University of Kentucky Medical School, Department of Physiology, Lexington, Kentucky 40536
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ABSTRACT |
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Epidermal growth factor
(EGF) and platelet-derived growth factor (PDGF) receptors have been
reported to signal via caveolin-containing membranes called caveolae.
In contrast, others report that EGF and PDGF receptors are exclusively
associated with caveolin-devoid membranes called rafts. Our subcellular
fractionation and coimmunoprecipitation studies demonstrate that, in
the absence of ligand, EGF and PDGF receptors are associated with
rafts. However, in the presence of ligand, EGF and PDGF receptors
transiently associate with caveolae. Surprisingly, pretreatment of
cells with EGF prevents PDGF-dependent phosphorylation of PDGF
receptors and extracellular signal-regulated kinase (ERK) 1/2 kinase
activation. Furthermore, cells pretreated with PDGF prevent
EGF-dependent phosphorylation of EGF receptors and ERK1/2 kinase
activation. Radioligand binding studies demonstrate that incubation of
cells with EGF or PDGF causes both EGF and PDGF receptors to be
reversibly sequestered from the extracellular space. Experiments with
methyl--cyclodextrin, filipin, and antisense caveolin-1 demonstrate
that sequestration of the receptors is dependent on cholesterol and
caveolin-1. We conclude that ligand-induced stimulation of EGF or PDGF
receptors can cause the heterologous desensitization of the other
receptor by sequestration in cholesterol-rich, caveolin-containing
membranes or caveolae.
caveolae; signaling; desensitization
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INTRODUCTION |
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THE REGULATION OF epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor activation, signaling, and desensitization is complex, and most likely several mechanisms exist to modulate these critical receptors. Both receptors have been reported to generate signals from coated pits (34), endosomes (34), plasma membranes (2), rafts (39), and caveolae (18). Although the specific details concerning the role of caveolae and rafts in EGF receptor and PDGF receptor signaling are contentious, a body of solid evidence is accumulating that clearly demonstrates that both domains are involved (10, 13, 38, 41). The purpose of the current studies was to determine if caveolae and/or rafts are involved in the desensitization of the EGF and PDGF receptors.
EGF receptors and PDGF receptors have been reported to be associated exclusively with caveolae (15, 16, 18, 19) and exclusively with rafts (39-41). The subcellular localization of EGF and PDGF receptors is important because the location of these receptors most likely dictates the mechanism for activation and desensitization. Liu et al. (16) and Mineo et al. (19) have used a Triton X-100-based subcellular fractionation procedure to demonstrate that EGF receptors and PDGF receptors are highly enriched in caveolae and that both receptors can generate signals from these microdomains. Liu et al. (14) used a detergent-free subcellular fractionation method to demonstrate that PDGF receptors are enriched in and signal from endothelial caveolae. In addition, Pike and Miller (23) used a carbonate-based subcellular fractionation method to demonstrate that EGF-stimulated phosphatidylinositol turnover occurs in caveolae. In contrast to these studies, Waugh et al. (39) first enriched for caveolae (caveolin containing) by subcellular fractionation and then further purified caveolin-containing membranes (caveolae) by immunoisolation. Surprisingly, caveolin and EGF receptors did not reside in the same membranes. The authors concluded that EGF receptors are in low-density lipid rafts that are not caveolae.
Although some of the literature suggests that EGF and PDGF receptors signal through caveolae, the studies of Couet et al. (5) demonstrate that caveolin directly binds to and inhibits the activity of EGF receptors. Caveolin is a 22-kDa protein that is a key component of caveolae, although the protein can be found in other subcellular locations (30, 32). Caveolin contains a "scaffolding domain" consensus sequence that binds to a "caveolin-binding motif" found in many signaling proteins (30). The in vitro binding of peptides corresponding to the caveolin scaffolding domain inhibits the kinase activity of EGF receptors, suggesting that caveolin negatively regulates EGF receptors (6). In agreement with the peptide-binding studies, Park et al. (21) have documented that upregulation of caveolin in senescent cells decreases EGF-mediated signaling. In addition, Zhang et al. (43) have shown that adenovirus-mediated overexpression of caveolin-1 will inhibit EGF-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells. Taken together, these data suggest that caveolae are sites of desensitization rather than activation.
