Heterologous desensitization of EGF receptors and PDGF receptors by sequestration in caveolae

Sergey V. Matveev and Eric J. Smart

University of Kentucky Medical School, Department of Physiology, Lexington, Kentucky 40536


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials. DMEM-low glucose, FBS, L-glutamine, trypsin-EDTA, OptiPrep, and penicillin/streptomycin were from Life Technologies (Grand Island, NY). Percoll, filipin, and methyl-beta -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 · W-1 · 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.

The above procedure enriches for both caveolae and rafts (20). Caveolae were further purified by immunoisolation (see below and Ref. 20). Both caveolae and rafts are defined functionally as cold Triton X-100-insoluble membranes (30). To ensure that the material isolated above was not contaminated with noncaveolae or nonrafts, the caveola/raft fraction was incubated with 1% Triton X-100 or 1% SDS for 20 min at 4°C. The material was then centrifuged at 100,000 g for 1 h to separate detergent-insoluble membranes (pellet) from detergent-soluble membranes (supernatant).

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) beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 1.   Epidermal growth factor (EGF) receptors, platelet-derived growth factor (PDGF) receptors, and caveolin cofractionate. Swiss 3T3 cells were cultured for 12 h in the absence of serum. EGF was then added to a final concentration of 100 ng/ml for 0, 10, or 60 min. A: cells were washed extensively and fractionated in the presence of 2 mM sodium orthovanadate. Equal amounts of protein (15 µg) were resolved by SDS-PAGE, transferred to nylon, and immunoblotted with IgGs for EGF receptors, PDGF receptors, caveolin-1, and phosphotyrosine (PhTyr). Immunoblots were developed by the method of chemiluminescence (60-s exposures). Similar data were obtained when the cells were starved of serum for 2 or 6 h (data not shown). Data are representative of 4 independent experiments. PNS, postnuclear supernatant; CYTO, cytosol; PM, plasma membrane; BPM, bulk plasma membrane; CM/raft, caveolae/rafts. B: caveola/raft fractions in A (EGF, 0 min) were incubated with 1% Triton X-100 or 1% SDS for 20 min at 4°C. The material was then centrifuged at 100,000 g for 1 h to separate detergent-insoluble membranes (pellet) from detergent-soluble membranes (supernatant). The pellets and supernatants were then resolved by SDS-PAGE and immunoblotted with IgGs for EGF receptors, PDGF receptors, and caveolin. Immunoblots were developed by the method of chemiluminescence (60-s exposures). Data are representative of 4 independent experiments. Similar data were obtained when the cells were starved of serum for 2 or 6 h (data not shown). P, pellet; S, supernatant.

Although it has been demonstrated that the caveola/raft isolation procedure used in these studies specifically isolates caveolae and rafts without contamination from other membranes, it is always possible to cross-contaminate subcellular fractions. Therefore, both caveolae and rafts are defined functionally as cold Triton X-100-insoluble membrane domains to ensure that the caveola/raft fraction was not contaminated, but they were incubated with 1% Triton X-100 or 1% SDS for 20 min at 4°C. The material was then centrifuged at 100,000 g for 1 h to separate detergent-insoluble membranes (pellet) from detergent-soluble membranes (supernatant). The pellets and supernatants were then resolved by SDS-PAGE and immunoblotted with IgGs for EGF receptors, PDGF receptors, and caveolin. Figure 1B demonstrates that all of the EGF receptors, PDGF receptors, and caveolin in the caveola/raft fraction were insoluble in 1% Triton X-100 (in the pellet). In contrast, treatment with 1% SDS solubilized all of the EGF receptors, PDGF receptors, and caveolin in the caveola/raft fraction (in the supernatant).

