©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Caveolin-3, a Novel Member of the Caveolin Gene Family Expressed Predominantly in Muscle (*)

(Received for publication, September 27, 1995; and in revised form, November 10, 1995)

ZhaoLan Tang (1) Philipp E. Scherer (1)(§) Takashi Okamoto (2)(¶) Kenneth Song (1) Caryn Chu (3) D. Stave Kohtz (3) Ikuo Nishimoto (4) Harvey F. Lodish (1) (5) Michael P. Lisanti (1)(**)

From the  (1)Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, the (2)Shriners Hospitals for Crippled Children, Massachusetts General Hospital, Department of Anesthesia, Harvard Medical School, Boston, Massachusetts 02114, the (3)Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029, the (4)Department of Medicine, Cardiovascular Research Center, Massachusetts General Hospital-East, Charlestown, Massachusetts 02129, and the (5)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second caveolin gene has been identified and termed caveolin-2. Here, we report the molecular cloning and expression of a third member of the caveolin gene family, caveolin-3. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are 65% identical and 85% similar. A single stretch of eight amino acids (FEDVIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a ``caveolin signature sequence'' that is characteristic of members of the caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, caveolin-3 is similar to caveolin-1: (i) caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) caveolin-3 colocalizes with caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the caveolin gene family may have implications for understanding the role of caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.


INTRODUCTION

Caveolae are flask-shaped plasma membrane invaginations(1) . They are most conspicuous in adipocytes, endothelial cells, muscle cells, and fibroblasts, but are thought to exist in most cell types(2) . The exact cellular function of caveolae remains unknown, although they have been implicated in endothelial transcytosis(3) , potocytosis(4) , and signal transduction (reviewed in (5) and (6) ). Purification of caveolae-enriched membrane fractions reveals several distinct classes of lipid-modified signaling molecules, including heterotrimeric G-proteins (alpha- and beta-subunits), Src-like kinases, and Ras-related GTPases(7, 8, 9, 10, 11, 12, 13) . Based on these observations, we have proposed the ``caveolae signaling hypothesis,'' which states that compartmentalization of certain cytoplasmic signaling molecules within caveolar membranes could allow rapid and efficient coupling of activated receptors to more than one effector system(5, 7) .

A 22-kDa integral membrane protein termed caveolin is a principal component of caveolae in vivo(14) . Caveolin may act as a scaffolding protein within caveolar membranes. Caveolin exists as a high molecular mass homo-oligomer (14-16 monomeric units per oligomer)(15, 16) , and these purified caveolin homo-oligomers have the capacity to self-associate into caveolae-like structures(15) . In this regard, caveolin could serve as an oligomeric docking site for organizing and concentrating certain caveolin-interacting proteins (such as heterotrimeric G-proteins) within caveolae(17) . Caveolin interacts directly with G-protein alpha-subunits. Residues 82-101 of caveolin are most critical for this interaction, and a caveolin-derived polypeptide encoding these residues functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins, acting as a GDP dissociation inhibitor (GDI) (^1)(17) . Also, caveolin rapidly undergoes tyrosine phosphorylation in response to insulin stimulation and thus could serve as a regulated docking site for SH2 domain-containing molecules(18) .

Caveolin appears to be important for the formation of caveolar membranes as caveolin expression levels correlate very well with the morphological appearance of caveolae. For example, (i) caveolin is most abundant in cell types that contain numerous caveolae, i.e. adipocytes, endothelial cells, smooth muscle cells, and fibroblasts (19) ; (ii) caveolin and caveolae are both induced 10-25-fold during the differentiation of 3T3-L1 fibroblasts to the adipocyte form(20, 21) ; and (iii) caveolin levels are dramatically reduced and caveolae are morphologically absent in cells transformed by various activated oncogenes, including v-abl and activated ras(22) . However, there are certain cell lines that morphologically contain caveolae, but fail to express caveolin(7) . This finding has suggested that other caveolin-related proteins may exist that are immunologically distinct from caveolin.