We used Swiss 3T3 cells, which endogenously express both EGF and PDGF receptors, to determine if caveolae were the sites of EGF and PDGF receptor desensitization. In unstimulated cells and cells stimulated for a short time with EGF or PDGF, the EGF and PDGF receptors associated with rafts and not caveolae. Longer stimulation with the ligands caused both receptors to become dephosphorylated and to associate with caveolae and not rafts. In addition, the stimulation and subsequent desensitization of one receptor promoted the heterologous desensitization of the other receptor (without prior activation of the other receptor) by sequestration in a compartment that was not accessible to external reagents. This compartment appears to be a cholesterol-rich, caveolin-containing membrane or caveola.
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EXPERIMENTAL PROCEDURES |
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Materials.
DMEM-low glucose, FBS, L-glutamine, trypsin-EDTA, OptiPrep,
and penicillin/streptomycin were from Life Technologies (Grand Island,
NY). Percoll, filipin, and methyl--cyclodextrin were from Sigma (St.
Louis, MO). 125I-labeled EGF (sp act 1,210 Ci/mmol) and
125I-labeled PDGF (sp act 569 Ci/mmol) were from New
England Nuclear (Boston, MA). The anti-caveolin-1 IgG (clone no. 2234)
and anti-phosphotyrosine IgG were from Transduction Laboratories
(Lexington, KY). The anti-EGF receptor IgG was from Santa Cruz (Santa
Cruz, CA). The anti-PDGF receptor, extracellular signal-regulated
kinase (ERK) 1/2, and activated ERK1/2 IgGs were from Upstate
Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated IgGs
were supplied by Cappel (West Chester, PA). Super Signal
chemiluminescent substrate was purchased from Pierce (Rockford, IL).
Buffers. Sample buffer (5×) consisted of 0.31 M Tris, pH 6.8, 2.5% (wt/vol) SDS, 50% (vol/vol) glycerol, and 0.125% (wt/vol) bromphenol blue. Tris-buffered saline (TBS) consisted of 20 mM Tris, pH 7.6, and 137 mM NaCl. Blotting buffer consisted of TBS plus 0.5% Tween 20 and 5% dry milk. Wash buffer consisted of TBS plus 0.5% Tween 20 and 0.2% dry milk. Tris-saline consisted of 50 mM Tris (pH 7.4) and 150 mM NaCl.
Cell culture. Swiss 3T3 cells were cultured in a monolayer and plated in a standard format. On day 0, ~5 × 105 cells were seeded in 100-mm dishes with 10 ml of DMEM supplemented with 100 U/ml penicillin/streptomycin, 0.5% (vol/vol) L-glutamine, and 10% (vol/vol) FBS. The cells were used on day 6.
Isolation of caveolae and rafts.
Caveola and raft membranes were enriched using a previously described
and well-characterized isolation procedure (35-37).
Cells were washed in 0.25 M sucrose, 1 mM EDTA, and 20 mM Tricine, pH 7.8 (buffer A), scraped off the dish, and collected by
centrifugation at 1,400 g for 5 min at 4°C. The cells were
suspended in 1 ml of buffer A, placed in a 2-ml Wheaton
tissue grinder, and homogenized with 20 strokes of the Teflon
homogenizer. The homogenate was centrifuged at 1,000 g for
10 min at 4°C to remove the nuclei and large mitochondria. The
resulting supernatant was designated the postnuclei supernatant (PNS).
The PNS was layered on 23 ml of 30% Percoll diluted in buffer
A and centrifuged at 84,000 g for 30 min at 4°C in a
Beckman Ti60 rotor. The plasma membrane fraction was a visible band
~5.7 cm from the bottom of the centrifuge bottle. The plasma membrane
fraction was collected and adjusted to a final volume of 2 ml with
buffer A and sonicated for 6 s two successive times
(total power, 50 J · W1 · s
1 each time). The
sonicate was mixed with OptiPrep (final concentration of 23% OptiPrep)
and placed in the bottom of a SW41 centrifuge tube. A liner
20-10% OptiPrep gradient was poured on top of the sample, and the
material was centrifuged at 52,000 g for 90 min at 4°C.
