Cell-type and Tissue-specific Expression of Caveolin-2

Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21–24-kDa integral membrane protein, is a principal structural component of caveolae membranein vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been re-termed caveolin-1. Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.

the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.
Several independent lines of evidence suggest that caveolin functions as a scaffolding protein within caveolae membranes. Caveolin forms a high molecular mass oligomeric complex (14,15) that is thought to represent the assembly unit of caveolae membranes (16), and recombinant expression of caveolin in caveolin-negative cells is sufficient to drive the formation of caveolae-sized vesicles (17)(18)(19). As caveolin interacts directly with cholesterol (20,21) and glyco-sphingolipids (22), it has been proposed that the caveolin-mediated selection of endogenous lipid components could provide the driving force for caveolae formation (18).
Loss or dramatic reduction of caveolin expression and caveolae occurs in NIH 3T3 cells transformed by activated oncogenes other than v-Src (23). Caveolin expression was monitored in normal NIH 3T3 cells and compared with NIH 3T3 cells transformed with known oncogenes, such as bcr-abl, v-abl, middle T antigen, and activated Ras. In all cases, quantitation of caveolin protein expression revealed that the caveolin levels were dramatically reduced, from 25-to 100-fold depending on the specific oncogene examined. Transformed cells that expressed little or no caveolin did not contain any caveolae, as visualized by transmission electron microscopy (23). In addition, caveolin expression levels correlated inversely with the ability of these cells to grow in soft agar. That is, the cells expressing the least amount of caveolin and containing no detectable caveolae formed the largest colonies in soft agar. These results identify caveolin as a candidate tumor suppressor gene (23).
Recently, we have expressed caveolin in oncogenically transformed cells under the control of an inducible expression system (19). Regulated induction of caveolin expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that the caveolin protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin expression in v-Abltransformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar, and resulted in the de novo formation of caveolae as seen by transmission electron microscopy (19). Consistent with its antagonism of Ras-mediated cell transformation, caveolin expression dramatically inhibited both Ras/mitogen-activated protein kinase-mediated and basal transcriptional activation of a mitogen-sensitive promoter (19). Using an established system to detect apoptotic cell death, it appears that the effects of caveolin may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells (19). In addition, we find that caveolin expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations (19).
Caveolin also interacts directly with signaling molecules, preferring their inactive conformation. Using a variety of domain-mapping approaches (deletion mutagenesis, glutathione S-transferase fusion proteins, and synthetic peptides), a region within caveolin has been defined that mediates the interaction of caveolin with itself and other proteins. This cytoplasmic 41-amino acid membrane proximal region of caveolin is sufficient to mediate the formation of caveolin homo-oligomers (14), and the C-terminal half of this region (20 amino acids; residues 82-101) mediates the interaction of caveolin with G-protein ␣ subunits, H-Ras, and Src-family tyrosine kinases (24 -26). This caveolin region preferentially recognizes the inactive conformation of these molecules, as mutationally activated G ␣ subunits (G s ; Q227L), v-Src, and H-Ras (G12V) fail to interact with caveolin (24 -26). As this caveolin domain (residues 82-101) is critical for caveolin homo-oligomerization and the interaction of caveolin with certain caveolae-associated proteins (G-proteins, H-Ras, and Src-family kinases), we have previously termed this protein domain the caveolin-scaffolding domain or CSD (25,27).
We have suggested that the caveolin scaffolding domain may function like other modular protein domains (SH-2, SH-3, PH, WW, and others) to generate membrane-bound oligomeric complexes that contain signaling molecules and cytoskeletal elements (2,27). In essence, caveolin may act as molecular "velcro" to nucleate the formation of signal transduction complexes, holding these molecules in the off state. These findings would also explain the ability of caveolin expression to abrogate the anchorage-independent growth of cancerous/transformed cells (19).
Recent studies have shown that caveolin is only the first member of a growing gene family of caveolin proteins; caveolin has been re-termed caveolin-1. Three different caveolin genes (Cav-1, Cav-2, and Cav-3) encoding four different subtypes of caveolin have been described thus far (2). There are two subtypes of caveolin-1 (Cav-1␣ and Cav-1␤) that differ in their respective translation initiation sites (28). The tissue distribution of caveolin-2 mRNA is extremely similar to caveolin-1 mRNA (5). In striking contrast, caveolin-3 mRNA and protein are expressed mainly in muscle tissue types (skeletal, cardiac, and smooth) (29 -31).
