERK2: expression vector for HA-tagged ERK2 (gift from M. Cobb).
The presence of correct mutations in the various constructs was verified by direct sequencing.
Antibodies
Murine monoclonal antibodies directed against phosphotyrosine
(4G10) were obtained from Upstate Biotechnology, Inc.
(Lake Placid, NY). Polyclonal antibodies against the myc epitope and
the insulin receptor ß-subunit were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibodies
against caveolin-1 (no. 13620) were obtained from Transduction Laboratories, Inc. (Lexington, KY). Phospho-MAPK antibodies were
obtained from New England Biolabs, Inc. (Beverly, MA) and
monoclonal antibodies against the HA epitope (HA-11) were obtained from
BabCO (Berkeley, CA).
Transient Transfection of Cos-7 Cells
Cos-7 cells were cultured in DMEM supplemented with 25
mM glucose, 20 mM glutamine, 100 U/ml
penicillin G, 100 µg/ml streptomycin, and 10% FCS at 37 C, 5%
CO2. Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD) was used to transfect cells at
approximately 80% confluency in six-well dishes according to the
manufacturers protocol.
Transient Transfection of Rat Adipose Cells and Assay for Cell
Surface Epitope-Tagged GLUT4
Isolated adipose cells from epididymal fat pads of male rats
(170–200 g, CD strain) were transfected by electroporation as
described previously (41, 44). To assess effects of insulin on
translocation of GLUT4, groups of cells were transfected with an empty
expression vector pCIS2 alone or cotransfected with GLUT4-HA and pCIS2,
Cav-WT, or Cav-Mut. Twenty hours after electroporation, adipose cells
were treated with insulin at 37 C for 30 min. Cell surface
epitope-tagged GLUT4 was quantified by using the anti-HA mouse
monoclonal antibody HA-11 in conjunction with 125I-labeled
sheep antimouse IgG as described (22).
Immunoprecipitation and Immunoblotting
For Cos-7 cells, lysates were prepared by washing cells with PBS
and then scraping the cells in ice-cold RIPA buffer on ice (20
mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 50
mM NaF, 1 mM sodium orthovanadate, 1% Triton
X-100 and 0.1% SDS, and a protease inhibitor cocktail (Complete
Tablet, Boehringer Mannheim, Mannheim, Germany)).
For some experiments, transfected cells were serum starved overnight
and then treated without or with insulin (100 nM, 2 min)
followed by freezing on liquid nitrogen. Cell lysates were then
prepared as described above. Lysates were centrifuged at 10,000 x
g for 10 min to pellet cellular debris. For
immunoprecipitation with the antiinsulin receptor ß-subunit antibody,
0.6 µg antibody and prewashed protein A-agarose (5% of the total
volume) were mixed with each sample (300 µg total protein) at 4 C on
a rotating wheel overnight. The immune complexes were washed three
times with RIPA buffer, and samples were pelleted by centrifugation and
eluted by boiling in Laemmli sample buffer for 5 min followed by
SDS-PAGE and immunoblotting with various antibodies. For adipose cells,
whole cell lysates and membrane fractions were prepared as previously
described (44). Immunoprecipitation with the antiinsulin receptor
ß-subunit antibody was carried out on membrane fractions as described
above. For immunoprecipitation of HA-ERK2, whole cell lysates (400 µg
total protein) were incubated with 7 µg HA-11 antibody and buffer
supplemented with 100 nM of okadaic acid at 4 C on a
rotating wheel. After 2 h, protein G-agarose was added (5% of
total volume) and the samples were incubated overnight at 4 C on a
rotating wheel. Samples were then treated as described above and
immunoblotted with antibodies against the HA-tag or phospho-MAPK.
Quantification of phospho-ERK2 was performed using a laser scanning
densitometer (Molecular Dynamics, Inc., Sunnyvale,
CA).
125I-Labeled Insulin Binding
Tracer insulin binding to the cell surface of Cos-7 cells
transfected with various insulin receptor constructs was assessed at 4
C as previously described (45).
Phosphorylation of Elk-1
The Path-Detect System (Stratagene) was used to
assess the effects of overexpression of caveolin-1 on
insulin-stimulated phosphorylation of Elk-1 in Cos-7 cells. In this
assay, phosphorylation of a transfected GAL4 binding domain/Elk-1
activation domain fusion protein results in activation of a
cotransfected GAL4 binding sequence/luciferase reporter plasmid
resulting in increased luciferase expression. The luciferase activity
was measured in cell lysates as previously described (46) and was
assumed to correlate with the level of Elk-1 phosphorylation.