Two discrete isoforms of caveolin are known to exist: alpha-caveolin contains residues 1-178, while beta-caveolin contains residues 32-178 (23) . These isoforms are derived from a single gene through alternate initiation during translation. More specifically, methionine 32 serves as an internal translation initiation site to generate beta-caveolin (23) . Although these two isoforms are functionally identical in many respects, they assume a distinct but overlapping subcellular distribution within a single cell(23) , and only beta-caveolin is phosphorylated on serine residues in vivo(21) . Thus, coexpression of alpha- and beta-caveolins may serve to generate or mark distinct subpopulations of caveolae that are differentially regulated by an unknown caveolin-associated serine kinase(23) .

A second mechanism exists for generating caveolin diversity. A novel caveolin-related protein has recently been identified. This protein, termed caveolin-2, is the product of a separate gene(24) . Thus, caveolin (re-termed caveolin-1) is the first member of a multigene family. As both caveolin-1 and caveolin-2 have a relatively restricted tissue distribution(24) , this suggested to us that other caveolin genes should exist.

Here, we report the molecular cloning, sequence, and tissue-specific expression of a third novel member of the caveolin gene family, caveolin-3. The current discovery of tissue-specific caveolin genes may end or at least alter the debate regarding the existence of caveolae without caveolin. Thus, a lack of caveolin expression (now known as caveolin-1) may simply reflect tissue-specific expression of different caveolin genes for which the appropriate probes have not yet been generated.


EXPERIMENTAL PROCEDURES

Materials

A monoclonal antibody(2297) directed against caveolin-1 was the generous gift of Dr. John R. Glenney (Transduction Laboratories, Lexington, KY). A rabbit polyclonal antibody directed against the C-terminal 44 amino acids of caveolin-1 (residues 135-178) was as characterized previously(23) . This antibody specifically recognizes both alpha- and beta-isoforms of caveolin-1, but does not recognize caveolin-2 or caveolin-3. The monclonal antibody 9E10 was provided by the Harvard Monoclonal Antibody Facility (Cambridge, MA). A variety of other reagents were purchased commercially: purified rat genomic DNA (Promega), a rat heart gt11 cDNA library (CLONTECH), fetal bovine serum (JRH Biosciences), prestained protein markers (Life Technologies, Inc.), and Slow-Fade anti-fade reagent (Molecular Probes, Inc., Eugene, OR). Peptide synthesis was performed by the Biopolymers Facility at the Massachusetts Institute of Technology.

Northern Analysis of Tissues and cDNA Cloning

mRNA isolation from tissues as well as agarose gel electrophoresis and transfer to nylon membranes were performed as described(25) . Hybridizations were performed in 50% formamide, 5 times SSC, 25 mM sodium phosphate, pH 7.0, 10 times Denhardt's solution, 5 mM EDTA, 1% SDS, 0.1 mg/ml poly(A) at 42 °C overnight and subsequently washed in 2 times SSC, 0.1% SDS and in 0.5 times SSC, 0.1% SDS at 50 °C. Radiolabeled DNA concentrations were at 2 times 10^6 cpm/ml. To obtain the rat caveolin-3 cDNA, a rat heart cDNA library (gt11) was screened using a polymerase chain reaction-generated probe (see ``Results'') essentially as described previously for caveolin-1(26) . Automated DNA sequencing was performed by the Molecular Biology Core Facility at the Dana-Farber Cancer Institute.