Although caveolins-1 and -3 are now well-characterized, the study of caveolin-2 has been hampered by a lack of caveolin-2specific antibody probes. Here, we have generated and characterized a novel mAb 1 probe that recognizes the caveolin-2 pro-tein but not other known members of the caveolin gene family. Using this novel mAb probe, we (i) characterize the cell-type and tissue-specific expression of the caveolin-2 protein, (ii) report is co-expression, co-localization, and co-immunoprecipitation with caveolin-1, a well established caveolar marker protein, and (iii) demonstrate that caveolin-2 is localized to caveolae membranes as seen by immunoelectron microscopy.
Hybridoma Production-A monoclonal antibody to caveolin-2 was generated by multiple immunizations of Balb/c female mice with a fusion protein encoding the full-length human caveolin-2 protein. Mice showing the highest titer of anti-caveolin-2 immunoreactivity were used to create fusions with myeloma cells using standard protocols (34). Positive hybridomas were cloned twice by limiting dilution and injected into mice to produce ascites fluid. IgGs were purified by affinity chromatography on protein A-Sepharose. These antibodies were produced in collaboration with Drs. Roberto Campos-Gonzalez and John R. Glenney, Jr. (Transduction Laboratories, Lexington, KY).
Tissue Western-Approximately 200 mg of various mouse tissues were lysed in immunoprecipitation buffer and homogenized on ice with a Polytron tissue grinder, as described (4). Equal amounts (100 g) were loaded on an SDS-PAGE gel (12% acrylamide). After transfer to nitrocellulose, the blot was probed with antibodies directed against caveolins-1, -2, -3 and GDI.
Immunofluorescence Microscopy-All reactions were performed at room temperature. 3T3-L1 fibroblasts were briefly washed three times with PBS and fixed for 45 min in PBS containing 3% paraformaldehyde. Fixed cells were rinsed with PBS and treated with 25 mM NH 4 Cl in PBS for 10 min to quench free aldehyde groups. Cells were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature and washed with PBS, four times at 10 min each. The cells were then successively incubated with PBS, 2% BSA containing: (i) a 1:200 dilution of anti-caveolin-2 IgG (mAb 65) and anti-caveolin-1 IgG (pAb; directed against caveolin-1 residues 2-21), and (ii) lissamine rhodamine B sulfonyl chloride-conjugated goat anti-mouse antibody (5 g/ml) and fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (5 g/ml). The first incubation was 30 min while primary and secondary antibody reactions were 60 min each. Cells were washed three times with PBS between incubations. Slides were mounted with Slow-Fade anti-fade reagent and observed under a Bio-Rad MR600 confocal fluorescence microscope.
Cell Culture Models of Adipocyte and Skeletal Muscle Differentiation-3T3-L1 mouse fibroblasts were propagated in 10-cm dishes and differentiated according to the conventional protocol (35). C2C12-3 cells (36) were derived from a single colony of C2C12 cells (37) cultured at clonal density and display a more stable phenotype than the parental cell line. C2C12-3 myoblasts were cultured as described elsewhere (36). Briefly, proliferating C2C12-3 cells were cultured in high mitogen medium (Dulbecco's modified Eagle's containing 15% fetal bovine serum and 1% chicken embryo extract) and induced to differentiate at confluence in low mitogen medium (Dulbecco's modified Eagle's containing 3% horse serum). Overt differentiation was indicated by the assembly of multi-nucleated syncytia, which commenced 36 -48 h after the cells were switched to low mitogen media.
Immunoblotting-Samples were separated by SDS-PAGE (15% acrylamide) and transferred to nitrocellulose. After transfer, nitrocellulose sheets were stained with Ponceau S to visualize protein bands and subjected to immunoblotting. For immunoblotting, incubation conditions were as described by the manufacturer (Amersham Life Science, Inc.), except that we supplemented our blocking solution with both 1% bovine serum albumin and 1% non-fat dry milk (Carnation).
Immunogold Electron Microscopy-The procedures used were as described previously (39). Briefly, samples were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for 24 h at room temperature. Cells were transferred in 0.2% paraformaldehyde, scraped, and collected. Samples were then processed for cryo-electron microscopy using a Leica ultra-cryomicrotome and Diatome diamond knife. Sections of 45 nm were cut at Ϫ125°C and collected with a mixture of sucrose and cellulose (40). Cryosections were incubated at room temperature with antibodies for 30 min, washed, and incubated for 20 min with protein A gold. Electron micrographs were made with a JEOL 1010 electron microscope at 80 kV.
Immunostaining of Human Skeletal Muscle Tissue-All solutions were prepared in PBS. Frozen sections (4 -6 m) from normal human muscle biopsies were fixed with 4% paraformaldehyde and blocked for 1 h with 5% horse serum and 5% non-fat dry milk. Primary antibodies were diluted in blocking solution (1:400) and incubated at 4°C overnight. After washing 4 times, sections were incubated with horseradish peroxidase-conjugated anti-mouse antibody (diluted 1:1,500) for 2 h at 4°C. To visualized bound secondary antibodies, sections were further incubated with 3,3Ј-diaminobenzidine (1 mg/ml) and 0.03% hydrogen peroxide. Note that endogenous peroxidase activities were inactivated after fixation and prior to antibody incubations.

