Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells

Franco Capozza,1,2 Alex W. Cohen,1,2 Michelle W.-C. Cheung,1,2 Federica Sotgia,1,2 William Schubert,1,2 Michela Battista,1,2 Hyangkyu Lee,1,2 Philippe G. Frank,1,2 and Michael P. Lisanti1,2

1Departments of Molecular Pharmacology and Medicine, Albert Einstein College of Medicine, and 2The Albert Einstein Cancer Center, Bronx, New York

Submitted 11 May 2004 ; accepted in final form 15 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-deficient mouse embryonic fibroblasts, in which we have stably expressed caveolin-1, -2, or -3, we find that caveolin-1 interacts with caveolin-2 in this setting, whereas caveolin-3 does not, in agreement with most published observations. Next, we used a transfected L6 myoblast cell system expressing all three caveolin proteins. Surprisingly, we found that caveolin-1, -2, and -3 all coimmunoprecipitate in this cell type, suggesting that this interaction is muscle cell specific. Similar results were obtained when the skeletal muscle of caveolin-1 transgenic animals was analyzed for caveolin-1 and caveolin-3 coimmunoprecipitation. Thus we conclude that all three caveolins can interact to form a discrete hetero-oligomeric complex, but that such complex formation is clearly muscle specific.

caveolae; caveolin-1; caveolin-2; caveolin-3


FIRST DESCRIBED in the 1950s as abundant plasma membrane invaginations, caveolae are unique well-defined organelles (24, 54). Although they lack an ultrastructurally identifiable coat protein like larger, more discrete clathrin-coated pits, their characteristic flask-shaped necks make them easily distinguishable by electron microscopy. Caveolae are found in many cell types, being most abundant in terminally differentiated cells such as epithelia, endothelia, adipocytes, and skeletal and cardiac myocytes (45). While these structures have been the subject of intense study for nearly 50 years, their multiple roles in cellular metabolism, trafficking, and signal transduction are just now beginning to be elucidated.

Caveolae are a subset of specialized liquid-ordered domains, referred to as lipid rafts, which are uniquely enriched in various membrane components, such as cholesterol, sphingolipids, and glycosyl phosphatidylinositol-anchored proteins (5, 17, 22). The identification of the principal structural components of caveolae, the caveolin protein family, has provided biochemical markers for the study of these microdomains (32, 37, 50). In recent years it has become well established that caveolae are multifunctional organelles, playing important roles in a variety of cellular processes mainly through interactions with one of the caveolin proteins and a multitude of known signaling molecules, such as heterotrimeric G proteins, Src family tyrosine kinases, H-Ras, endothelial nitric oxide synthase, and the insulin receptor (23, 30, 51).

Caveolar biogenesis, or the formation of invaginations from otherwise flat plasma membrane lipid rafts, has been the subject of intense study. It is well known that this process is highly dependent on at least two molecules, cholesterol and caveolin, because treatments to eliminate either one of these players result in the loss of identifiable caveolae (42). The specific caveolin proteins involved in the formation of caveolae are known to be tissue specific, with caveolin-3 (Cav-3) being expressed in all myocytic cells, whereas Cav-1 and Cav-2 are coexpressed in most other cell types (36, 50). Expression of either Cav-1 or -3 is sufficient to drive caveolae formation, whereas the sole expression of Cav-2 is not only insufficient to support caveolar biogenesis but also insufficient to support its own stability. For example, it has been demonstrated that Cav-2 requires the expression of Cav-1; in the absence of Cav-1, Cav-2 is rapidly degraded by proteasomal mechanisms (28). Both Cav-1 and -3 form functional oligomeric complexes that insert into the plasma membrane, driving the formation of these flask-shaped organelles (29).

Interestingly, smooth muscle cells are the only adult cell type that coexpresses all three caveolin isoforms (Cav-1, -2, and -3) (47). However, only arterial smooth muscle cells coexpress all three caveolin isoforms; in contrast, venous smooth muscle cells specifically lack Cav-3 expression, but retain the coexpression of Cav-1 and -2 (43). On the basis of these findings, Sessa and colleagues (43) have postulated that Cav-3 may have a very specialized function in smooth muscle cells, possibly to maintain the contractile phenotype of arterial vascular smooth muscle cells. In accordance with this hypothesis, uterine smooth muscle cells fail to express detectable levels of Cav-3 in the nonpregnant state (13). However, bladder smooth muscle cells coexpress all three caveolins (53) and perform an essential repeated contractile function.

While the interaction between Cav-1 and -2 has been explored and verified and is now readily accepted, the ability of Cav-3 to interact with either Cav-1 or -2 has remained controversial. Several attempts have been made to analyze the potential Cav-1, -2, and-3 interaction in cell culture models, with varied results. Das and colleagues (2) demonstrated that while Cav-2 can be coimmunoprecipitated with antibodies directed against Cav-1 in Cos-7 cells, similar results could not be obtained with the immunoprecipitation (IP) of Cav-3; thus Cav-2 failed to coimmunoprecipitate with Cav-3 in this cellular context. Furthermore, it has also been shown that whereas transient transfection of Cav-2 causes an upregulation of endogenous Cav-1 in Chinese hamster ovary cells, the same is not true when Cav-3 is transfected (25). This finding supports the contention that Cav-2 interacts with and can stabilize Cav-1, whereas Cav-3 does not form a complex with Cav-1.

More recently, however, findings reported by Rybin et al. (33) challenge this notion, as these authors found Cav-2 expression in cultured cardiac myocytes. They further asserted that previous reports on the subject were premature in dismissing the presence of Cav-2 in myocyte cultures as originating from contaminating fibroblasts, and that Cav-2 was indeed expressed in their cardiac muscle cell cultures. Furthermore, these authors reported that Cav-2 coimmunoprecipitates with Cav-3 as well as cofractionates with Cav-3 by sucrose density centrifugation, thus indicating that these two caveolin proteins are capable of interacting in cultured myocytes (33). While this is the first report showing a Cav-2/-3 interaction, it is in direct contention with most other findings on the subject and thus leaves the issue unresolved.

In the current study, we employ three distinct genetic approaches to analyze the potential Cav-1/-2/-3 interaction. We first fully explored the interaction between Cav-1, -2, and -3 in Cav-1-null 3T3 mouse embryonic fibroblasts (MEFs), a fibroblast cell line deficient in the Cav-1 gene, using a variety of methods. We show that whereas stable transfection of these cells with Cav-3 does not rescue expression of Cav-2, stable transfection with Cav-1 does rescue Cav-2 levels, as predicted. Also, with the use of well-established assay systems, such as sucrose density centrifugation, coimmunoprecipitation, and solubility in cold Triton X-100, we demonstrate that Cav-3 does not interact with either Cav-1 or -2 in these fibroblastic cells.