The top 5 ml of the first OptiPrep gradient was collected, placed in a
fresh SW41 centrifuge tube, and mixed with 4 ml of 50% OptiPrep
solution, containing 83% OptiPrep, 42 mM sucrose, 1 mM EDTA, and 20 mM
tricine, pH 7.8. The sample was overlaid with 1 ml of 15% OptiPrep
solution and 0.5 ml of 5% OptiPrep solution, prepared by diluting 50%
OptiPrep solution with buffer A, and centrifuged at 52,000 g for 90 min at 4°C. A distinct opaque band was present at
both interfaces. The band at the 5% interface was collected and
designated caveola/raft membranes. We typically obtained 30-50
µg of protein in this band. The top 2 ml of the Percoll gradient were
collected and centrifuged at 350,000 g for 1 h, and the
resulting supernatant was defined as the cytosol. All of the Percoll
fractions below the plasma membrane band were pooled and centrifuged at
100,000 g for 1 h, and the resulting pellet was
collected and defined as the intracellular membranes. The bulk plasma
membranes are obtained by pooling the bottoms of both OptiPrep gradients.
Immunoprecipitations. Protein A-Sepharose beads were first blocked by incubation for 4 h at 4°C with Chinese hamster ovary cell lysate (200 µg/ml) plus 30 mg/ml BSA in immunoprecipitation buffer (150 mM NaCl, 0.5% Triton X-100, and 50 mM Tris, pH 8.0). Blocked beads were then used to preclear the experimental fractions that had been adjusted to 0.5% (vol/vol) Triton X-100. Precleared fractions were incubated for 2 h at 4°C with the appropriate antibody before addition of blocked Protein A-Sepharose beads and incubation for an additional 1 h at 4°C. The beads were collected by centrifugation, washed four times in high-salt (500 mM NaCl) immunoprecipitation buffer, and then placed in Laemmli sample buffer. Immunoprecipitated proteins were detected by immunoblotting.
Electrophoresis and immunoblots.
Cellular fractions were dissolved in 0.015% (wt/vol) deoxycholate,
concentrated by precipitation with 7% (wt/vol) TCA, and washed in
acetone. Pellets were suspended in 1× sample buffer plus 1.2%
(vol/vol) -mercaptoethanol and heated to 95°C for 5 min
immediately before loading. Proteins were separated on a 12.5% polyacrylamide gel at 50 mA (constant current) and subsequently transferred to a polyvinylidene difluoride membrane at 50 volts (constant voltage) for 2 h. Membranes were blocked with blotting buffer for 60 min at 22°C. Primary antibodies were diluted in blotting buffer and incubated with blocked membranes for 60 min at
22°C. Membranes were washed four times for 10 min in wash buffer. Horseradish peroxidase-conjugated IgGs directed against the appropriate host IgGs were diluted and incubated with membranes as described for
primary antibodies. Membranes were washed four times for 10 min in wash
buffer and visualized using chemiluminescence.
Sequestration assay. The sequestration assay was done as previously described (29). At the beginning of the experiment, the cells were washed in PBS and then subjected to the indicated experimental protocol. Once the protocol was completed, cells were chilled on ice for 20 min and washed two times with ice-cold PBS. Externally exposed radioligand was released when cells were incubated on ice for 30 s in the presence of acid saline (0.15 M NaCl, adjusted to pH 3.0 with glacial acetic acid). Sequestered radioligand was the amount of radiation that remained associated with the acid/saline-treated cells. The cells were lysed in 0.1 N NaOH (15 min, room temperature), and the material was collected to quantify cell-associated radioligand. Radioactivity was measured in a Packard Instruments gamma counter. Nonspecific binding was measured by adding 100-fold excess unlabeled ligand and was <1% of specific binding. Degradation of the radiolabeled ligand was estimated by determining the amount of radiation that could not be precipitated by TCA. Less than 3% of the label was degraded in any of the experimental conditions.