Generation and Characterization of a mAb Probe Specific for
Caveolin-2-Caveolins-1, -2, and -3 are distinct gene products with different molecular masses, all in the range of ϳ18 -24 kDa (5,28,30,31). Currently, there are no available antibody probes that selectively recognize caveolin-2. Thus, a fusion protein carrying the full-length human caveolin-2 protein was used to generate a caveolin-2 specific monoclonal antibody probe. Fig. 1 (bottom panel) demonstrates the specificity of this novel mAb probe; it selectively recognizes caveolin-2, but does not recognize caveolin-1 or -3.
Thus, this novel mAb probe can be used, in conjunction with other published antibodies directed against caveolins-1 and -3 (28,29,41), to study the function and differential expression of distinct caveolin gene family members.
Cell-type and Tissue-specific Expression of the Caveolin-2 Protein-To identify model cell systems to study caveolin-2, we examined the expression of caveolin-2 in a variety of commonly used cell lines and primary cultured cells (Fig. 2). The expression patterns of caveolins-1 and -3 are shown for comparison; antibodies to the ubiquitously expressed GDI were used to confirm equal loading. Note that caveolin-2 is most widely expressed, whereas caveolin-1 shows a more restricted distribution, and caveolin-3 is found only within a cell line derived from skeletal muscle. More specifically, caveolin-2 is most abundant in endothelial cells, smooth muscle cells, skeletal myoblasts (L6 and BC3H1), fibroblasts, and 3T3-L1 adipocytes. Thus, the expression of caveolin-2 protein most closely parallels the distribution of caveolin-1. Also, it is important to note that L6 myoblasts are the only cell line that expresses all three caveolins simultaneously.
To establish the tissue distribution of caveolin-2 protein, we prepared extracts from a number of different murine tissues (Fig. 3A). The tissue distribution of caveolins-1 and-3 are shown for comparison; again, to ensure equal protein loading in all lanes, we also probed these blots with anti-GDI antibodies. Caveolin-2 is detected mainly in adipose and lung tissues, although longer exposures demonstrate a lower level of caveolin-2 expression in most tissue types. This is consistent with (i) our previous work demonstrating that caveolin-2 mRNA is most abundant in white adipose tissue, differentiated 3T3-L1 adipocytes, and lung tissue (5) and (ii) with previous morphological evidence suggesting that differentiated 3T3-L1 adipocytes are a rich source of caveolae (3,4).
Caveolin-2 Protein Is Induced during Adipocyte Differentiation-Cultured 3T3-L1 fibroblasts offer a convenient system to study adipocyte differentiation (3). These cells can be induced to differentiate over a period of 8 days from precursor fibroblasts into adipocytes. Caveolin-2 protein is strongly induced between 2 and 10 days of differentiation (Fig. 3B, top panel). Note that the expression of caveolin-2 is induced ϳ10 -20-fold. The expression of an adipocyte-specific secretory protein (Acrp30; Ref. 42) is included as a positive control for the differentiation process (Fig. 3B, bottom panel). Caveolins-1, -2, and -3 are distinct gene products with different molecular masses, all in the range of ϳ18 -24 kDa. C-terminal myctagged forms of caveolins-1, -2, and -3 were transiently expressed in 293T cells. Lysates were generated and used to determine the specificity of caveolin antibody probes by immunoblotting. As a control for equal loading, immunoblotting was first performed with mAb 9E10 that recognizes the myc-epitope; this antibody reveals all three myc-tagged caveolin gene products (top panel). Note that mAb 65 only recognizes caveolin-2 (bottom panel). Molecular weight markers are as indicated.

Caveolins-1 and -2 Form a Stable
Hetero-oligomeric Complex-Given the similarity between the tissue and cellular distributions of caveolins 1 and 2, we wondered whether these two distinct caveolin gene products interact in vivo. To address this issue, we performed a series of co-immunoprecipitation experiments. 3T3-L1 adipocytes were lysed and subjected to immunoprecipitation with a mAb directed against caveolin-1 (2234) that recognizes a unique N-terminal epitope that is not found in other caveolin family members (28). These immunoprecipitates were then probed by Western analysis using anti-caveolin-2 IgG (mAb 65). Conversely, lysates were also immunoprecipitated with IgGs directed against caveolin-2 and then probed by Western analysis using anti-caveolin-1 IgG (mAb 2297). Fig. 4 demonstrates that mAb 2234 directed against caveolin-1 (28) can be used to co-immunoprecipitate both caveolins-1 and -2. In addition, mAb 65 directed against caveolin-2 can be used to co-immunoprecipitate both caveolins-1 and -2. This is despite the fact that these antibodies are monospecific as determined by Western blot analysis (Ref. 28, and this report).