Recent studies have demonstrated that subcellular fractionation methods used to isolate caveolin-containing membranes (caveolae) also isolate caveolin-devoid membranes (rafts; see Ref. 20), calling into question which membrane(s) EGF and PDGF receptors associate with. To determine if EGF and PDGF receptors associate with caveolae and/or rafts, we first isolated a subcellular fraction enriched in caveolae/rafts as described for Fig. 1. With the use of conditions that do not disrupt caveola membranes (20), antibodies to caveolin-1, EGF receptors, and PDGF receptors were used to immunoprecipitate intact membrane domains. As a control to ensure that caveolae were separated from rafts, we also immunoblotted the precipitated material with CD55 antibodies. CD55 is a glycosylphosphatidylinositol (GPI)-anchored protein, and non-cross-linked GPI-anchored proteins have been shown to be associated with rafts but not caveolae (17, 28). Figure 2A demonstrates that caveolin never coprecipitated CD55, indicating that rafts were not precipitated in the assay. In unstimulated cells (no ligand) or cells stimulated with 100 ng/ml EGF for 10 min, caveolin-1 IgG precipitated membranes that contained only minor amounts of EGF or PDGF receptors. However, EGF receptor antibodies precipitated membranes that contained PDGF receptors, and CD55 and PDGF receptor antibodies precipitated membranes that contained EGF receptors and CD55. After 60 min of treatment with EGF, each antibody was capable of precipitating membranes that contained caveolin-1, EGF receptor, and PDGF receptor but not CD55. Immunoblots of the immunoprecipitation supernatant (material that did not precipitate) and densitometric quantification of all the immunoblots (data not shown) indicated that <3% of the EGF and PDGF receptors were in membranes that could be precipitated with caveolin-1 IgG in unstimulated cells. In contrast, 87-94% of the EGF and PDGF receptors was in membranes that precipitated with caveolin-1 IgG after 60 min of growth factor incubation.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2.   Immunoprecipitation of caveolae and phosphorylation of EGF receptor (EGFR) and PDGF receptor (PDGFR). Swiss 3T3 cells were cultured for 12 h in the absence of serum. EGF was then added to a final concentration of 100 ng/ml for the indicated times. A: cells were washed extensively and fractionated to isolate caveolae/rafts. Equal amounts of caveolae/rafts (30 µg) were immunoprecipitated with 5 µg/ml of IgG for caveolin-1 (clone no. 2234), EGF receptor, or PDGF receptor. The precipitates were resolved by SDS-PAGE, transferred to nylon, and immunoblotted with IgGs for PDGF receptor, EGF receptor, caveolin-1, or CD55. Immunoblots were developed by the method of chemiluminescence (60-s exposures). Data are representative of 3 independent experiments. IP, immunoprecipitate. B: Swiss 3T3 cells were cultured for 12 h in the absence of serum and then incubated with EGF (100 ng/ml), PDGF (67 ng/ml), or buffer for the indicated times. Equal amounts of protein from each cell lysate were resolved by SDS-PAGE and immunoblotted with IgGs for EGF receptor, PDGF receptor, or phosphotyrosine. Note that nonphosphorylated EGF receptor, phosphorylated EGF receptor, and PDGF receptor (nonphosphorylated and phosphorylated have the same apparent molecular weight) resolve at different apparent molecular weights. The immunoblots were developed by the method of chemiluminescence (60-s exposures). Data are representative of 3 independent experiments.

To confirm that the addition of EGF was causing phosphorylation of EGF receptors, we used phosphotyrosine antibodies to determine if the receptors were phosphorylated. Figure 2B demonstrates that, in unstimulated cells, neither EGF (~170 kDa) nor PDGF (~185 kDa) receptors were phosphorylated. Treatment of cells with EGF for 10 min resulted in phosphorylation of EGF receptors (~178 kDa), but after 60 min of EGF treatment the EGF receptors were no longer phosphorylated. Note that, after 60 min of exposure to EGF, the EGF receptors were not degraded and were still present in the cells. In addition, treatment of cells with PDGF for 10 min resulted in the phosphorylation of PDGF receptors. In this system, the addition of EGF did not promote PDGF receptor phosphorylation, and the addition of PDGF did not promote the phosphorylation of EGF receptors (Fig. 2). The nonphosphorylated and phosphorylated receptors were identified by specific IgGs and apparent molecular weight in SDS-PAGE.

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.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 3.   Pretreatment with EGF prevents EGF- and PDGF-induced receptor phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 kinase activation. Swiss 3T3 cells were cultured for 12 h in the absence of serum. The cells were incubated with 100 ng/ml EGF or medium for 60 min at 37°C. Cells were then rapidly washed and incubated with medium, 100 ng/ml EGF, or 67 ng/ml PDGF for 10 min at 37°C. Cells were washed, and a cell lysate was prepared in the presence of 2 mM sodium vanadate. Equal amounts of protein (15 µg) were resolved by SDS-PAGE, transferred to nylon, and immunoblotted with IgGs for phosphotyrosine, EGF receptor, PDGF receptor, phospho-ERK1/2 (PhERK1/2), ERK1/2, or actin. Immunoblots were developed by the method of chemiluminescence (60-s exposures). Data are representative of 6 independent experiments. MAPK, mitogen-activated protein kinase.