Caveolin transfections. Human caveolin cDNA consisting of just the coding region was subcloned in the expression plasmid pCI-neo (Promega, Madison, WI), using EcoR I sites. PCR analysis and restriction digest analysis were used to identify a clone containing caveolin in the antisense orientation. This type of antisense construct has been used before to decrease the cellular expression of caveolin (9). The construct was transfected to Swiss 3T3 cells with Lipofectamine Plus (Life Technologies), and stable clones expressing different levels of caveolin were obtained by selecting the cells in 0.5 mg/ml G-418.
Statistical analysis. Least-squares ANOVA was used to evaluate the data with respect to cell fraction, time, and their interaction using the ANOVA procedure of SigmaPlot. When appropriate, fractions were compared within a given time using the Tukey's Honest Significant Difference test. Means were considered different at P < 0.01.
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RESULTS |
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Localization of EGF and PDGF receptors.
To determine if EGF and PDGF receptors cofractionate with caveolae
and/or rafts, Swiss 3T3 cells were deprived of serum for 12 h
before treatment with 100 ng/ml EGF for 0, 10, or 60 min. The serum
starvation did not affect the morphology or viability of the cells
(data not shown). The cells were then processed to obtain a subcellular
fraction enriched in caveolae and rafts. Figure
1A demonstrates that EGF and
PDGF receptors cofractionated with caveolin both in the absence and
presence of EGF. A phosphotyrosine band corresponding to the molecular
weight of phosphorylated EGF receptor was detected in the caveolae
fraction after 10 min of ligand stimulation but not in the absence of
ligand or after 60 min of treatment with ligand.
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Heterologous desensitization.
Because both receptors were in the same membrane domain, we next
determined if the activation of one receptor affected the activation of
the other receptor. To do this, we serum starved cells for 12 h
and then pretreated the cells with 100 ng/ml of EGF or medium only for
60 min. The cells were then incubated with 100 ng/ml EGF or 67 ng/ml
PDGF for an additional 10 min. Cell lysates were analyzed by SDS-PAGE
and immunoblot. Figure 3 demonstrates that the relative amount of EGF and PDGF receptors was not affected by
any of the treatments. Cells pretreated with medium only and then
incubated with EGF or PDGF for 10 min contained tyrosine-phosphorylated receptors; however, pretreatment of cells with EGF prevented both EGF
and PDGF from promoting the phosphorylation of the receptors. It was
possible that the phosphorylation of EGF and PDGF receptors did not
initiate a signaling cascade, so we also determined whether ERK1/2
kinase (mitogen-activated protein kinase) was phosphorylated. ERK1/2 kinases are downstream effectors of EGF and PDGF
receptor-mediated signaling and are activated by phosphorylation
(4, 11). Figure 3 demonstrates that the relative levels of
ERK1/2 kinases were not changed throughout the study; however, 10 min
of EGF or PDGF stimulation greatly induced the phosphorylation of
ERK1/2 kinases. In addition, lack of EGF or PDGF receptor
phosphorylation (EGF pretreatment) correlated with a decrease in ERK1/2
kinase phosphorylation.
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Sequestered compartment.
The immunoisolation of membrane domains that contain caveolin-1, EGF
receptors, and PDGF receptors along with the finding that both
receptors are sequestered after 60 min of exposure to ligand suggested
that the receptors might be sequestered in a caveolin-containing
membrane. To begin to address this possibility, we determined whether
filipin and methyl--cyclodextrin, pharmacological reagents that
inhibit caveola internalization, would prevent the heterologous
desensitization of the receptors. Swiss 3T3 cells were pretreated with
100 ng/ml EGF for 60 min in the presence or absence of 1 µg/ml
filipin or 5 mM methyl-
-cyclodextrin for 30 min. Without washing the
cells, 100 ng/ml EGF, 67 ng/ml PDGF, or medium only was added to the
pretreated cells for 10 min. The cells were then washed and lysed, and
the amount of phosphorylated PDGF receptor was determined by
phosphotyrosine immunoblot analysis. Figure
7 demonstrates that pretreatment with EGF
for 60 min greatly reduces the amount of PDGF-inducible tyrosine
phosphorylation of the PDGF receptor. However, if the cells were also
pretreated with filipin or methyl-
-cyclodextrin, to prevent caveola
internalization, then the addition of PDGF promoted the phosphorylation
of PDGF receptors. Similar data were obtained when the order of the
growth factor additions was reversed (data not shown).