Thus, it appears that caveolins 1 and 2 form a stable complex in vivo.
To estimate the amount of caveolin-2 that forms a complex with caveolin-1, a 3T3-L1 adipocyte lysate was divided into two parts. Part A was loaded directly onto an SDS-PAGE gel to quantitate the total amount of caveolin-2 in the extract. Part B was immunoprecipitated with anti-caveolin-1 IgG (mAb 2234). Fig. 5 shows that immunoprecipitation of the lysate with anticaveolin-1 IgG (mAb 2234) resulted in a dramatic reduction of the caveolin-2 signal by Ͼ90%. These results clearly demonstrate that under steady-state conditions the bulk of caveolin-2 is associated with caveolin-1.
Immunolocalization of Caveolin-2 to the Plasma Membrane and Intracellular Membranes: Co-localization with Caveolin-1-To further examine whether caveolins 1 and 2 are physically associated as a discrete complex in intact cells, we performed double-labeling with mAb 65 (caveolin-2-specific) and an anti-caveolin-1-specific polyclonal IgG directed against a unique N-terminal region of caveolin-1 (residues 2-21). These two primary antibodies were chosen for double-labeling experiments as they were elicited in different animal species (mouse versus rabbit), minimizing possible cross-reaction of the individual primary antibodies with distinctly tagged secondary antibodies. Immunostaining was visualized by traditional fluorescence microscopy.
The immunostaining pattern obtained in 3T3-L1 fibroblasts with caveolin-2 is very similar to immunostaining patterns observed previously for caveolins-1 and -3 (data not shown) (5, 12, 13, 28, 30 -32). Many small micro-patches are present throughout the cell and along the cell surface. In addition, double-labeling experiments employing 3T3-L1 fibroblasts that co-express caveolin-1 and caveolin-2 demonstrate significant co-localization of these two distinct gene products (not shown). The intense immunostaining may represent the leading edge of the cell as caveolae are known to be morphologically concentrated in this area of the cell (12).
As our initial experiments using traditional fluorescence microscopy showed co-localization of caveolins 1 and 2, we used confocal laser scanning microscopy to more stringently assess their co-localization. Fig. 6A shows a series of optical sections taken from the top (panel 1) to the bottom (panel 10) of a single 3T3-L1 fibroblast. A stacked composite of these images is presented in Fig. 6B. Note that in all the optical planes examined, caveolins-1 and -2 demonstrate the same pattern of localization, indicating that these two caveolins co-exist within the same regions of a given cell.
Ultrastructural Localization of Caveolin-2 to Caveolae Membranes by Immunoelectron Microscopy-Using ultrathin cryosections, we next explored the ultrastructural localization of caveolin-2 by immunoelectron microscopy. Fig. 7 shows the distribution of caveolin-2 in a fibroblastic cell line (CHO cells, panel A) and endothelial cells (panel B) derived from a human skin biopsy. In CHO cells, immunogold labeling is specifically associated with caveolae (see arrows). In endothelial cells, caveolae on both luminal and basolateral sides of the cell were stained by immunogold labeling with anti-caveolin-2 IgG (mAb 65). In contrast, mitochondria, endoplasmic reticulum, the nu- cleus, endosomes, intermediate filaments, the basal lamina, and erythrocytes remained completely unlabeled by this technique. Immunogold labeling of caveolae within endothelial cells is consistent with our observation that caveolin-2 is abundantly expressed in endothelial cells by Western blot analysis (Fig. 2).