The binding of ligands to receptors often promotes the internalization of the receptor-ligand complexes. Because EGF and PDGF receptors reside in the same membrane domain in these cells, one possible mechanistic explanation for the above data is that stimulation of EGF receptors with EGF induced the sequestration of both EGF and PDGF receptors from the extracellular environment. To determine whether receptors were sequestered or exposed to the extracellular space, we used radiolabeled ligands to label the receptors. Receptor-ligand complexes that are exposed to the extracellular space can be disrupted by a brief incubation with acid-saline, and sequestered receptor-ligand complexes will not be disrupted by an acid-saline wash (29, 31). Figure 4 demonstrates that temperature (4 or 37°C) did not affect the total amount of 125I-EGF or 125I-PDGF associated with cells. When the radioligands were incubated at 4°C to prevent the formation of sequestered compartments, all of the radiolabeled ligand was stripped by acid-saline (extracellular). In contrast, when the radioligands were incubated at 37°C, the radiolabeled ligand was not stripped by acid-saline (sequestered).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Sequestration of EGF receptors and PDGF receptors. Swiss 3T3 cells were cultured for 12 h in the absence of serum. One set of cells was chilled to 4°C (A and B), and one set of cells was placed at 37°C (C and D). 125I-labeled EGF (A and C) or 125I-labeled PDGF (B and D) was incubated with the cells for 60 min at either 4 or 37°C. Cells were then processed to remove externally exposed ligand, as described in EXPERIMENTAL PROCEDURES. Cells were collected to determine the amount of radiolabel that was resistant to removal by acid-saline. Excess unlabeled ligand prevented the binding of the corresponding radiolabeled ligand, and the ligands were not degraded (data not shown). Findings were confirmed in 3 independent experiments (mean ± SE). dpm, Disintegrations/min.

To determine if the addition of one growth factor could induce both growth factor receptors to be sequestered, we incubated cells with either 100 ng/ml EGF or 67 ng/ml PDGF for 0, 10, or 60 min at 37°C, washed the cells, and chilled the cells to 4°C. Cells that were originally treated with EGF were incubated with 125I-PDGF, and cells originally treated with PDGF were incubated with 125I-EGF for 60 min at 4°C. Figure 5 demonstrates that a 0- or 10-min pretreatment with one ligand was not sufficient to sequester the other receptor (acid-saline sensitive). In contrast, a 60-min pretreatment with unlabeled ligand eliminated binding of the other radiolabeled ligand, which is consistent with sequestration of both receptors (Fig. 5).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   A 60-min pretreatment with either EGF or PDGF promotes the sequestration of both EGF receptors and PDGF receptors. Swiss 3T3 cells were cultured for 12 h in the absence of serum. EGF (100 ng/ml; A) or PDGF (67 ng/ml; B) was then added for 0, 10, or 60 min at 37°C. 125I-PDGF (A) or 125I-EGF (B) was then incubated with the cells for 60 min at 4°C. Cells were processed to remove externally exposed ligand as described in EXPERIMENTAL PROCEDURES. Cells were collected to determine the amount of radiolabel that was resistant to removal by acid-saline. Excess unlabeled ligand prevented the binding of the corresponding radiolabeled ligand, and the ligands were not degraded (data not shown). Findings were confirmed in 3 independent experiments (mean ± SE). * P < 0.01 with respect to 0 min of treatment.