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DISCUSSION |
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Subcellular fractionation methods that isolate caveolae also isolate rafts (20), making it difficult to determine whether EGF and PDGF receptors are associated with caveolae or rafts. We used a previously described and well-characterized isolation procedure (33, 35-37) to demonstrate that EGF and PDGF receptors coisolate with caveolae and rafts (Fig. 1). However, when caveolae were further purified by immunoisolation, EGF and PDGF receptors did not immunoisolate with caveolae (Fig. 2) but remained with the raft fraction. The GPI-anchored protein, CD55, was used as a marker for rafts. GPI-anchored proteins normally associate with rafts and only associate with caveolae when they have been cross-linked by a ligand or multivalent antibodies (17, 28). Caveolin and CD55 were never found in the same fraction after immunoprecipitation, indicating that rafts and caveolae were separated (20). Another critical control was demonstrating that the material isolated in the caveola/raft fraction was cold Triton X-100 insoluble because rafts are functionally defined as cold Triton X-100-insoluble membrane domains (30). These findings show the necessity of immunoisolating caveola membranes (containing caveolin) when subcellular fractionation methods are employed to study caveolae. It is also important to emphasize that the immunoisolation studies presented here do not necessarily indicate protein-protein interactions because entire membrane domains were isolated and not individual proteins. Significantly, antibodies to either EGF or PDGF receptors precipitated membranes that contained both receptors, consistent with the earlier work of Liu and Anderson (15), demonstrating that the receptors are physically located in the same membrane domains. The fact that the receptors are physically in close proximity to each other is a critical component of the putative mechanism of heterologous receptor desensitization presented below.
Short-term treatment of cells (10 min) with either EGF or PDGF promoted receptor phosphorylation but not association with caveolae. These findings are consistent with those of Waugh et al. (40), who demonstrated that, in unstimulated cells and cells stimulated for a short time (2 min), EGF receptors were not associated with caveolae. However, in the present study, we extended these findings to reveal that after 60 min of incubation with ligand both receptors were associated with membranes that precipitated with caveolin-1 IgG, and, importantly, the receptors no longer associated with CD55 (a raft marker). In unstimulated cells, EGF and PDGF receptors were found in rafts, the receptors were not phosphorylated, and ERK1/2 kinases, downstream effectors of growth factor receptor signaling, were inactive. The addition of ligand caused receptor phosphorylation and ERK1/2 kinase phosphorylation but not the immediate association with caveolae. After 60 min in the presence of ligand, EGF and PDGF receptors were associated with caveolae, the receptors were dephosphorylated, and ERK1/2 kinase was inactive. In contrast, these data are in apparent contradiction to those of Mineo et al. (18) who demonstrated that EGF receptors copurify with caveolin in quiescent cells and do not copurify with caveolin after treatment of the cells with EGF. The authors interpreted these data to mean that 1) EGF receptors are present in caveolae in the absence of EGF and 2) the EGF receptors move to a noncaveola plasma membrane location after binding EGF. With respect to the first inconsistency, the subcellular fractionation procedure used by Mineo et al. (18) isolates both caveolae and rafts, making it impossible to determine if the EGF receptors are indeed localized to caveolae. In regard to the second discrepancy, Mineo et al. (18) demonstrated that EGF receptors no longer copurify with caveolin after EGF treatment. The authors stated that the receptors were not internalized; however, the immunoblot data document that the EGF receptors did not appear in the noncaveola membrane fraction, leaving the final localization of the receptors undetermined. The explanation for the lack of EGF receptor immunoreactivity after EGF treatment is unclear, but the negative data preclude the conclusion that EGF receptors are no longer associated with caveolae or rafts. One possible explanation for the lack of plasma membrane-associated immunoreactivity is that the EGF receptors were sequestered/internalized, which would be consistent with our current data. Overall, the studies of Mineo et al. (18) are inconclusive with respect to the subcellular localization of EGF receptors and are in opposition to the widespread concept that caveolae are sites where growth factor receptors are in an active state. In contrast, our data indicate that EGF and PDGF receptors interact with both rafts and caveolae in a ligand- and time-dependent manner and are consistent with other studies demonstrating that caveolin is involved in the negative regulation of growth factor receptor signaling (for review, see Ref. 30).