To further dissect this issue, we next explored potential Cav-1, -2, and -3 interactions in a Cav-3-transfected myoblast cell line. Careful examination reveals that Cav-2, as well as Cav-1, coimmunoprecipitate with Cav-3 in this setting. These observations suggest that an interaction does indeed exist between Cav-1, -2, and -3 and that this interaction is muscle cell-type specific. To extend these observations to an in vivo situation, we next studied skeletal muscle samples from Cav-1 transgenic (Tg) mice, which overexpress Cav-1 in all tissues, including skeletal muscle. Our coimmunoprecipitation experiments clearly demonstrate that Cav-1 does indeed form a tight complex with Cav-3, thus confirming that a muscle-specific interaction occurs between the caveolins.

These data, indicating a cell-type-specific interaction between the caveolin protein family members, hold numerous functional implications for muscle cell development and differentiation (see DISCUSSION).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Antibodies and their sources were as follows: anti-Cav-1 monoclonal antibodies (MAb) (clones 2297 and 2234), anti-Cav-2 MAb (clone 65), anti-Cav-3 MAb (clone 26) (gifts of Dr. Roberto Campos-Gonzalez, BD Pharmingen/Transduction Laboratories) (36, 38, 47); anti-Flotillin-1 MAb (BD Pharmingen/Transduction Laboratories) (52); anti-Cav-1 (N-20) polyclonal antibody (PAb) and anti-c-Myc MAb and PAb (Santa Cruz Biotechnology); anti-Cav-2 PAb and anti-Cav-3 PAb (gift of Dr. Brent Rollman, Affinity Bioreagents); anti-actin MAb and Hoechst-33258 were from Sigma. Cell culture reagents were from GIBCO-BRL.

MEF culture. Primary MEFs were obtained from day 13.5 embryos and immortalized as previously described (28). Immortalized 3T3 MEFs were grown in complete medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO-BRL).

Cav-1, -2, and -3 and retroviral infection. The cDNAs encoding full-length Cav-1, -2, and -3 genes were subcloned into the pBabe-Puro retroviral expression vector (21) using a standard PCR-based strategy. It is important to note that these cDNAs were not epitope tagged. Stable infection of 3T3 MEFs and rat L6 myoblasts was conducted essentially as previously described (12, 14). Briefly, pBabe vectors were transiently transfected into the ecotrophic packaging cell line, Phoenix, using a modified calcium phosphate method (12, 14). Forty-eight hours after transfection, the viral supernatant was collected, filtered, and added to the target cells. Two infection cycles were carried out (every 12 h). After the last cycle of infection, cells were selected for 5 days in complete medium containing puromycin at a final concentration of 2.5 µg/ml. Stable expression in the target cell population was confirmed by Western blot analysis.

Transmission electron microscopy. 3T3 MEFs were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixed with OsO4, and stained with uranyl acetate and lead citrate. After being processed, the samples were examined under a transmission electron microscope (model 1200EX; JEOL) and photographed at a magnification of x16,000. Caveolae were identified by their characteristic flask shape, size (50–100 nm), and location at or proximal to the plasma membrane (15, 19).

Immunoblot analysis. Cells were cultured in complete medium and allowed to reach 80–90% confluence. Subsequently, they were washed with PBS and incubated with lysis buffer (10 mM Tris, pH 7.5; 50 mM NaCl; 1% Triton X-100; 60 mM n-octylglucoside) containing protease inhibitors (Roche Molecular Biochemicals). When necessary, protein concentrations were determined using the bicinchoninic acid reagent (Pierce). Proteins were separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S, followed by immunoblot analysis. For all subsequent washing, the buffers contained 10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20, which was supplemented with 1% bovine serum albumin (BSA) and 4% nonfat dry milk (Carnation) for the blocking solution and 1% BSA for the antibody dilution. Horseradish peroxidase-conjugated secondary antibodies were used to visualize bound primary antibodies with the Supersignal chemiluminescence substrate (Pierce).

Triton X-100 insolubility. Extraction of Triton X-100 soluble proteins was performed essentially as we described previously in detail (39, 40). Briefly, cells were grown to confluence in 35-mm-diameter dishes and washed twice with ice-cold PBS. An ice-cold buffer (25 mM Mes, pH 6.5, 150 mM NaCl) containing 1% Triton X-100 plus protease inhibitors (1 ml) was gently added to the cells. After a 30-min incubation period on ice without agitation, the soluble fraction was then collected from the edge of the dish (without scraping the cells) using a 1-ml micropipette (blue tip). An equal volume of 2% SDS (1 ml) was then added to the plate to dissolve the remaining Triton X-100-insoluble material. The SDS fraction was collected using a cell scraper and briefly sonicated to decrease its viscosity. Equal volumes of the Triton X-100 soluble and insoluble fractions were then each separated by SDS-PAGE and subjected to immunoblot analysis, as described above.

Purification of caveolae-enriched membrane fractions. Caveolae enriched membrane fractions were purified essentially as previously described (35, 40). Cells grown to confluence in two 100-mm-diameter plates were washed twice in ice-cold PBS, scraped into 750 µl of Mes-buffered saline (MBS) (25 mM Mes, pH 6.5, 150 mM NaCl) containing 1% Triton X-100, passed five times through a tightly fitting Dounce homogenizer, and mixed with an equal volume of 80% sucrose prepared in MBS lacking Triton X-100. The sample was then transferred to a 4.5-ml ultracentrifuge tube and overlaid with a discontinuous sucrose gradient (1.5 ml of 30% sucrose, 1.5 ml of 5% sucrose, both prepared in MBS lacking detergent). The samples were then subjected to centrifugation at 200,000 g (44,000 rpm in a Sorval rotor TH-660) for 18 h. A light-scattering band was observed at the 5%/30% sucrose interface. Twelve 375-µl fractions were collected, and 50-µl aliquots of each fraction were subjected to SDS-PAGE and immunoblot analysis.

Velocity gradient centrifugation. Velocity gradient centrifugation was conducted as we have described (34, 37). Cells were grown to confluence in 10-cm dishes and dissociated in MBS (25 mM Mes, pH 6.5, 0.15 M NaCl) containing 60 mM n-octylglucoside. Soluble material was loaded on top of a 5–40% linear sucrose gradient and centrifuged at 50,000 rpm for 10 h in a SW 60 rotor (Beckman). Gradient fractions were collected from above and subjected to immunoblot analysis as described.

Immunofluorescence microscopy. Immunofluorescence was performed as described previously (11). For coimmunolocalization of Cav-2 and -3, Myc-tagged murine Cav-2 was transiently transfected into Cav-1-null cells stably expressing Cav-3 using Lipofectamine Plus (Invitrogen). Forty-eight hours after transfection, cells were processed for immunofluorescence. 3T3 MEFs were grown on glass coverslips, washed three times with PBS, and fixed for 30 min at room temperature with 2% paraformaldehyde. Fixed cells were washed with PBS and permeabilized in washing buffer (PBS containing 0.1% Triton X-100 and 0.2% BSA) for 10 min. The cells were then treated with 25 mM NH4Cl in PBS for 10 min at room temperature to quench any free aldehyde groups. The cells were rinsed with PBS and incubated with the primary antibody for 1 h at room temperature. After three washes with washing buffer (10 min each), the cells were incubated with the secondary antibody for 1 h at room temperature. Finally, the cells were washed three times with washing buffer (10 min each wash) and counterstained with Hoechst-33258 (1 µg/ml) for 15 min to visualize the nucleus. After extensive washing in PBS, slides were mounted with the Slow-Fade reagent (Molecular Probes, Eugene, OR) and observed under a confocal microscope (model MR 600; Bio-Rad). All microscopy was performed at the Analytical Imaging Facility of the Albert Einstein College of Medicine.