Culture and Differentiation of C2C12 Skeletal Myoblasts

C2C12-3 cells (27) were derived from a single colony of C2C12 cells (28) cultured at clonal density and display a more stable phenotype than the parental cell line. C2C12-3 myoblasts were cultured as described elsewhere(27) . Briefly, proliferating C2C12-3 cells were cultured in high mitogen medium (Dulbecco's modified Eagle's medium 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 medium containing 3% horse serum). Overt differentiation was indicated by the assembly of multinucleated syncytia, which commenced 36-48 h after the cells were switched to low mitogen medium. For Northern analysis, mRNA was isolated from proliferating C2C12-3 myoblasts in high mitogen medium at 50% confluence or from differentiated cells cultured at confluence in low mitogen medium for 48 h(27) . Total cellular RNA was extracted with guanidine isothiocyanate and isolated by cesium chloride density centrifugation(29) . mRNA was enriched by oligo(dT)-cellulose affinity chromatography and resolved by formaldehyde gel electrophoresis(30) .

Recombinant Expression and Selection of Stable Cell Lines

An epitope-tagged form of caveolin-3 was subcloned into the MCS of the vector pCB7 (containing the hygro^R marker; gift of J. Casanova, Massachusetts General Hospital) for expression in COS-7 or MDCK cells. The myc epitope tag was incorporated into the C terminus (caveolin-3-GGEQKLISEEDLN) of the cloned rat caveolin-3 cDNA using polymerase chain reaction primers; GG was placed as a spacer between the epitope and the caveolin-3 coding sequences, as performed previously for caveolin-1 and caveolin-2(23, 24, 31, 32) . Constructs were transiently transfected into COS-7 cells by the DEAE-dextran method(33) . In addition, MDCK cells were stably transfected using a modification of the calcium phosphate precipitation procedure, as we described previously(13, 23) . After selection in medium supplemented with 400 µg/ml hygromycin B, resistant colonies were picked by trypsinization using cloning rings. Individual clones were screened by immunofluorescence and immunoblotting. Caveolin-3 expression in MDCK cells was detected using monoclonal antibody 9E10, which recognizes the myc epitope (EQKLISEEDLN).

Velocity Gradient Centrifugation

Estimation of the molecular mass of caveolin-3 was performed as described previously for caveolin-1(15) . Briefly, samples were dissociated by incubation with 500 µl of Mes-buffered saline (MBS; 25 mM Mes, pH 6.5, 0.15 M NaCl) plus 60 mM octyl glucoside. Solubilized material was then loaded atop a 5-40% linear sucrose gradient (4.3 ml) and centrifuged at 50,000 rpm (340,000 times g) for 10 h in an SW 60 rotor (Beckman Instruments). Note that the entire gradient was prepared in MBS plus 60 mM octyl glucoside. After centrifugation, gradient fractions were collected from the top. Molecular mass standards for velocity gradient centrifugation were as follows: carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), beta-amylase (200 kDa), and apoferritin (443 kDa) (Sigma).

Immunofluorescence

All reactions were performed at room temperature. COS-7 cells (cotransfected with cDNAs for caveolin-1 and caveolin-3) were briefly washed three times with PBS and fixed for 45 min in PBS containing 3% paraformaldehyde, 10 mM NaIO(4), and 70 mM lysine HCl. Fixed cells were rinsed with PBS and treated with 100 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 either at room temperature or on ice and washed with PBS (four times, 10 min each). The cells were then successively incubated with PBS, 2% bovine serum albumin containing the following: (i) 50 µg/ml each of normal goat IgG and donkey IgG, (ii) a 1:300 dilution of monoclonal antibody 9E10 and 40 µg/ml anti-caveolin-1 C terminus-specific polyclonal IgG, and (iii) lissamine rhodamine B sulfonyl chloride-conjugated goat anti-mouse antibody (5 µg/ml) and fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (5 µg/ml). The first incubation was for 30 min, while primary and secondary antibody reactions were for 60 min each. Cells were washed three time with PBS between incubations. Slides were mounted with Slow-Fade anti-fade reagent and observed under a Bio-Rad MR600 confocal fluorescence microscope.