We next determined whether sequestration of the receptors was a reversible process, that is, if the receptors were reexposed to the extracellular environment. The cells were incubated with 50 µg/ml cycloheximide (to inhibit protein synthesis) 60 min before the addition of either 100 ng/ml EGF or 67 ng/ml PDGF for 60 min. The ligands were then removed, and the cells were incubated in medium containing cycloheximide for 10, 30, 60, 90, and 120 min. At the end of the incubation, the cells were chilled to 4°C and then incubated with 125I-EGF (prior treatment with PDGF) or 125I-PDGF (prior treatment with EGF) for 60 min at 4°C. The radioligands would only bind if previously sequestered receptors are exposed to the extracellular environment. Radiolabeled ligands began to associate with cells after 60 min at 37°C and reached control levels by 120 min (Fig. 6). Importantly, cycloheximide was present throughout the experiment so that new protein synthesis was inhibited, thereby suggesting that sequestration is a reversible process.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Sequestration of the receptors is reversible. Swiss 3T3 cells were cultured for 12 h in the absence of serum. Cells were pretreated with 50 µg/ml cycloheximide for 60 min at 37°C and then treated with either 100 ng/ml EGF or 67 ng/ml PDGF for 60 min at 37°C in the continuous presence of cycloheximide. The ligands were then removed, and the cells were incubated in medium containing cycloheximide for 10, 30, 60, 90, and 120 min at 37°C. At the end of the incubation, the cells were chilled to 4°C and then incubated with 125I-EGF (prior treatment with PDGF) or 125I-PDGF (prior treatment with EGF) for 60 min at 4°C. The cells were then processed to remove externally exposed ligand as described in EXPERIMENTAL PROCEDURES. Cells were collected to determine the amount of radiolabel that was resistant to removal by acid-saline. Excess unlabeled ligand prevented the binding of the corresponding radiolabeled ligand, and the ligands were not degraded (data not shown). Findings were confirmed in 3 independent experiments (mean ± SE). * P < 0.01 with respect to 10 min.

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-beta -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-beta -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-beta -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).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Filipin and methyl-beta -cyclodextrin maintain ligand-induced receptor phosphorylation. Swiss 3T3 cells were cultured for 12 h in the absence of serum. Cells were pretreated with medium or 100 ng/ml EGF for 60 min at 37°C. Without removing the EGF, the cells were then incubated for an additional 30 min in the presence or absence of 1 µg/ml filipin or 5 mM methyl-beta -cyclodextrin. Without washing the cells, 100 ng/ml EGF, 67 ng/ml PDGF, or medium only was added for 10 min. Cells were then washed and lysed in the presence of sodium orthovanadate, and the amount of phosphorylated PDGF receptor was determined by phosphotyrosine immunoblot analysis. Findings were confirmed in 3 independent experiments (mean ± SE). * P < 0.01, compare lanes 3-5 with lane 1, lane 10 with lanes 8 or 9, and lane 12 with lanes 8 or 11.

To further confirm that caveolin-containing membranes or caveolae are involved in heterologous receptor sequestration, we generated Swiss 3T3 lines with reduced levels of caveolin-1. The control cell line, 12G2, was stably transfected with the vector containing a nonsensical or "scrambled" caveolin-1 sequence, whereas cell lines 12G4 and 12F6 were transfected with caveolin-1 antisense cDNA. Immunoblot analysis demonstrated that caveolin-1 was reduced ~50% in 12G4 cells and barely detectable in 12F6 cells with respect to control 12G2 cells (Fig. 8A). To determine whether caveolin-1 is involved in EGF and/or PDGF receptor sequestration, the cells were serum starved for 12 h and then incubated with 125I-EGF or 125I-PDGF for 60 min at 4 or 37°C. The cells were then processed to quantify the amount of radiolabeled ligand that was sensitive to an acid wash and the amount of radiolabeled ligand that was resistant to an acid wash (Fig. 8B). The three cell lines associated with similar amounts of acid-sensitive radiolabeled ligand at 4°C. Incubation at 37°C permitted the radiolabeled ligands to become acid resistant in cells containing caveolin (12G2), whereas the radiolabeled ligands remained sensitive to an acid wash in cells expressing minimal amounts of caveolin (12F6). In contrast, about one-half of the radiolabeled ligand became acid resistant in 12G4 cells. Importantly, the expression of caveolin in 12G4 cells was reduced by ~50% compared with control cells.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 8.   Caveolin-1-deficient Swiss 3T3 cells do not sequester EGF receptors or PDGF receptors in the presence of ligands. A: the coding region of caveolin-1 was subcloned in a pCI-neo vector in the antisense orientation. The construct was transfected in Swiss 3T3 cells and cells containing the vector grown in G-418. Selected cells were lysed, and proteins were resolved by SDS-PAGE; the cells were transferred to nylon and immunoblotted with IgG for EGF receptors, PDGF receptors, and caveolin-1. Cell line 12G2 contains the vector and a scrambled caveolin cDNA. Cell lines 12G4 and 12F6 contain the antisense caveolin construct. The immunoblots were developed by the method of chemiluminescence (60-s exposures). The data are representative of 3 independent experiments. B: to determine whether caveolin-deficient cells could sequester EGF and/or PDGF receptors, the cells were cultured for 12 h in the absence of serum then incubated with 125I-EGF (a and c) or 125I-PDGF (b and d) for 60 min at 4°C (a and b) or 37°C (c and d). Cells were then processed to quantify the amount of radiolabeled ligand that was sensitive to an acid wash and the amount of radiolabeled ligand that was resistant to an acid wash. Excess unlabeled ligand prevented the binding of the corresponding radiolabeled ligand, and the ligands were not degraded (data not shown). Findings were confirmed in 3 independent experiments (mean ± SE). * P < 0.01 with respect to cell line 12G2. Open bars, cell line 12G2; hatched bars, cell line 12G4; crosshatched bars, cell line 12F6.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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-beta -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-beta -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-beta -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-beta -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.