Differential Expression of Caveolins-1 and -2 in Oncogenically Transformed NIH 3T3 Cells-Modification and/or inacti-vation of caveolin-1 expression appears to be a common feature of the transformed phenotype. Historically, caveolin-1 was first identified as a major v-Src substrate in Rous sarcoma virustransformed cells (9). Based on this observation, it has been proposed that caveolin-1 may represent a critical target during cell transformation (9,10). In direct support of this notion, caveolin-1 mRNA and protein expression are reduced or absent in NIH 3T3 cells transformed by a variety of activated oncogenes (such as v-Abl and H-Ras (G12V)); caveolae organelles are also missing from these transformed cells (23). However, it remains unknown whether caveolin-2 is down-regulated in response to oncogenic transformation. Fig. 8A shows that while caveolin-1 expression is dramatically down-regulated in v-Abl and H-Ras (G12V)-transformed FIG. 3. Western blot analysis of the tissue distribution of caveolin-2: induction of caveolin-2 protein during adipocyte differentiation. A, tissue Western. Extracts of mouse tissues were prepared as described under "Experimental Procedures". In addition, a lane containing differentiated 3T3-L1 adipocytes was included as a comparison. After SDS-PAGE and transfer to nitrocellulose, blots were probed with anti-caveolin IgG. First panel, caveolin-1 (mAb 2297); second panel, caveolin-2 (mAb 65); third panel, caveolin-3 (mAb 26); and fourth panel, GDI polyclonal IgG (as a control for equal loading). Caveolins-1 and -2 are most abundantly expressed in adipose tissue and 3T3-L1 adipocytes. Note that the lane containing skeletal muscle is underloaded as judged by the low signal obtained for GDI immunoblotting. B, adipocyte differentiation. 50 g of total cellular protein extracted from 3T3-L1 fibroblasts and 3T3-L1 adipocytes (days 2, 4, 6, 8, and 10) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-caveolin-2 (mAb 65; top panel) or anti-Acrp30 (rabbit polyclonal IgG; bottom panel). Note that caveolin-2 and Acrp30 (an adipocyte-specific secretory protein) are both dramatically induced during adipocyte differentiation. Acrp30 served as a positive control for differentiation. FIG. 4. Caveolins 1 and 2 form a stable hetero-oligomeric complex. 3T3-L1 fibroblasts were lysed and subjected to immunoprecipitation with anti-caveolin-1 IgG (mAb 2234) that recognizes a unique N-terminal epitope that is not found in other caveolin family members (28). These immunoprecipitates were then probed by Western analysis using anti-caveolin-2 IgG (mAb 65). Conversely, lysates were also immunoprecipitated with IgGs directed against caveolin-2 (mAb 65) and then probed by Western analysis using anti-caveolin-1 IgG (mAb 2297). Immunoprecipitation with anti-caveolin-3 IgG (mAb 26) was included as a negative control as the expression of caveolin-3 is muscle specific. Note that anti-caveolin-1 IgG (mAb 2234) can be used to co-immunoprecipitate both caveolins-1 and -2. In addition, mAb 65 directed against caveolin-2 can be used to co-immunoprecipitate both caveolins-1 and -2. Thus, it appears that caveolins 1 and 2 form a stable complex in vivo.

FIG. 5. A significant fraction of total caveolin-2 is associated
with caveolin-1 at steady-state. One 10-cm plate of 3T3-L1 adipocytes was lysed as described under "Experimental Procedures." Prior to the immunoprecipitation, 5% of the total lysate was removed (T, Total). The remaining 95% was subsequently immunoprecipitated with anticaveolin-1 IgG (mAb 2234); 5% of the resulting immunoprecipitate (P, Pellet) and 5% of the remaining supernatant after the extract was immunodepleted for caveolin 1 (S, Supernatant) were then analyzed by SDS-PAGE/Western blotting and probed with anti-caveolin-2 IgG. Note that Ͼ90% of caveolin-2 was recovered with anti-caveolin-1 IgG (mAb 2234). NIH 3T3 cells, caveolin-2 expression remains virtually unaffected in v-Abl transformed cells and is slightly induced (ϳ2fold) in H-Ras (G12V) transformed cells. As we have previously demonstrated that these transformed cells do not contain detectable caveolae (23), it appears that expression of caveolin-2 is not sufficient to drive caveolae formation. Thus, caveolin-2 can be expressed within cells that lack morphologically distinguishable caveolae.
As caveolin-1 forms a high molecular mass oligomeric complex (14, 15), we wondered if low levels of caveolin-1 expression would affect the oligomeric state of caveolin-2. Thus, we compared the size of caveolin-2 complexes in normal and v-Abl transformed NIH 3T3 cells (Fig. 8B). Our results indicate that in v-Abl transformed cells that lack normal levels of caveolin-1 expression, a significant fraction of caveolin-2 was present in the monomeric or dimeric state. This is consistent with our previous results demonstrating that recombinant over-expression of caveolin-2 in Cos-7 cells yields primarily dimeric and monomeric caveolin-2 (5). However, in normal NIH 3T3 cells that express caveolin-1, all of the caveolin-2 was present within large oligomeric complexes (Ͼ150 kDa). These results suggest that caveolin-1 co-expression facilitates the formation of high molecular mass complexes that contain both caveolins 1 and 2.
The distribution of recombinantly over-expressed caveolin-2 after transient expression in Cos-7 cells is shown for comparison; recombinant caveolin-2 was detected using mAb 9E10 that recognizes the myc-epitope (Fig. 8B). In contrast to endogenous caveolin-2, recombinantly over-expressed caveolin-2 behaves mainly as a dimer in these velocity gradients (5).