Coimmunoprecipitation studies. IP was performed as previously described (41). Briefly, 3T3 MEFs and L6 myoblasts grown to near confluence were washed twice with cold PBS and scraped into 1 ml of IP buffer [10 mM Tris (pH 8.0), 150 mM NaCl, 1% (vol/vol) Triton X-100, 60 mM n-octylglucoside], supplemented with protease inhibitors. For IP from skeletal muscle, mouse tissue was harvested, minced with scissors, homogenized in a Polytron tissue grinder for 30 s, and solubilized in IP buffer containing protease and phosphatase inhibitors. After incubation on ice for 30 min, debris was removed by centrifugation at 13,000 rpm for 10 min. Proteins (500 µg) were precleared by incubation with 30 µl of 1:1 slurry of protein A-Sepharose (Amersham Pharmacia Biotech) for 45 min at 4°C and then transferred to tubes containing fresh protein A-Sepharose and IP buffer. Anti Cav-1 and -3 MAbs were added to the mixture. For coimmunoprecipitation studies, rabbit anti-GFP IgG (FL) or mouse anti-c-Myc IgG were used as negative controls. After a 4-h or overnight incubation at 4°C, immune complexes were collected by centrifugation, washed five times in 1 ml of IP buffer, washed four times with 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1% (vol/vol) Triton X-100, and disrupted by boiling in 1% (wt/vol) SDS. Immune complexes were then resolved by SDS-PAGE and processed for immunoblotting as described above.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Retroviral transduction of wild-type and Cav-1-null MEFs. Embryonic fibroblast cell lines were developed from wild-type and Cav-1-null mouse embryos and subsequently immortalized using a defined 3T3 protocol (28); these cells are henceforth referred to as 3T3 MEFs to indicate their immortalization. Because Cav-1-null 3T3 MEFs are deficient in Cav-1, have been shown to express very low levels of Cav-2 (a 95% reduction compared with WT levels), and do not express Cav-3, they provide a novel system in which to explore complementation studies with either Cav-1 or -3 (28, 49).

However, preliminary studies (28) have indicated that these cells, like other MEF cell lines, are resistant to standard transfection protocols, thus limiting their usefulness. Therefore, we chose to overcome this issue by using a highly efficient retroviral expression vector, which allows for stable integration into the target cells. Thus the full-length untagged cDNAs encoding Cav-1, -2, or -3 were subcloned into the pBabe-puro retroviral expression vector and transfected into the ecotrophic packaging cell line, Phoenix. Virus-containing supernatants were filter-purified from infected cells and used to transduce wild-type and Cav-1-null 3T3 MEFs (12, 14). Stable cell lines were generated by selection in medium containing puromycin and analyzed for integration and expression of the desired vector by Western blot analysis (Fig. 1). Note that similar levels of expression were observed for all three proteins. In addition, stable expression of Cav-3 in wild-type 3T3 MEFs did not affect the expression of endogenous Cav-1 (data not shown).



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Fig. 1. Stable retroviral transduction of wild-type (WT) and caveolin-1 (Cav-1) null 3T3 mouse embryonic fibroblasts (MEFs). WT and Cav-1-deficient (knockout; KO) 3T3 MEFs were used to generate stable cell lines recombinantly expressing either Cav-1, -2, or -3 by standard retroviral techniques. After selection in puromycin, stable expression was analyzed by Western blot (WB) analysis with antibodies directed against Cav-1, Cav-2, and Cav-3. Note that recombinant expression of Cav-1 and -2 is at physiological levels, that is, equivalent to that of WT 3T3 MEFs.

 
Expression of Cav-1 or -3 but not of Cav-2 restores the ability of Cav-1-null 3T3 MEFs to form caveolae. It has previously been demonstrated that primary MEFs derived from Cav-1-null animals, as well as NIH3T3 cells transfected with anti-sense Cav-1, or overexpressing Ha-Ras or v-Abl (conditions that downregulate Cav-1 expression) are unable to form caveolae (8, 28). In addition, recombinant expression of Cav-1 or -3 in the Sf21 insect cells has been shown to drive the formation of caveolae-like vesicles (1618).

Thus we next examined the plasma membrane of confluent transfected 3T3 Cav-1-null MEFs for the presence of caveolae by transmission electron microscopy. As expected, Cav-1-null 3T3 MEFs do not form recognizable caveolae, whereas the introduction of either Cav-1 or Cav-3 is sufficient to restore the formation of these organelles (Fig. 2, arrows). In addition, overexpression of Cav-2 in Cav-1-null 3T3 MEFs is not sufficient to drive caveolae biogenesis (Fig. 2).



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Fig. 2. Electron photomicrographs of Cav-1-null 3T3 MEFs transduced with Cav-1, -2, or -3. Stable cell lines were grown to confluence on 60-mm dishes and prepared for transmission electron microscopy, as described in MATERIALS AND METHODS. All analyses were performed at a magnification of x6,000. Note that the recombinant expression of Cav-1 (KO + Cav-1) and Cav-3 (KO + Cav-3) restores the ability of Cav-1 null cells (KO) to form caveolae, whereas the expression of Cav-2 (KO + Cav-2) does not allow for the formation of caveolae. Arrows indicate caveolae organelles. Arrowheads indicate the more dense clathrin-coated vesicles/pits.

 
Cav-2 expression is selectively rescued by Cav-1, but not by Cav-3, in Cav-1-null 3T3 MEFs. It has previously been reported that primary MEFs derived from Cav-1-null mice demonstrate greatly reduced levels of the Cav-2 protein (28). This was shown to be due to enhanced proteasomal degradation of Cav-2, as inhibition of this machinery rescues the expression of Cav-2; thus Cav-1 expression is necessary for the stabilization of Cav-2 (28).

To determine whether Cav-3 could functionally play a similar role in rescuing Cav-2 from degradation, we analyzed the Cav-2 protein content of Cav-1-null 3T3 MEFs recombinantly overexpressing Cav-3. Western blot analysis of these cells demonstrates that overexpression of Cav-3 is not sufficient to rescue Cav-2 protein levels (Fig. 3). Importantly, control experiments, in which Cav-1 was reintroduced into Cav-1-null 3T3 MEFs, demonstrate that Cav-1 overexpression does indeed result in the rescue of Cav-2 (Fig. 3). Western blot analysis for Cav-2 in Cav-2-overexpressing cells is shown for comparison. Note that in this case a near normal level of Cav-2 is achieved; however, Cav-2 is still confined to the Golgi as described below (Fig. 8B).