Statistical Analysis
Paired Student’s t-tests were used for comparing
individual points where appropriate. P < 0.05 was
considered to signify statistical difference. The insulin dose-response
curves for GLUT4 translocation were fit using a nonlinear least squares
method and were compared by multiple ANOVA (MANOVA) as described
(44).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication June 17, 1999. Revision received August 25, 1999. Accepted for publication September 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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subunits, and H-Ras share a common membrane-anchored scaffolding
protein, caveolin. Caveolin binding negatively regulates the
auto-activation of Src tyrosine kinases. J Biol Chem 271:29182–29190, 
),
conventional (, ß) and novel (
, 
) protein kinase C isoforms
on insulin-stimulated translocation of epitope-tagged GLUT4 glucose
transporters in rat adipocytes: specific interchangeable effects of
protein kinases C- and C-. Biochem J 337:461–470
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P. F. Pilch, R. P. Souto, L. Liu, M. P. Jedrychowski, E. A. Berg, C. E. Costello, and S. P. Gygi Cellular spelunking: exploring adipocyte caveolae J. Lipid Res., October 1, 2007; 48(10): 2103 - 2111. [Abstract] [Full Text] [PDF]
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S. A. Predescu, D. N. Predescu, and A. B. Malik Molecular determinants of endothelial transcytosis and their role in endothelial permeability Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L823 - L842. [Abstract] [Full Text] [PDF]
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 K. Kabayama, T. Sato, K. Saito, N. Loberto, A. Prinetti, S. Sonnino, M. Kinjo, Y. Igarashi, and J.-i. Inokuchi Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance PNAS, August 21, 2007; 104(34): 13678 - 13683. [Abstract] [Full Text] [PDF]
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 Z.-g. Li, W. Zhang, and A. A.F. Sima Alzheimer-Like Changes in Rat Models of Spontaneous Diabetes Diabetes, July 1, 2007; 56(7): 1817 - 1824. [Abstract] [Full Text] [PDF]
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 C. A. Syme, L. Zhang, and A. Bisello Caveolin-1 Regulates Cellular Trafficking and Function of the Glucagon-Like Peptide 1 Receptor Mol. Endocrinol., December 1, 2006; 20(12): 3400 - 3411. [Abstract] [Full Text] [PDF]
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 A. K. Nevins and D. C. Thurmond Caveolin-1 Functions as a Novel Cdc42 Guanine Nucleotide Dissociation Inhibitor in Pancreatic beta-Cells J. Biol. Chem., July 14, 2006; 281(28): 18961 - 18972. [Abstract] [Full Text] [PDF]
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 M. M. Vihanto, C. Vindis, V. Djonov, D. P. Cerretti, and U. Huynh-Do Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase J. Cell Sci., June 1, 2006; 119(11): 2299 - 2309. [Abstract] [Full Text] [PDF]
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X. Lu, F. Kambe, X. Cao, T. Yoshida, S. Ohmori, K. Murakami, T. Kaji, T. Ishii, D. Zadworny, and H. Seo DHCR24-Knockout Embryonic Fibroblasts Are Susceptible to Serum Withdrawal-Induced Apoptosis Because of Dysfunction of Caveolae and Insulin-Akt-Bad Signaling Endocrinology, June 1, 2006; 147(6): 3123 - 3132. [Abstract] [Full Text] [PDF]
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 M. Inoue, S.-H. Chiang, L. Chang, X.-W. Chen, and A. R. Saltiel Compartmentalization of the Exocyst Complex in Lipid Rafts Controls Glut4 Vesicle Tethering Mol. Biol. Cell, May 1, 2006; 17(5): 2303 - 2311. [Abstract] [Full Text] [PDF]
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L. C. Matthews, M. J. Taggart, and M. Westwood Effect of Cholesterol Depletion on Mitogenesis and Survival: The Role of Caveolar and Noncaveolar Domains in Insulin-Like Growth Factor-Mediated Cellular Function Endocrinology, December 1, 2005; 146(12): 5463 - 5473. [Abstract] [Full Text] [PDF]
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R. Veluthakal, I. Chvyrkova, M. Tannous, P. McDonald, R. Amin, T. Hadden, D. C. Thurmond, M. J. Quon, and A. Kowluru Essential Role for Membrane Lipid Rafts in Interleukin-1{beta}-Induced Nitric Oxide Release From Insulin-Secreting Cells: Potential Regulation by Caveolin-1+ Diabetes, September 1, 2005; 54(9): 2576 - 2585. [Abstract] [Full Text] [PDF]