    ACKNOWLEDGEMENTS

We thank the Cardiovascular Research Group and William Everson for invaluable advice and assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Bowen-Pope, DF, Dicorleto PE, and Ross R. Interactions between the receptors for platelet-derived growth factor and epidermal growth factor. J Cell Biol 96: 679-683, 1983[Abstract].

2.   Carpenter, G. The EGF receptor: a nexus for trafficking and signaling. Bioessays 22: 697-707, 2000[ISI][Medline].

3.   Chang, WJ, Rothberg KG, Kamen BA, and Anderson RG. Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate. J Cell Biol 118: 63-69, 1992[Abstract].

4.   Conway, A, Rakhit S, Pyne S, and Pyne NJ. Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-src tyrosine kinases and phosphoinositide 3-kinase. Biochem J 337: 171-177, 1999[ISI][Medline].

5.   Couet, J, Li S, Okamoto T, Ikezu T, and Lisanti MP. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272: 6525-6533, 1997[Abstract/Free Full Text].

6.   Couet, J, Sargiacomo M, and Lisanti MP. Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities. J Biol Chem 272: 30429-30438, 1997[Abstract/Free Full Text].

7.   Fra, AM, Williamson E, Simons K, and Parton RG. De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin. Proc Natl Acad Sci USA 92: 8655-8659, 1995[Abstract].

8.   Furuchi, T, and Anderson RG. Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK). J Biol Chem 273: 21099-21104, 1998[Abstract/Free Full Text].

9.   Galbiati, F, Volonté D, Engelman JA, Watanabe G, Burk R, Pestell RG, and Lisanti MP. Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade. EMBO J 17: 6633-6648, 1998[Abstract/Free Full Text].

10.   Jang, IH, Kim JH, Lee BD, Bae SS, Park MH, Suh PG, and Ryu SH. Localization of phospholipase C-gamma 1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation. FEBS Lett 491: 4-8, 2001[ISI][Medline].

11.   Jost, M, Huggett TM, Kari C, and Rodeck U. Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway. Mol Biol Cell 12: 1519-1527, 2001[Abstract/Free Full Text].

12.   Kapeller, R, Chakrabarti R, Cantley L, Fay F, and Corvera S. Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton. Mol Cell Biol 13: 6052-6063, 1993[Abstract].

13.   Lehto, VP. EGF receptor: which way to go? FEBS Lett 491: 1-3, 2001[ISI][Medline].

14.   Liu, J, Oh P, Horner T, Rogers RA, and Schnitzer JE. Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains. J Biol Chem 272: 7211-7222, 1997[Abstract/Free Full Text].

15.   Liu, P, and Anderson RG. Spatial organization of EGF receptor transmodulation by PDGF. Biochem Biophys Res Commun 261: 695-700, 1999[ISI][Medline].

16.   Liu, P, Ying Y, and Anderson RG. Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae. Proc Natl Acad Sci USA 94: 13666-13670, 1997[Abstract/Free Full Text].

17.   Mayor, S, Rothberg KG, and Maxfield FR. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264: 1948-1951, 1994[ISI][Medline].

18.   Mineo, C, Gill GN, and Anderson RG. Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem 274: 30636-30643, 1999[Abstract/Free Full Text].

19.   Mineo, C, James GL, Smart EJ, and Anderson RG. Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane. J Biol Chem 271: 11930-11935, 1996[Abstract/Free Full Text].

20.   Oh, P, and Schnitzer JE. Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage. J Biol Chem 274: 23144-23154, 1999[Abstract/Free Full Text].