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Fig. 3. Cav-2 protein expression is rescued by Cav-1 expression, but not by Cav-3 expression, in Cav-1 null 3T3 MEFs. Cav-1 null 3T3 MEFs (KO) recombinantly expressing Cav-1, -2, or -3 were analyzed for the expression of Cav-2. Western blot analysis demonstrates that in KO MEFs, Cav-2 expression is greatly decreased, whereas reintroduction of Cav-1 (KO + Cav-1) results in a rescue of Cav-2 expression to levels similar to those seen in WT cells. In contrast, recombinant expression of Cav-3 in these cells (KO + Cav-3) does not rescue Cav-2 expression, because the levels of Cav-2 are comparable to those seen in Cav-1 null cells. Cav-2-overexpressing cells (KO + Cav-2) are shown for comparison. Actin immunoblotting is shown as a control for equal protein loading.

 


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Fig. 8. Expression of Cav-1, but not Cav-3, shifts Cav-2 immunostaining from the perinuclear region to the plasma membrane. WT, KO, and Cav-1 null 3T3 MEFs expressing Cav-1 (KO + Cav-1), Cav-2 (KO + Cav-2), or Cav-3 (KO + Cav-3) were fixed and stained with antibodies as indicated. Similarly, WT 3T3 MEFs recombinantly expressing Cav-3 (WT + Cav-3) were also processed in parallel. All images were obtained using confocal microscopy, unless otherwise indicated. A: WT and KO. Note that Cav-1 (green) and -2 (red) demonstrate extensive colocalization at the plasma membrane in WT cells and that in Cav-1 null cells, Cav-2 shows perinuclear staining, indicative of its being trapped in the Golgi, as expected. B: KO + Cav-1 or Cav-2. The reintroduction of Cav-1 (green) into Cav-1 null cells rescues Cav-2 (red) from its perinuclear confines and allows plasma membrane localization (arrows). In contrast, retroviral transduction of Cav-2 into Cav-1 null MEFs has no effect on the perinuclear distribution of Cav-2. C: KO + Cav-3. Unlike Cav-1, transduction of Cav-3 (green) into Cav-1 null cells does not cause redistribution of Cav-2 (red) to the plasma membrane, indicating that Cav-2 and -3 do not interact in MEFs. Top, confocal images; bottom, images obtained using a charge-coupled device (CCD) camera. In the CCD images, the nucleus is labeled with Hoechst-33258 (blue). D: WT + Cav-3. Dual labeling for Cav-1 (green) and Cav-3 (red) in WT MEFs recombinantly expressing Cav-3 reveals that these two proteins show extensive colocalization, at the level of the plasma membrane. E: WT + Cav-3, same as in D, except a series of four individual confocal slices are shown, from the bottom to the top of the cell. In AE, the nuclei are labeled N. Merged images are also shown, where appropriate.

 
Expression of Cav-3 does not confer Triton X-100 insolubility upon Cav-2. An important distinguishing characteristic of caveolae, lipid rafts, and their constitutive protein components is their insolubility in the detergent Triton X-100 at 4°C. This physical property reflects the high concentration of saturated sphingolipids and cholesterol in these microdomains (4, 22). Cav-1 and -3 can independently target to caveolae, whereas Cav-2 cannot; thus Cav-2 generally remains soluble in Triton X-100 at 4°C, unless chaperoned to plasmalemmal caveolae by Cav-1.

To determine whether Cav-3 could functionally substitute for Cav-1 in this regard, we performed Triton X-100 solubility measurements on Cav-1-null 3T3 MEFs stably expressing Cav-3. Figure 4A shows that in these cells, Cav-2 remains in the soluble fraction, whereas in Cav-1-null 3T3 MEFs overexpressing Cav-1, as well as in wild-type MEFs, a major portion of Cav-2 localizes to the Triton X-100 insoluble fraction. As expected, Cav-1 and Cav-3 are both insoluble, thus indicating that Cav-2 follows the behavior of Cav-1, but not Cav-3 (Fig. 4B). However, Cav-3 is not quite as Triton insoluble as Cav-1; this may be due to differences in the primary sequence of Cav-3 that alters its affinity for cholesterol-rich lipid raft membrane domains. Most importantly, these data demonstrate that, in a fibroblast system, Cav-3 does not confer Triton X-100 insoluble characteristics upon Cav-2.



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Fig. 4. Expression of Cav-1, but not Cav-3, recruits Cav-2 to the Triton X-100 insoluble fraction. WT, KO, and Cav-1 null 3T3 MEFs expressing Cav-1 (KO + Cav-1) or Cav-3 (KO + Cav-3) were grown to confluence and lysed in ice-cold buffer containing Triton X-100. After incubation on ice for 30 min, the Triton X-100 soluble and insoluble fractions were separated and resolved by SDS-PAGE. A: Western blot analysis of WT cell lysates with anti-Cav-2 antibodies shows the normal distribution of this protein in both the soluble and insoluble fraction. In Cav-1 deficient cells, Cav-2 is found only in the soluble fraction. Note that in Cav-1 null cells, recombinant expression of Cav-1, but not Cav-3, results in the recruitment of Cav-2 into the Triton-insoluble fraction. B: Western blot analysis with anti-Cav-1 or anti-Cav-3 IgG reveals that Cav-1 and Cav-3 are found to localize predominantly to the Triton X-100 insoluble fraction, as expected.

 
Cav-2 is not targeted to lipid rafts/caveolae in the presence of Cav-3. Isolation of caveolae membrane microdomains and their resident protein components can be achieved by ultracentrifugation on an equilibrium sucrose density gradient because of their relative buoyant properties or their ability to "float." It has previously been shown that caveolae-enriched fractions isolated in this manner exclude markers for non-caveolar membranes, such as the Golgi apparatus, mitochondria, and the endoplasmic reticulum (19, 48). Similar to its Triton X-100 solubility, the targeting of Cav-2 to the buoyant membrane fraction is dependent upon its ability to localize to these domains, and is normally determined by an interaction with Cav-1. To determine whether Cav-3 possesses similar chaperoning characteristics and is able to confer buoyant properties upon Cav-2, we performed sucrose density centrifugation on Cav-1-null 3T3 MEFs recombinantly overexpressing Cav-3.

The results presented in Fig. 5 indicate that Cav-2 correctly targets to caveolae (fractions 5 and 6) in wild-type cells or Cav-1-null 3T3 MEFs overexpressing Cav-1, as expected (Fig. 5, A and C). Furthermore, in Cav-1-null 3T3 MEFs, Western blot analysis for Cav-2 reveals that this protein does not "float" without the presence of Cav-1, as expected (Fig. 5B). In addition, the overexpression of Cav-2 is not sufficient to localize this protein to buoyant membranes (Fig. 5D). In contrast to the results obtained with stable transfection of Cav-1, overexpression of Cav-3 in Cav-1-null 3T3 MEFs demonstrates that Cav-2 is not recruited to caveolae membrane microdomains by Cav-3 (Fig. 5E). Taken together with the results presented above, these data strongly argue that, in 3T3 MEFs, Cav-3 does not interact with Cav-2 in mouse embryonic fibroblasts.