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 F. Capozza, A. W. Cohen, M. W.-C. Cheung, F. Sotgia, W. Schubert, M. Battista, H. Lee, P. G. Frank, and M. P. Lisanti Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells Am J Physiol Cell Physiol, March 1, 2005; 288(3): C677 - C691. [Abstract] [Full Text] [PDF]
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 K. Kabayama, T. Sato, F. Kitamura, S. Uemura, B. W. Kang, Y. Igarashi, and J.-i. Inokuchi TNF{alpha}-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3 Glycobiology, January 1, 2005; 15(1): 21 - 29. [Abstract] [Full Text] [PDF]
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 S. Eash, W. Querbes, and W. J. Atwood Infection of Vero Cells by BK Virus Is Dependent on Caveolae J. Virol., November 1, 2004; 78(21): 11583 - 11590. [Abstract] [Full Text] [PDF]
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 A. W. Cohen, R. Hnasko, W. Schubert, and M. P. Lisanti Role of Caveolae and Caveolins in Health and Disease Physiol Rev, October 1, 2004; 84(4): 1341 - 1379. [Abstract] [Full Text] [PDF]
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A. Balbis, G. Baquiran, C. Mounier, and B. I. Posner Effect of Insulin on Caveolin-enriched Membrane Domains in Rat Liver J. Biol. Chem., September 17, 2004; 279(38): 39348 - 39357. [Abstract] [Full Text] [PDF]
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J. Oshikawa, K. Otsu, Y. Toya, T. Tsunematsu, R. Hankins, J.-i. Kawabe, S. Minamisawa, S. Umemura, Y. Hagiwara, and Y. Ishikawa Insulin resistance in skeletal muscles of caveolin-3-null mice PNAS, August 24, 2004; 101(34): 12670 - 12675. [Abstract] [Full Text] [PDF]
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 H. P. KIM, X. WANG, F. GALBIATI, S. W. RYTER, and A. M. K. CHOI Caveolae compartmentalization of heme oxygenase-1 in endothelial cells FASEB J, July 1, 2004; 18(10): 1080 - 1089. [Abstract] [Full Text] [PDF]
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A. W. Cohen, B. Razani, W. Schubert, T. M. Williams, X. B. Wang, P. Iyengar, D. L. Brasaemle, P. E. Scherer, and M. P. Lisanti Role of Caveolin-1 in the Modulation of Lipolysis and Lipid Droplet Formation Diabetes, May 1, 2004; 53(5): 1261 - 1270. [Abstract] [Full Text] [PDF]
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P. Langlais, L. Q. Dong, F. J. Ramos, D. Hu, Y. Li, M. J. Quon, and F. Liu Negative Regulation of Insulin-Stimulated Mitogen-Activated Protein Kinase Signaling By Grb10 Mol. Endocrinol., February 1, 2004; 18(2): 350 - 358. [Abstract] [Full Text] [PDF]
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M. Kanzaki, S. Mora, J. B. Hwang, A. R. Saltiel, and J. E. Pessin Atypical protein kinase C (PKC{zeta}/{lambda}) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways J. Cell Biol., January 19, 2004; 164(2): 279 - 290. [Abstract] [Full Text] [PDF]
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S. Uhles, T. Moede, B. Leibiger, P.-O. Berggren, and I. B. Leibiger Isoform-specific insulin receptor signaling involves different plasma membrane domains J. Cell Biol., December 22, 2003; 163(6): 1327 - 1337. [Abstract] [Full Text] [PDF]
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 A. W. Cohen, T. P. Combs, P. E. Scherer, and M. P. Lisanti Role of caveolin and caveolae in insulin signaling and diabetes Am J Physiol Endocrinol Metab, December 1, 2003; 285(6): E1151 - E1160. [Abstract] [Full Text] [PDF]
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H. Thorn, K. G. Stenkula, M. Karlsson, U. Ortegren, F. H. Nystrom, J. Gustavsson, and P. Stralfors Cell Surface Orifices of Caveolae and Localization of Caveolin to the Necks of Caveolae in Adipocytes Mol. Biol. Cell, October 1, 2003; 14(10): 3967 - 3976. [Abstract] [Full Text] [PDF]
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A. W. Cohen, B. Razani, X. B. Wang, T. P. Combs, T. M. Williams, P. E. Scherer, and M. P. Lisanti Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue Am J Physiol Cell Physiol, July 1, 2003; 285(1): C222 - C235. [Abstract] [Full Text] [PDF]