The most novel aspect of these studies is the putative mechanism whereby the activation of one growth factor receptor can influence the activation of the other growth factor receptor. We found that a 60-min pretreatment with one growth factor completely inhibited the other growth factor from promoting receptor phosphorylation or ERK1/2 kinase activation (Fig. 3). Earlier studies by other investigators have demonstrated that the binding of PDGF to PDGF receptors can decrease the binding of EGF to EGF receptors (42). This process is called transmodulation (27). It has been suggested that the mechanism of decreased ligand binding is the result of a change in the affinity of the receptor for its ligand (1). Our current data suggest an additional mechanism, namely sequestration of the receptors in a compartment that prevents receptor-ligand interactions. We used a classic acid-sensitivity assay (29) to determine if the receptors were exposed to the extracellular space or sequestered from the extracellular environment (Fig. 4). If the receptor-ligand complex is exposed to the extracellular environment, then a brief acid-saline wash will strip the ligand from the receptor. However, if the receptor-ligand complex is in a sequestered compartment, the acid-saline will not strip the ligand from the receptor. When radiolabeled ligand was added to the cells at 4°C, all of the radiolabeled ligand remained in an acid- and saline-sensitive pool; however, when the radiolabeled ligand was added for 60 min at 37°C, all of the radiolabeled ligand was resistant to an acid-saline wash. These data indicate that the receptors were initially exposed to the cell surface, and after 60 min in the presence of ligand the receptors were quantitatively sequestered from the cell surface. Significantly, the addition of one ligand (EGF or PDGF) for 60 min at 37°C prevented the association of the other ligand (PDGF or EGF) with its receptor (Fig. 5). These data are consistent with the idea that binding of one ligand to its receptor causes both receptors to be sequestered.
EGF and PDGF appeared to specifically bind the appropriate receptors because control binding studies done at 4°C demonstrated that addition of both radiolabeled ligands at the same time increased total binding by the sum of each individual binding (data not shown). In addition, degradation of the heterologous receptor does not seem to be involved in desensitization because the steady-state amount of the receptors, as determined by immunoblot analysis, did not vary throughout the experiments. To further confirm that the receptors were not being degraded but were being recycled to the cell surface, we designed a resensitization assay (Fig. 6). By using cycloheximide to inhibit protein synthesis, we demonstrated that the same receptors that were heterologously desensitized were reexposed to the extracellular environment and that they were capable of binding ligand. In contrast, other studies have shown that EGF and PDGF receptors are internalized and degraded by clathrin-coated pits upon ligand binding (12, 34). The molecular details of what regulates these distinct regulatory mechanisms are unclear. The studies described here used Swiss 3T3 cells, which express relatively small amounts of both EGF and PDGF receptors and large amounts of caveolin. It is possible, although speculative, that the relative amounts of caveolae, clathrin-coated pits, and receptors influence the predominant regulatory mechanism in any given cell.