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Fig. 5. The buoyant density characteristics of Cav-2 are dependent on the expression of Cav-1, but not Cav-3, in 3T3 MEFs. Lysates from WT, Cav-1-null, and Cav-1-null 3T3 MEFs expressing Cav-1, -2, or -3 were prepared and subjected to sucrose density gradient centrifugation to assess interactions between the caveolin proteins. Twelve equal fractions were collected, of which fractions 5 and 6 are considered caveolar, whereas fractions 8–12 are considered to be of noncaveolar origin. Western blot analysis reveals that, in WT (A) and Cav-1 null cells reexpressing Cav-1 (C), Cav-1 and -2 target to the caveolar fractions, as expected. In Cav-1 null cells (B), as well as Cav-1 null 3T3 MEFs expressing Cav-2 (D), Cav-2 does not target to caveolar fractions, also as expected. Most importantly, expression of Cav-3 in Cav-1 null cells does not recruit Cav-2 into the caveolar fractions (E), thus indicating that these two proteins do not stably interact. In addition, note that flotillin-1 always targets the lipid raft/caveolar fractions (5 and 6), independently of caveolin expression (AE). Note that in B and E, lanes showing Cav-2 expression in KO alone and KO + Cav-3 cells are intentionally overexposed. This was done to allow the reader to better compare the buoyancy of Cav-2 relative to the other genotypes.

 
Recently, flotillins have been described as a new family of proteins associated with caveolae (1, 9). Overexpression of these proteins in insect cells can drive the formation of caveolae-like vesicles, which are larger than caveolae (52). Like caveolae, these related domains are dramatically enriched in cholesterol, sphingolipids, and lipid modified signaling molecules (44). In the present study, we examined the effect of caveolin proteins on flotillin localization by sucrose density centrifugation. Our results indicate that flotillin-1 always targets to the lipid raft/caveolae fractions (fractions 5 and 6), independent of the presence of Cav-1, -2, or -3 (Fig. 5, AD). In addition, recombinant expression of Cav-1, -2, or -3 in Cav-1-null 3T3 MEFs did not affect the overall expression levels of flotillin-1 (data not shown).

Oligomerization of Cav-2 is partially rescued by the introduction of Cav-3 in Cav-1-null 3T3 MEFs. Another important property of the caveolin proteins is their ability to form functional oligomeric complexes (34). After synthesis in the endoplasmic reticulum, Cav-1 can form either homo-oligomers or hetero-oligomers with Cav-2 composed of 14–16 individual caveolin monomers. Cav-3 normally forms only homo-oligomeric complexes, as it is generally thought to be the sole caveolin expressed in muscle cells. In the Golgi apparatus, adjacent hetero-oligomers of Cav-1 and -2 undergo a second stage of oligomerization through contacts between the COOH- terminal domains of Cav-1 proteins (2, 36, 39, 48). This second step of oligomerization leads to the formation of a caveolin-rich scaffold. Studies (2, 36, 39, 48) employing site-directed mutagenesis have demonstrated that residues 61–101 of Cav-1 are involved in the first step of oligomerization, whereas residues 168–178 are involved in the second step. It remains unknown however, whether Cav-3, which shares 85% homology to Cav-1, can induce the formation of Cav-2-containing high molecular mass complexes. The oligomeric state of any caveolin protein can be assessed using a well-established velocity gradient centrifugation system (34, 37, 50).

Here, we utilize this system to determine whether overexpression of Cav-3 in Cav-1-null 3T3 MEFs can lead to the formation of oligomeric Cav-2-containing complexes. Figure 6, A and C, shows that Cav-2 is found in high molecular mass oligomers of 200–400 kDa (fractions 6 and 7) in presence of Cav-1 (in wild-type 3T3 MEFs and Cav-1-overexpressing Cav-1-null 3T3 MEFs), as expected. In the absence of Cav-1, endogenous Cav-2 fails to form high molecular mass oligomers, also as expected (Fig. 6B). In addition, overexpression of Cav-2 is not sufficient to induce the formation of high molecular mass oligomers (Fig. 6D). Interestingly, we observe that recombinant expression of Cav-3 in Cav-1-null 3T3 MEFs partially restores the ability of endogenous Cav-2 to form high molecular mass oligomers (Fig. 6E).



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Fig. 6. Oligomerization of Cav-2 is partially rescued by the expression of Cav-3. Velocity gradient sedimentation was used to analyze the oligomerization state of the various caveolins in WT, Cav-1 null, and transfected Cav-1-null 3T3 MEFs. As expected, high-mass Cav-2-containing oligomers are observed in WT (A) and Cav-1 null 3T3 MEFs reexpressing Cav-1 (C), but not in Cav-1 null cells (B) or Cav-1 null 3T3 MEFs stably expressing Cav-2 (D). Surprisingly, overexpression of Cav-3 results in partial rescue of Cav-2 oligomerization (E), indicating that Cav-3 allows for Cav-2 high molecular mass complex formation to occur. However, this does not appear to be due to a stable interaction, as revealed by coimmunoprecipitation studies (Fig. 7). Note that in B and E, lanes showing Cav-2 expression in KO alone and KO + Cav-3 cells are intentionally overexposed. This was done to allow the reader to better compare the oligomeric state of Cav-2, relative to the other genotypes.

 
One possible explanation for this partial complementation of Cav-2 oligomerization is that Cav-3 transiently interacts with Cav-2 at the level of the endoplasmic reticulum/Golgi. However, all of the other parameters tested indicate that this is not a stable interaction (i.e., failure to rescue Cav-2 total protein expression, Triton insolubility, and caveolar targeting, as well as immunolocalization and coimmunoprecipitation studies; see Figs. 3, 4A, 5E, 7, A and B, and 8C).



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Fig. 7. Cav-2 coimmunoprecipitates (co-IPs) with Cav-1 but not Cav-3. Lysates from wild-type MEFs (WT), WT transfected with Cav-3 (WT + Cav-3), and Cav-1-null MEFs transfected with Cav-1 (KO + Cav-1) were immunoprecipitated with antibodies directed against either Cav-1 or Cav-3 and then subjected to Western blot analysis. A: immunoblotting of WT, KO + Cav-1, and KO + Cav-3 lysates with anti-Cav-2 antibodies demonstrates that Cav-1, but not Cav-3, forms measurable protein complexes with Cav-2. B: IP and Western blot analysis of lysates derived from WT and WT + Cav-3 cells reveals that Cav-1 and Cav-3 do not interact in MEFs.

 
Alternatively, Cav-3 expression may alter cholesterol transport in Cav-1-null cells, thereby indirectly influencing Cav-2 oligomerization. In support of this hypothesis, caveolins are known cholesterol-binding proteins (18, 22) that can promote cholesterol transport from the endoplasmic reticulum to the plasma membrane (46). In addition, we (6) have previously demonstrated that cholesterol loading of cells stabilizes the Cav-1 protein product, dramatically increasing its steady-state expression levels in transiently transfected cells. Similarly, cholesterol loading of cells also stabilizes the Cav-2 and Cav-3 protein products (unpublished observations). Finally, Kurzchalia and colleagues (20) have shown that cholesterol promotes and/or stabilizes the formation of Cav-1/2 hetero-oligomers.