Caveolin-1 Embeds Caveolin-2 Tightly within a Hetero-oligomeric Complex-During the course of the current studies, we noticed that there are higher levels of caveolin-2 in immunoprecipitates generated with anti-caveolin-1 IgG than in immunoprecipitates generated with caveolin-2 IgG directly. Given that the anti-caveolin-1 mAb does not cross-react with caveolin-2, we found this observation quite surprising.
A possible explanation for this phenomenon is epitope masking, in which caveolin-1 binding to caveolin-2 would block access to the epitope recognized by the anti-caveolin-2 IgG. To test this hypothesis, we took advantage of a v-Abl-transformed cell line that harbors a copy of the caveolin-1 cDNA under the control of the lacZ promoter (19). While these cells express very low levels of endogenous caveolin-1 due to transformation, caveolin-1 expression levels can be dramatically induced in the presence of IPTG. Fig. 9 shows that in the absence of IPTG, i.e. very low levels of caveolin-1, small amounts of caveolin-2 can be recovered with anti-caveolin-1 IgG (first lane). Upon induction of caveolin-1, increased levels of caveolin-2 can be recovered with anticaveolin-1 IgG in agreement with increased incorporation of caveolin-2 into caveolin-1 containing complexes (second lane). However, induction of caveolin-1 decreases the amount of caveolin-2 signal that can be recovered with anti-caveolin-2 IgG (third and fourth lanes).
This observation is in line with our hypothesis that caveolin-1 binding to caveolin-2 masks the epitope recognized by the caveolin-2 IgG. In addition, control experiments confirmed that total caveolin-2 levels remained constant before and after induction of caveolin-1 (not shown). Hence, we conclude that caveolin-2 molecules are tightly embedded within the caveolin-1 oligomer.
Caveolin-2 Is Constitutively Expressed in C2C12 Myoblasts and Myotubes-Cultured C2C12 cells offer a convenient system to study skeletal myoblast differentiation. These cells can be induced to differentiate from myoblasts into myotubes bearing an embryonic phenotype in low mitogen medium over a period of 2 days (36,37). We and others have previously shown that caveolin-3 mRNA and protein are undetectable in precursor myoblasts and are strongly induced during myoblast differentiation (29 -31). In contrast, no caveolin-1 expression was detected in either precursor myoblasts or differentiated myotubes (29,30). These results are consistent with the selective expression of caveolin-3 in skeletal muscle and other muscle tissues (29 -31) and suggest that caveolin-3 may function in muscle from the earliest stages of its development.
As caveolin-2 was expressed in L6 and BC3H1 skeletal myo-FIG. 6. Immunolocalization of caveolin-2 in 3T3-L1 fibroblasts by laser scanning confocal microscopy. A, cells were doubly immunostained with a mouse mAb directed against caveolin-2 (mAb 65) and a rabbit polyclonal antibody directed against the unique N terminus of caveolin-1 (residues 2-21). Bound primary antibodies were visualized by incubation with distinctly tagged fluorescent secondary antibodies; see "Experimental Procedures." Note that caveolins-1 and -2 are colocalized to the same areas of the plasma membrane and internal membranes. A series of ten optical sections from the top (panel 1) to the bottom (panel 10) of a single cell are shown. Each individual panel shows: left, caveolin-1 immunostaining (FITC, fluorescein isothiocyanate); right, caveolin-2 immunostaining (LRSC, lissamine rhodamine B sulfonyl chloride). Note the dramatic co-localization within each optical plane. B, a stacked summation of all ten sections is shown. blasts (Fig. 2), we assessed whether caveolin-2 is induced during differentiation of C2C12 cells from myoblasts to myotubes. Fig. 10 shows that caveolin-2 levels remained constant during this process of differentiation. As a positive control for the differentiation process, we also assessed the induction of caveolin-3 within the same samples. In contrast, caveolin-3 was dramatically induced during the transition from myoblasts to myotubes. These results suggest that the expression of caveolins 2 and 3 are independently regulated within skeletal muscle fibers.