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R. P. Souto, G. Vallega, J. Wharton, J. Vinten, J. Tranum-Jensen, and P. F. Pilch Immunopurification and Characterization of Rat Adipocyte Caveolae Suggest Their Dissociation from Insulin Signaling J. Biol. Chem., May 9, 2003; 278(20): 18321 - 18329. [Abstract] [Full Text] [PDF]
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S. Shigematsu, R. T. Watson, A. H. Khan, and J. E. Pessin The Adipocyte Plasma Membrane Caveolin Functional/Structural Organization Is Necessary for the Efficient Endocytosis of GLUT4 J. Biol. Chem., March 14, 2003; 278(12): 10683 - 10690. [Abstract] [Full Text] [PDF]
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 B. Razani, S. E. Woodman, and M. P. Lisanti Caveolae: From Cell Biology to Animal Physiology Pharmacol. Rev., September 1, 2002; 54(3): 431 - 467. [Abstract] [Full Text] [PDF]
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L. Vargas, B. F. Nore, A. Berglof, J. E. Heinonen, P. T. Mattsson, C. I. E. Smith, and A. J. Mohamed Functional Interaction of Caveolin-1 with Bruton's Tyrosine Kinase and Bmx J. Biol. Chem., March 8, 2002; 277(11): 9351 - 9357. [Abstract] [Full Text] [PDF]
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P. E. Bickel Lipid rafts and insulin signaling Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E1 - E10. [Abstract] [Full Text] [PDF]
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L. V. Ravichandran, H. Chen, Y. Li, and M. J. Quon Phosphorylation of PTP1B at Ser50 by Akt Impairs Its Ability to Dephosphorylate the Insulin Receptor Mol. Endocrinol., October 1, 2001; 15(10): 1768 - 1780. [Abstract] [Full Text] [PDF]
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 F. C. Bender, M. A. Reymond, C. Bron, and A. F. G. Quest Caveolin-1 Levels Are Down-Regulated in Human Colon Tumors, and Ectopic Expression of Caveolin-1 in Colon Carcinoma Cell Lines Reduces Cell Tumorigenicity Cancer Res., October 1, 2000; 60(20): 5870 - 5878. [Abstract] [Full Text]
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S. Parpal, M. Karlsson, H. Thorn, and P. Stralfors Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control J. Biol. Chem., March 23, 2001; 276(13): 9670 - 9678. [Abstract] [Full Text] [PDF]
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S. Shigematsu, S. L. Miller, and J. E. Pessin Differentiated 3T3L1 Adipocytes Are Composed of Heterogenous Cell Populations with Distinct Receptor Tyrosine Kinase Signaling Properties J. Biol. Chem., April 27, 2001; 276(18): 15292 - 15297. [Abstract] [Full Text] [PDF]
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S. Le Lay, S. Krief, C. Farnier, I. Lefrere, X. Le Liepvre, R. Bazin, P. Ferre, and I. Dugail Cholesterol, a Cell Size-dependent Signal That Regulates Glucose Metabolism and Gene Expression in Adipocytes J. Biol. Chem., May 11, 2001; 276(20): 16904 - 16910. [Abstract] [Full Text] [PDF]
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B. Razani, T. P. Combs, X. B. Wang, P. G. Frank, D. S. Park, R. G. Russell, M. Li, B. Tang, L. A. Jelicks, P. E. Scherer, et al. Caveolin-1-deficient Mice Are Lean, Resistant to Diet-induced Obesity, and Show Hypertriglyceridemia with Adipocyte Abnormalities J. Biol. Chem., March 1, 2002; 277(10): 8635 - 8647. [Abstract] [Full Text] [PDF]
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R. T. Watson, S. Shigematsu, S.-H. Chiang, S. Mora, M. Kanzaki, I. G. Macara, A. R. Saltiel, and J. E. Pessin Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation J. Cell Biol., August 20, 2001; 154(4): 829 - 840. [Abstract] [Full Text] [PDF]
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). Results shown are the means ±
SEM of six independent experiments. The best-fit curve for
the control group had an ED50 = 0.07 nM.
The best-fit curve for the Cav-WT group had an ED50 =
0.12 nM. The difference in the two curves was statistically
significant by multivariate ANOVA (P < 1 x
10-5). B, Recruitment of epitope-tagged GLUT4 to the cell
surface of cells cotransfected with Cav-Mut/GLUT4-HA (•) was
decreased compared with the control group (