Neither Cav-1 nor -2 coimmunoprecipitates with Cav-3 in 3T3 MEFs. To further explore possible interactions between the three caveolin proteins, we next performed coimmunoprecipitation studies. Protein lysates were prepared from wild-type, untransfected Cav-1-null, and Cav-1-null 3T3 MEFs overexpressing each of the caveolin proteins. Lysates were immunoprecipitated with either an anti-Cav-1 or anti-Cav-3 IgG and then subjected to SDS-PAGE and subsequent immunoblot analysis with antibodies directed against Cav-2.

The results presented in Fig. 7A demonstrate that endogenous Cav-2 is coimmunoprecipitated with antibodies directed against Cav-1 in wild-type 3T3 MEFs, as well as in Cav-1-null 3T3 MEF stably overexpressing Cav-1. However, in striking contrast, Cav-2 does not coimmunoprecipitate when an antibody directed against Cav-3 is used in Cav-1-null 3T3 MEFs overexpressing Cav-3. This again indicates that Cav-2 and -3 do not interact in this fibroblast system. As important negative controls, protein A-sepharose beads alone or an irrelevant IgG antibody showed no coimmunoprecipitation of Cav-2 (Fig. 7A).

In this experiment, we also evaluated a possible interaction between Cav-1 and -3 by coimmunoprecipitation. Protein lysates from wild-type 3T3 MEFs and wild-type 3T3 MEFs recombinantly expressing Cav-3 were immunoprecipitated with monoclonal antibodies directed against either Cav-1 or -3. Western blot analysis showed that no interaction between Cav-1 and -3 is detectable (Fig. 7B). Thus, in the setting of a fibroblast cell line, Cav-1 does not interact with Cav-3.

Recombinant expression of Cav-3 in Cav-1-null 3T3 MEFs does not rescue the intracellular retention of Cav-2. Because it has been shown that the absence of Cav-1 causes intracellular retention of Cav-2 in Cav-1-null 3T3 MEFs (28), we next attempted to determine the subcellular localization of Cav-2 in wild-type 3T3 MEFs and Cav-1-null 3T3 MEFs recombinantly overexpressing Cav-1, -2, or- 3. In Fig. 8A, we show that in wild-type cells, Cav-2 and -1 follow a similar expression pattern, indicative of colocalization. Furthermore, loss of Cav-1 causes a characteristic redistribution of Cav-2 to the perinuclear region (see nuclei, labeled as N). We further demonstrate that reintroduction of Cav-1 in Cav-1-null 3T3 MEFs restores the plasma membrane localization of Cav-2 (Fig. 8B). In addition, Fig. 8B demonstrates that overexpression of Cav-2 does not compensate for the absence of Cav-1, as Cav-2 staining remains perinuclear.

We next examined the subcellular localization of Cav-2 in Cav-1-null 3T3 MEFs recombinantly overexpressing Cav-3. In these studies, Myc-tagged Cav-2 was transiently transfected into Cav-3-overexpressing Cav-1-null 3T3 MEFs. These cells were then fixed and immunostained with antibodies directed against the Myc epitope tag and Cav-3. Consistent with the biochemical data above, we find that Cav-3 is correctly targeted to the plasma membrane, whereas Cav-2 is retained in the perinuclear compartment (Fig. 8C). These data again confirm that expression of Cav-3 is not sufficient to confer membrane targeting upon Cav-2 in mouse embryonic fibroblasts. In this cell, the nucleus is labeled with Hoechst-33258 (blue). Identical experiments employing the transient expression of Cav-1 demonstrated that Myc-tagged Cav-2 was properly targeted to the plasma membrane (data not shown).

To assess the relationship between Cav-1 and -3, we next sought to determine the subcellular localization of these two proteins in wild-type 3T3 MEFs recombinantly overexpressing Cav-3. Figure 8D demonstrates that when coexpressed, Cav-1 and Cav-3 show a diffuse overlapping distribution with extensive colocalization at the level of the plasma membrane, indicating that they both colocalize to lipid raft microdomains at the cell surface.

Cav-1, -2, and -3 interact in undifferentiated L6 myoblasts recombinantly expressing Cav-3. Because all of the findings discussed above were made in a fibroblast cell line, we next decided to verify these data in a different cellular system. For this purpose, we chose the L6 myoblast cell line because, under proliferating conditions, these cells coexpress Cav-1 and Cav-2, but not Cav-3. Induction of Cav-3 expression generally occurs when this cell line is forced to differentiate by changes in serum media concentrations, such as serum starvation. Therefore, we generated an L6 myoblast cell line recombinantly overexpressing Cav-3 (termed L6/Cav-3 cells) by using standard retroviral techniques (Fig. 9A) similar to those described above for the 3T3 MEFs. Interestingly, recombinant expression of Cav-3 in L6 cells causes a ~2-fold increase in the expression levels of endogenous Cav-2, suggesting that Cav-3 expression stabilizes the Cav-2 protein product. However, no changes in Cav-1 levels were observed.



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Fig. 9. Cav-1, -2, and -3 interact in L6 myoblasts transduced with Cav-3 (L6/Cav-3). Stable retroviral transduction of L6 myoblasts with Cav-3 was performed similar to that described for 3T3 MEFs. Recombinant expression of Cav-3 causes an increase in the expression levels of endgenous Cav-2 (A). As seen by coimmunoprecipation, all three caveolins (Cav-1, -2, and -3) form a complex in Cav-3-transfected L6 myoblasts (B). Note also that Cav-3 expression increases the efficiency of caveolar targeting for both Cav-1 and Cav-2 in L6 myoblasts (C). Finally, Cav-3 expression also positively affects the oligomeric state of Cav-1 and Cav-2 (D). A: recombinant expression. Cell lysates were prepared from L6 cells stably transfected with Cav-3 and subjected to Western blot analysis with antibodies directed against Cav-1, Cav-2, and Cav-3 as indicated. Each lane contains an equivalent amount of total protein. Immunoblotting with {beta}-actin IgG was performed as a control for equal loading. Note that recombinant expression of Cav-3 causes a ~2- to 2.5-fold increase in the expression levels of Cav-2, suggesting that Cav-3 expression stabilizes the Cav-2 protein product. pBABE, vector alone; Cav-3, transduced with pBABE-Cav-3. B: coimmunoprecipitation studies. Cell lysates were prepared from L6 cells stably transfected with Cav-3 and subjected to coimmunoprecipitation with antibodies directed against Cav-1 or Cav-3 and immunoblotted with antibodies, as indicated. In contrast to the results obtained in MEFs, note that Cav-1, -2, and -3 all coimmunoprecipitate in this setting. These results demonstrate that a muscle-specific interaction occurs between all three caveolin isoforms. C: caveolar targeting. L6 myoblasts stably expressing Cav-3 were subjected to sucrose density gradient centrifugation to assess the caveolar targeting of Cav-1 and Cav-2. Untransfected and vector alone L6 myoblasts were processed in parallel. Note that Cav-3 expression increases the caveolar targeting of both Cav-1 and Cav-2 (see *; fraction 5). –, untransfected; +, Cav-3 transfected. D: oligomerization. L6 myoblasts stably expressing Cav-3 were subjected to velocity gradient centrifugation, to assess the oligomeric state of Cav-1 and Cav-2. Untransfected and vector alone L6 myoblasts were processed in parallel. Cav-3 expression modestly increases the oligomeric state of both Cav-1 and Cav-2. For example, note the disappearance of oligomers in fractions 5 and 6 (see *) in Cav-3 transfected L6 cells. –, untransfected; +, Cav-3 transfected. In AD, vector alone controls behaved the same as the untransfected parental L6 cell line.