Localization of Caveolin-2 within Bona Fide Skeletal Muscle Tissue-Given that caveolin-2 was constitutively expressed in three distinct myoblast cell lines (L6, BC3H1, and C2C12) and within differentiated C2C12 myotubes, we next examined the localization of caveolin-2 within human skeletal muscle tissue. Fig. 11 shows that caveolin-2 is primarily expressed within the endothelial cells that line the blood vessels that run between the muscle fibers but not within the myofibers themselves. The distribution of caveolin-3 is shown for comparison. Note that caveolin-3 immunostaining is confined to the sarcolemma (plasma membrane) of the myofibers and is not detected within any other cell types. Thus, co-expression of caveolins-2 and -3 in myoblasts and myotubes does not reflect the state of their expression within human adult skeletal muscle tissue. DISCUSSION Caveolins-1, -2, and -3 are a family of cytoplasmic membrane-anchored scaffolding proteins that (i) help to sculpt caveolae membranes from the plasma membrane proper and FIG. 8. Expression of caveolin-2 in normal and transformed NIH 3T3 cells. A, lysates were prepared from normal and transformed (v-Abl and H-Ras (G12V)) NIH 3T3 cells and subjected to immunoblot analysis with anti-caveolin-1 IgG (mAb 2297) or anti-caveolin-2 IgG (mAb 65). Note that caveolin-1 expression is dramatically down-regulated in v-Abl and H-Ras (G12V)-transformed NIH 3T3 cells, whereas caveolin-2 expression remains virtually unaffected in v-Abl transformed cells and is slightly induced (ϳ2-fold) in H-Ras (G12V) transformed cells. Each lane contains ϳ50 g of protein lysate. B, velocity gradient analysis of caveolin-2. Normal and v-Abl-transformed NIH 3T3 cells were solubilized, loaded atop a 5-40% sucrose density gradient and subjected to centrifugation for 10 h, as we described previously for caveolin-1 (14). The distribution of endogenous caveolin-2 was detected by immunoblot analysis with anti-caveolin-2 IgG (mAb 65). Arrows mark the positions of molecular mass standards. The distribution of recombinantly over-expressed caveolin-2 after transient expression in Cos-7 cells is shown for comparison; recombinant caveolin-2 was detected using mAb 9E10 that recognizes the myc-epitope. In contrast to endogenous caveolin-2, recombinantly over-expressed caveolin-2 behaves as a dimer in these velocity gradients as we have shown previously (5).
FIG. 9. Induction of caveolin-1 protein expression promotes complex formation between caveolins 1 and 2. v-Abl transformed NIH-3T3 cells were transfected with caveolin-1 under the control of an IPTG-inducible promoter (19). In this cell line, addition of 5 mM IPTG for 24 h dramatically induces the expression of caveolin-1, as we have recently described (19). v-Abl cells were lysed before or after induction of caveolin-1 expression and subjected to immunoprecipitation with IgG directed against either caveolin-1 (mAb 2234) or caveolin-2 (mAb 65). After SDS-PAGE and transfer to nitrocellulose, these immunoprecipitates were analyzed by Western blotting to detect the presence of caveolin-2 within these complexes. Note that induction of caveolin-1 expression (i) enhances the ability of anti-caveolin-1 IgG to co-immunoprecipitate caveolin-2; and (ii) inhibits the ability of anti-caveolin-2 IgG to immunoprecipitate caveolin-2. These results suggest that complex formation between caveolins-1 and -2 can mask the antibody epitope that is recognized by anti-caveolin-2 IgG (mAb 65). (ii) participate in the sequestration of inactive signaling molecules (reviewed within Ref. 2). Although caveolins-1 and -3 are now well-characterized, the study of caveolin-2 has been hampered by a lack of caveolin-2-specific antibody probes. Only the distribution of caveolin-2 mRNA was previously studied, and a recombinant epitope-tagged form of the protein has been expressed in cultured cells (5).
Here, we have generated and characterized a novel mAb probe that recognizes the native endogenous caveolin-2 protein but not other known members of the caveolin gene family. This novel probe will greatly facilitate the study of caveolae in adipocytes, endothelial cells, and smooth and striated muscle cells as caveolin-2 is the most widely expressed caveolin family member observed to date. Caveolin-2 is highly expressed in many cell lines that fail to express caveolin-1, suggesting that caveolin-2 does not absolutely require caveolin-1 for its expression.
Immunolocalization of caveolin-2 in 3T3-L1 fibroblasts reveals that caveolin-2 is localized to the plasma membrane and internal membranes; this immunostaining pattern strictly coincides with the subcellular distribution of caveolin-1 within the same cell. In line with these observations, co-immunoprecipitation experiments clearly demonstrate that caveolins 1 and 2 form a stable hetero-oligomeric complex in vivo. This is consistent with our previous report demonstrating that the mRNA's for caveolins 1 and 2 are co-expressed within the same cell types and both mRNA species are co-induced during adipocyte differentiation (5).
We have previously described the existence of caveolin-1 homo-oligomeric complexes (14). This was at a time when we had no concrete evidence for the existence of additional caveolin genes. A host of experiments prompted us to conclude that caveolin-1 exists as a homo-oligomeric complex. These studies were performed mainly in MDCK cells. Interestingly, MDCK cells are peculiar in that they express caveolin-1 but very little if any caveolin-2 (See Fig. 2). As such, they are an exception since we find here that caveolins-1 and -2 are co-expressed in most other cell systems. However, this does explain our initial findings that caveolin-1 forms a homo-oligomeric complex in MDCK cells (14). In addition, we have shown that purified recombinant caveolin-1 expressed in E. coli and Sf 21 insect cells is sufficient to form caveolin-1 homo-oligomers of the same size as native endogenous caveolin-1 (14,18,21).