21.   Park, WY, Park JS, Cho KA, Kim DI, Ko YG, Seo JS, and Park SC. Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells. J Biol Chem 275: 20847-20852, 2000[Abstract/Free Full Text].

22.   Peiro, S, Comella JX, Enrich C, Martin-Zanca D, and Rocamora N. PC12 cells have caveolae that contain TrkA. J Biol Chem 275: 37846-37852, 2000[Abstract/Free Full Text].

23.   Pike, LJ, and Miller JM. Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover. J Biol Chem 273: 22298-22304, 1998[Abstract/Free Full Text].

24.   Pol, A, Lu A, Pons M, Peiro S, and Enrich C. EGF-mediated caveolin recruitment to early endosomes and MAPK activation. Role of cholesterol and actin-cytoskeleton. J Biol Chem 275: 30566-30572, 2000[Abstract/Free Full Text].

25.   Rothberg, KG, Heuser JE, Donzell WC, Ying Y, Glenney JR, and Anderson RGW Caveolin, a protein component of caveolae membrane coats. Cell 68: 673-682, 1992[ISI][Medline].

26.   Rothberg, KG, Ying Y, Kamen BA, and Anderson RGW Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol 111: 2931-2938, 1990[Abstract].

27.   Schlessinger, J. The epidermal growth factor receptor as a multifunctional allosteric protein. Biochemistry 27: 3119-3123, 1988[ISI][Medline].

28.   Schnitzer, JE, McIntosh DP, Dvorak AM, Liu J, and Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins. Science 269: 1435-1439, 1995[ISI][Medline].

29.   Smart, EJ, Foster DC, Ying YS, Kamen BA, and Anderson RG. Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae. J Cell Biol 124: 307-313, 1994[Abstract].

30.   Smart, EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, and Lisanti MP. Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol 19: 7289-7304, 1999[Free Full Text].

31.   Smart, EJ, Mineo C, and Anderson RG. Clustered folate receptors deliver 5-methyltetrahydrofolate to cytoplasm of MA104 cells. J Cell Biol 134: 1169-1177, 1996[Abstract].

32.   Smart, EJ, Ying Y, Donzell WC, and Anderson RG. A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane. J Biol Chem 271: 29427-29435, 1996[Abstract/Free Full Text].

33.   Smart, EJ, Ying YS, Mineo C, and Anderson RG. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci USA 92: 10104-10108, 1995[Abstract].

34.   Sorkina, T, Bild A, Tebar F, and Sorkin A. Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors. J Cell Sci 112: 317-327, 1999[Abstract/Free Full Text].

35.   Uittenbogaard, A, Shaul PW, Yuhanna IS, Blair A, and Smart EJ. High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae. J Biol Chem 275: 11278-11283, 2000[Abstract/Free Full Text].

36.   Uittenbogaard, A, and Smart EJ. Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae. J Biol Chem 275: 25595-25599, 2000[Abstract/Free Full Text].

37.   Uittenbogaard, A, Ying Y, and Smart EJ. Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking. J Biol Chem 273: 6525-6532, 1998[Abstract/Free Full Text].

38.   Wang, XJ, Liao HJ, Chattopadhyay A, and Carpenter G. EGF-dependent translocation of green fluorescent protein-tagged PLC-gamma 1 to the plasma membrane and endosomes. Exp Cell Res 267: 28-36, 2001[ISI][Medline].

39.   Waugh, MG, Lawson D, and Hsuan JJ. Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains. Biochem J 337: 591-7, 1999[ISI][Medline].

40.   Waugh, MG, Lawson D, Tan SK, and Hsuan JJ. Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes. J Biol Chem 273: 17115-17121, 1998[Abstract/Free Full Text].

41.   Waugh, MG, Minogue S, Anderson JS, Dos Santos M, and Hsuan JJ. Signalling and non-caveolar rafts. Biochem Soc Trans 29: 509-11, 2001[ISI][Medline].

42.   Wrann, M, Fox CF, and Ross R. Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derived growth factor. Science 210: 1363-1365, 1980[ISI][Medline].

43.   Zhang, W, Razani B, Altschuler Y, Bouzahzah B, Mostov KE, Pestell RG, and Lisanti MP. Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). J Biol Chem 275: 20717-20725, 2000[Abstract/Free Full Text].


Am J Physiol Cell Physiol 282(4):C935-C946
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society