 
To assess interactions between the caveolins, cellular lysates were prepared from proliferating L6/Cav-3 cells and subjected to IP with an anti-Cav-1 antibody or an anti-Cav-3 antibody. The immune complexes were then separated by SDS-PAGE and subjected to Western blot analysis. The results presented in Fig. 9B clearly demonstrate that endogenous Cav-1 and Cav-2, as well as recombinantly expressed Cav-3, can be coimmunoprecipitated with antibodies directed against Cav-1 or Cav-3. These results stand in striking contrast to those obtained above using the 3T3 MEF system, thus suggesting that an interaction between Cav-1, -2, and -3 can occur, but in a muscle cell-specific manner. As important negative controls, we also demonstrate that protein A-Sepharose beads alone or irrelevant IgG does not immunoprecipitate any of the caveolins.

To examine whether the introduction of recombinant Cav-3 could affect the caveolar targeting of the endogenous Cav-1 and Cav-2, we next performed sucrose density centrifugation. For this purpose, we compared the behavior of L6 cells, with and without Cav-3 expression (L6 vs. L6/Cav-3). The results presented in Fig. 9C clearly demonstrate that recombinant expression of Cav-3 in L6 myoblasts increases the caveolar localization (see especially fraction 5) of endogenous Cav-1 and Cav-2.

Next, using velocity gradient centrifugation, we analyzed the oligomeric state of endogenous Cav-1 and Cav-2 in the presence of recombinant Cav-3. Again, we directly compared the behavior of L6 cells, with and without Cav-3 expression (L6 vs. L6/Cav-3). Figure 9D shows that the presence of Cav-3 favors the oligomerization of endogenous Cav-1 and Cav-2. Note the disappearance of the oligomers from fractions 5 and 6 in L6/Cav-3 cells. These results provide independent support for our coimmunoprecipitation studies, showing that all three caveolins (Cav-1, -2, and -3) form a stable complex in L6 cells.

Cav-1 and Cav-3 colocalize in proliferating L6/Cav-3 cells. To further examine the interaction between the caveolins, we next performed immunofluorescence microscopy on L6/Cav-3 cells to determine the subcellular localization of these proteins. Cells were fixed and dually immunostained with antibodies against Cav-1 and Cav-3. Microscopic analysis of these cells demonstrates the well-defined colocalization of Cav-1 and -3, at the level of the plasma membrane (Fig. 10). Therefore, in undifferentiated myoblasts, like in the mouse embryonic fibroblasts above, Cav-1 and Cav-3 are directed to the plasma membrane. More important, however, are the findings that all three caveolin proteins interact and are part of the same complex, and that this interaction occurs only in muscle cells.



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Fig. 10. Cav-1 and -3 colocalize in transfected L6 myoblasts. A: L6 myoblasts recombinantly expressing Cav-3 were grown on glass coverslips, fixed, and double labeled with antibodies directed against Cav-1 (green) and Cav-3 (red). Note that Cav-1 and Cav-3 demonstrate extensive colocalization along the surface of the cells. B: same as A, except a series of four individual confocal slices are shown, from the bottom to the top of the cell. Merged images are also shown, where appropriate.

 
Cav-1 and Cav-3 interact in the skeletal muscle of Cav-1 Tg mice. Our laboratory has previously generated a Cav-1 transgenic (Tg) mouse model in which the Cav-1 transgene is ubiquitously expressed in all tissues using standard Tg methodology. Preliminary analysis of this mouse has shown that the expression of Cav-1 in skeletal muscle does not cause any overtly identifiable abnormalities (10). An interesting aspect of this mouse is that it allows for the in vivo examination of Cav-1 and Cav-3 interactions in skeletal muscle. It is important to note that this approach using Tg expression in mice is a completely different and independent experimental approach from the pBABE retroviral transduction system that we employed for the stable transfection of 3T3 fibroblasts and L6 myoblasts.

Thus we next focused our attention on this Tg mouse model to extend our findings with L6 myoblasts, i.e., that Cav-1 and -3 interact in a muscle-specific manner. Skeletal muscle extracts from wild-type and Cav-1 Tg mice were prepared and subjected to IP with an anti-Cav-1 antibody or an anti-Cav-3 antibody. Immunoblot analysis of the resultant protein complexes reveals that Cav-1 and Cav-3 coimmunoprecipitate with antibodies directed against either Cav-1 or Cav-3 (Fig. 11). Thus these in vivo data verify our results obtained in L6/Cav-3 myoblasts and further indicate that Cav-1 and Cav-3 are capable of interacting, but that this interaction is dependent on the cell type in which the two proteins are coexpressed. One limitation of this system, however, is that skeletal muscle does not normally express Cav-2, and thus we were unable to ascertain whether this protein also interacts with Cav-3 in this setting. Thus complex formation between Cav-1 and Cav-3 does not require coexpression with Cav-2.



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Fig. 11. Cav-1 and -3 coimmunoprecipitate in skeletal muscle derived from Cav-1 transgenic (Tg) mice. Protein lysates from control and Cav-1 Tg mice were prepared from skeletal muscle samples and subjected to IP with antibodies against either Cav-1 or Cav-3. Western blot analysis of the resultant immune complexes demonstrates that Cav-1 and Cav-3 coimmunoprecipitate in muscle samples from Cav-1 Tg mice, but not from normal control mice. This again indicates that a muscle-specific interaction exists between the caveolin protein family members.

 

    DISCUSSION
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 DISCUSSION
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In this study, we have explored the relationship between the three caveolin family members using recombinant expression systems in both myocytic and nonmyocytic cells. Using 3T3 mouse embryonic fibroblasts derived from wild-type and caveolin-1 (Cav-1)-null animals, we first confirm that Cav-1 and -2 interact normally in both wild-type and Cav-1-null cells recombinantly overexpressing Cav-1 (an important positive control). Next, we demonstrate that Cav-3 does not interact with Cav-1 or -2 in these cells via a variety of approaches. First, we show that whereas Cav-1 is able to confer Triton X-100 insolubility on Cav-2, Cav-3 does not possess this property. Similarly, by sucrose density centrifugation, we demonstrate that unlike Cav-1, Cav-3 does not recruit Cav-2 to buoyant membrane compartments and thus does not allow Cav-2 to "float" (lipid raft targeting). However, the addition of Cav-3 to Cav-1-null MEFs does cause some changes in the properties of Cav-2 in that, in Cav-3-overexpressing cells, the oligomerization of Cav-2 is partially rescued, albeit not to the extent as in Cav-1-overexpressing cells. Furthermore, coimmunoprecipitation experiments in these 3T3 MEF cells reveal that Cav-2 can only be found in complexes with Cav-1 and that Cav-1 and -3 do not form complexes detectable by this approach. With the use of immunofluorescence microscopy, we demonstrate that the membrane distribution pattern of Cav-2 is dependent upon the presence of Cav-1, whereas overexpression of Cav-3 does not rescue Cav-2 from the confines of the perinuclear region. Taken together, these data strongly argue that Cav-3 does not interact with Cav-2 or Cav-1 in mouse embryonic fibroblasts.