Perhaps surprisingly, we find that the co-expression of caveolins 1 and 2 is uncoupled by cellular transformation by activated oncogenes, such as v-Abl and activated H-Ras (G12V). While caveolin-1 mRNA and protein levels are downregulated in response to cellular transformation (23), caveolin-2 protein levels remain relatively unchanged. While these cells continue to express caveolin-2 protein (this report), they fail to contain detectable caveolae, as we have reported previously (23). These observations suggest that caveolin-2 expression alone is not sufficient to drive the formation of morphologically detectable caveolae. However, detergent-insoluble caveolin-2-rich domains still exist (in the absence of caveolin-1) that are not distinguishable as "invaginated caveolae" by conventional transmission electron microscopy. These observations suggest that caveolin-rich microdomains may be more versatile structures with greater plasticity than previously imagined. In support of this idea, we have recently expressed caveolin-2 in Sf 21 insect cells using baculo-virus-based vectors. Caveolin-2 expression in these insects cells fails to drive the formation of caveolae-like vesicles 2 while recombinant expression of caveolin-1 within the same cell system is sufficient to drive the formation of hundreds of uniform caveolae-like vesicles (50 -100 nm in diameter) (18). These results are consistent with the idea that caveolin-2 may function as an "accessory protein" in conjunction with caveolin-1.
In many experiments we observed a slightly less abundant and faster migrating form of caveolin-2 (See Figs. 2, 3, and 4). This may represent a proteolytic degradation product of caveolin-2 or may be a translationally produced isoform. As a single caveolin-1 mRNA gives rise to caveolin-1␣ and caveolin-1␤ via alternate translation initiation (28), we favor the possibility that the faster migrating form of caveolin-2 is also generated as a consequence of alternate translation initiation. In support of this hypothesis, further sequencing and analysis of the 5Ј end of the human caveolin-2 cDNA reveals an additional initiator methionine 13 amino acids upstream (MGLETEKADVQLFM-DD......) of the previously reported initiator methionine. The updated cDNA sequence can be found under GenBank™ accession number U32114. We are currently investigating the significance of this upstream initiation site. However, one possibility is that this upstream initiator may allow for myristoylation of caveolin-2 as an N-terminal cytoplasmic MG sequence is a co-translational consensus site for N-myristoylation. In addition, these two methionines may serve as alternate translation initiation sites to generate two distinct isoforms of caveolin-2 (Cav-2␣ and Cav-2␤). If this is the case, then only the longer isoform of caveolin-2 (Cav-2␣) would be expected to undergo myristoylation.
Caveolin-3 is a muscle-specific caveolin-related protein that is expressed in striated muscle cell types (cardiac and skeletal) (29 -31). Unlike caveolin-3, caveolin-1 is not expressed in striated muscle cells. It was, therefore, surprising to observe caveolin-2 expression in three skeletal myoblast cell lines (L6, BC3H1, and C2C12) and in differentiated C2C12 myotubes. As caveolins-1 and -3 are most closely related based on primary sequence homology and caveolin-2 is most distant (30), caveolin-2 may also function in embryonic muscle as a complex with caveolin-3. However, we have been unable to demonstrate a 2 M. P. Lisanti, unpublished observations. FIG. 11. Immunolocalization of caveolins 2 and 3 within bona fide skeletal muscle tissue. Two consecutive parallel sections derived from a human skeletal muscle biopsy were immunostained with either anti-caveolin-2 IgG (mAb 65; top panel) or anti-caveolin-3 IgG (mAb 26; bottom panel). Bound primary antibodies were visualized with the appropriate horseradish peroxidase-conjugated secondary antibody. Note that caveolin-2 is primarily expressed within the endothelial cells that line the blood vessels, while caveolin-3 is localized to the sarcolemma (plasma membrane) of the myofibers. stable association between caveolins 2 and 3 by co-immunoprecipitation (data not shown), as caveolin-2 is not expressed within adult skeletal muscle fibers (See Fig. 11). Caveolins 1 and 3 may have originated from a common ancestor as we have recently identified only two caveolins within C. elegans, termed Cav ce -1 and Cav ce -2 (43). Cav ce -1 is most closely related to mammalian caveolins-1 and -3; Cav ce -2 is most closely related to mammalian caveolin-2 (43). Thus, caveolin-2 appears to be structurally and functionally conserved from worms to man, suggesting an important evolutionary role for caveolin-2 in the regulation of caveolae membranes.