Even though the radiolabeled ligand-binding studies were consistent
with sequestration in a compartment, the data were also consistent with
earlier reports of a change in receptor affinity, albeit with a much
longer time course (42). Because the receptors associated
with a caveolin-containing membrane, or caveola, after 60 min of ligand
treatment, we used reagents that have been demonstrated to prevent
caveola internalization to determine if sequestration in caveolae was
involved. Filipin and methyl--cyclodextrin alter the amount of
cholesterol associated with caveolae and cause the microdomains to
flatten within the plane of the membrane, thereby preventing
sequestration (3, 8, 26). Previous electron microscopic
studies have conclusively demonstrated that these reagents cause
caveolae to flatten and that caveolin remains associated with these
structures (25). In addition, we used subcellular fractionation methods to confirm that these reagents do not alter the
localization of caveolin or the growth factor receptors in this system
(data not shown). In the presence of filipin or
methyl-
-cyclodextrin, incubation with EGF could not prevent
PDGF-induced receptor phosphorylation, and incubation with PDGF could
not prevent EGF-induced receptor phosphorylation (Fig. 7). It is
important to note that the filipin and methyl-
-cyclodextrin
experiments shown in Fig. 7 do not address any downstream signaling
events. These reagents were only used to look at receptor sequestration
by a methodology not involving radiolabeled ligand binding. Filipin and
methyl-
-cyclodextrin have been used in other studies to examine
growth factor receptor signaling (14, 22, 24), but these
studies did not directly examine the effect of these reagents on
receptor phosphorylation or on heterologous desensitization of multiple
growth factor receptors. Furthermore, the studies that used filipin and
methyl-
-cyclodextrin to study receptor signaling are contradictory,
with some studies showing activation (8) and some
studies showing inactivation (6). The data obtained with
these relatively nonspecific reagents must be interpreted cautiously
because of the potential for substantial nonspecific affects; however,
our data are consistent with a mechanism that prevents ligand binding
to the heterologous receptor by sequestering the receptor from the
extracellular environment.
Radiolabeled ligand-binding studies and pharmacological disruption studies suggested that EGF and PDGF receptors could be sequestered in caveolae. To further address this putative mechanism, we used an antisense approach to reduce the level of caveolin in Swiss 3T3 cells (Fig. 8). Cells that received a scrambled version of caveolin-1 were able to sequester the same amount of radiolabeled ligand as untreated cells (compare Figs. 4 and 8). Cells that only expressed a very small amount of caveolin could not sequester the receptors in an acid-resistant compartment, which is consistent with reports demonstrating that caveolin is required to form invaginated caveolae (7, 32). In addition, the cell line that had about a 50% reduction in caveolin expression could only sequester about one-half of the receptors. Importantly, these studies were independent of any direct effects caveolin may have on growth factor receptor signaling because we only measured the accessibility of the receptors to ligand.
We have presented data demonstrating the heterologous desensitization of EGF and PDGF receptors by sequestration in a cholesterol- and caveolin-dependent manner. The studies described here are consistent with the concept that caveolin and caveolae are involved in inhibiting growth factor receptor signaling. Caveolin-mediated sequestration (the present studies) and direct binding of caveolin to the receptors [other studies (5, 6)] are not necessarily exclusive mechanisms. In fact, it is possible, although speculative, that the interaction of the receptors with caveolin may promote sequestration. In addition, we have provided evidence suggesting that growth factor receptors signal via rafts and not caveolae. Our findings may potentially explain some of the confusion in the literature concerning growth factor localization to caveolae because it is now clear that the available subcellular fractionation methods do not separate bona fide caveolae (contain caveolin) from generic lipid raft-type membranes (caveolin devoid). In addition, the localization of the receptors depends both on the presence of ligand and the length of time the ligand is bound to the receptors. Many questions remain to be answered. Do the receptors move to caveolae or do caveolae form around the receptors? What happens to the bound ligand upon sequestration? What is the relationship between caveola sequestration and clathrin-coated pit endocytosis? These questions and others should be addressed in future studies.
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ACKNOWLEDGEMENTS |
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We thank the Cardiovascular Research Group and William Everson for invaluable advice and assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-62844, HL-58475, and HL-64056 (E. J. Smart).
Address for reprint requests and other correspondence: E. J. Smart, Univ. of Kentucky, Dept. of Physiology, 800 Rose St., MS 508 C, Lexington, KY 40536 (E-mail: ejsmart{at}uky.edu).
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.
10.1152/ajpcell.00349.2001
Received 26 July 2001; accepted in final form 4 December 2001.
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