We next attempted to extend these results in a different cellular system using L6 myoblasts recombinantly overexpressing Cav-3 that also express endogenous Cav-1 and -2. In these cells, coimmunoprecipitation experiments demonstrate that, contrary to our results in fibroblasts, Cav-1, -2, and -3 all coimmunoprecipitate. These data indicate that a muscle cell-type-specific interaction occurs between the caveolin protein family members. To confirm these findings in vivo, we next performed similar experiments on Cav-1 Tg mouse skeletal muscle samples. In these experiments, we demonstrate that Tg Cav-1 and endogenous Cav-3 do indeed coimmunoprecipitate. Therefore, this study shows for the first time that a muscle-specific interaction occurs between Cav-1, -2, and -3.

The tissue distribution pattern of the Cav-1, -2 and -3 has long been the subject of intense study. While no comprehensive report has ever been published, a careful review of the literature provides a consensus indicating that Cav-1 and -2 are found in most cell types with the exception of myocytes, whereas Cav-3 is confined to myocytic cells (36, 50). It is well known that both Cav-1 and -3 are capable of driving caveolae formation, whereas Cav-2 does not possess this property. Cav-2 is also known to require Cav-1 as a chaperone protein to localize it to the plasma membrane, because ablation of Cav-1 results in Golgi retention and the rapid proteasomal degradation of Cav-2 (28). With the recent publication of findings indicating that Cav-2 and -3 are coexpressed in ventricular myocardium (33), the question of whether Cav-3 can also act as a molecular chaperone for Cav-2 has taken on new importance. Sequence analysis of Cav-1 and Cav-3 reveals that these two proteins share 85% homology and 65% identity as well as an identical COOH-terminal palmitoylation pattern (3). However, it has previously been shown in Chinese hamster ovary and Cos-7 cells that Cav-2 and Cav-3 do not interact as determined by coimmunoprecipitation experiments (2, 25). To further address this issue, we undertook the present study using molecular genetic approaches in both myocytes and fibroblasts. Thus we are able to clearly demonstrate that the interaction between the caveolin proteins is cell-type specific.

Cav-2 has long been considered an accessory protein for Cav-1, with little ascribed functional role. In fact, the recent generation of Cav-1- and Cav-2-null mice has shown that all but one of the phenotypes recognized in the Cav-1-null mouse are due to a loss of Cav-1, not the subsequent Golgi retention and degradation of Cav-2 (27, 28, 31). However, the role of Cav-2 in the myocardium has not been addressed, as it was thought that this protein was not expressed in this tissue. The presence of Cav-2 in isolated cardiac myocytes has now been confirmed (33). Yet, as Cav-2 remains trapped in the Golgi complex without a molecular chaperone and it has been reproducibly shown that Cav-2 and -3 do not interact, it does not follow that these two proteins should be coexpressed in myocytes. Our new data resolve this conundrum, providing evidence that Cav-3 can interact with Cav-2, but only when these two proteins are coexpressed in myocytes.

What is the nature of the muscle-specific interaction of all three caveolin isoforms? One possibility is that a muscle-specific accessory protein acts as a bridge to allow the interaction of Cav-3 oligomers with Cav-1/2 hetero-oligomers. Alternatively, a muscle-specific chaperone protein may allow the hetero-oligomerization of all three caveolins (Cav-1, -2, and -3) at the level of the endoplasmic reticulum/Golgi. In this case, we would envision a transient interaction and the chaperone would not be part of the final caveolin hetero-oligomeric complex. Finally, in either case, such a factor is probably expressed in myoblasts before terminal muscle differentiation/fusion, as recombinant expression of Cav-3 in undifferentiated L6 myoblasts allows the formation of mixed caveolin oligomers (containing Cav-1, -2, and -3). However, this factor is apparently not expressed in fibroblastic cells. Thus our current studies provide a systematic basis for identifying such a muscle-specific accessory protein(s) or chaperone(s) that mediate caveolin hetero-oligomerization.

These data, indicating a cell-type-specific interaction between the caveolin protein family members, hold numerous functional implications for muscle cell development and differentiation. For instance, it is well known that caveolin protein family expression is limited to Cav-1 and -2 in cultured skeletal myoblasts during the proliferative portion of the life cycle (26). Conversely, in terminally differentiated myocytes, Cav-1 and -2 protein expression is nearly undetectable (26), whereas Cav-3 is highly expressed (47). However, during a significant portion of the differentiation process, from myoblast to myocyte, all three caveolin proteins are detectable in the same cell (7, 26). Perhaps, rather than just coincidental coexpression, the concomitant expression of all three caveolin protein family members may play a necessary role in muscle cell development and the terminal differentiation process.

In further support of the findings outlined here, it has recently been shown that in smooth muscle cells of the murine bladder, Cav-1, -2, and -3 are all coexpressed and can be coimmunoprecipitated (53). While it has long been thought that all three caveolin proteins were expressed in this cell type, the functional significance of this phenomenon remained elusive. However, in this report, it was also shown that a selective loss of Cav-3 (as in Cav-3-null mice) leads to a ~2-fold increase in morphologically identifiable plasmalemmal caveolae in bladder smooth muscle cells (53). This finding strongly indicates that Cav-3 coexpression may normally act to suppress Cav-1-driven caveolar invagination in smooth muscle cells. Conversely, a selective loss of Cav-1 (as in Cav-1-null mice) leads to a dramatic reduction (~6.5-fold) of plasmalemmal caveolae in bladder smooth muscle cells (53). In contrast, ablation of Cav-2 expression (as in Cav-2-null mice) had little or no effect on caveolae formation, assessed morphologically. Finally, dual ablation of Cav-1 and Cav-3 expression (as in Cav-1/-3 double-knockout mice) was required for the complete ablation of caveolae in these smooth muscle cells.


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 ABSTRACT
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This work was supported by National Institutes of Health (NIH) grants, the American Heart Association, the Susan G. Komen Breast Cancer Foundation, and a Hirschl/Weil-Caulier Career Scientist Award (all to M. P. Lisanti). A. W. Cohen was supported by NIH Medical Scientist Training Grant T32-GM07288. H. Lee was supported by NIH Graduate Training Program Grant T32-K07513. P. G. Frank was the recipient of a Scientist Development Grant from the American Heart Association.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. P. Lisanti, Depts. of Molecular Pharmacology and Medicine, Albert Einstein College of Medicine, and The Albert Einstein Cancer Center, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: lisanti{at}aecom.yu.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.


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