Cell Fractionation

MDCK cells (recombinantly expressing caveolin-3) were grown to confluence in 150-mm dishes and used to prepare caveolin-enriched membrane fractions essentially as described (7, 8, 21, 34) . Briefly, MDCK cells from a confluent 150-mm dish were scraped into 2 ml of MBS containing 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride. Homogenization was carried out initially with 10 strokes of a loose-fitting Dounce homogenizer, followed by a Polytron tissue grinder (three 10-s bursts; Brinkmann Instruments). The homogenate was adjusted to 40% sucrose by addition of 2 ml of 80% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. A 5-30% linear sucrose gradient was formed above the homogenate and centrifuged at 39,000 rpm for 16-20 h in an SW 41 rotor (Beckman Instruments). A light-scattering band confined to the 15-20% sucrose region was harvested, diluted 3-fold with MBS, and pelleted in a microcentrifuge (14,000 times g, 15 min, 4 °C). The majority of protein remained within the 40% sucrose region of the gradient. Approximately 4-6 µg of caveolin-enriched domains were obtained from one 150-mm dish of MDCK cells representing 10 mg of protein, a yield of 0.05% relative to the homogenate. We (7, 8, 13, 21) and others (34, 35) have demonstrated that these domains exclude a variety of organelle-specific membrane markers (for the endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria, and non-caveolar plasma membrane), but are dramatically enriched 2000-fold in caveolin-1, a caveolar marker protein.

Immunoblotting of Gradient Fractions

Gradient fractions were separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide) and transferred to nitrocellulose. After transfer, nitrocellulose sheets were stained with Ponceau S to visualize protein bands and subjected to immunoblotting with anti-caveolin-1 IgG (monoclonal antibody 2297; 1:400) or with 9E10 ascites (1:500) to visualize myc-tagged caveolin-3. For immunoblotting, incubation conditions were as described by the manufacturer (Promega and Amersham Corp.), except we supplemented our blocking solution with both 1% bovine serum albumin and 1% non-fat dry milk (Carnation). The amount of caveolin-1 and caveolin-3 that remains associated with caveolin-enriched fractions was estimated by immunoblotting as described(8) . Quantitation was performed with a Molecular Dynamics computing densitometer. To ensure that these estimates were made in the linear range, we used multiple autoradiographic exposures and monitored their linearity using the densitometer essentially as described (36) .

GTP Hydrolysis Assays

Trimeric G purified from bovine spleen was provided by T. Asano(37) . Trimeric G(o) purified from bovine brain was provided by T. Haga(38) . Steady-state GTP hydrolysis activity was examined as we described previously(39) . Briefly, the assay was performed for 20 min at 37 °C in the presence of 20 µM Mg with 10 nM G-protein. The caveolin-3-derived polypeptide contained the sequence DGVWRVSYTTFTVSKYWCYR, corresponding to amino acids 55-74 of caveolin-3.


RESULTS

Identification and Molecular Cloning of the cDNA for Caveolin-3

To identify other putative members of the caveolin gene family, the protein sequence of caveolin-1 was used to search existing data bases. Through this approach, a rat genomic sequence (350 nucleotides in length; GenBank accession number U15280) was identified that could potentially encode part of a novel caveolin-related protein. This short genomic sequence was present 3` to the last exon of the rat oxytocin receptor gene and in the opposite orientation to the coding sequence for the oxytocin receptor. Oligonucletide primers (5`-CCGGCCGAATTCATGATGTGATTGCGGACCGCGAG-3` and 5`-CCGGCCGGATCCATCACCTTAATGTTGCTCCAGAC-3`; EcoRI and BamHI restriction sites are in boldface, and target sequences are underlined) were designed and used to amplify this nucleotide sequence from purified rat genomic DNA by polymerase chain reaction. A fragment of the expected size was subcloned into the MCS of pBluescript II KS, and its identity was verified by DNA sequencing.

Fig. 1shows the tissue distribution of an 1.2-kilobase mRNA species that specifically hybridizes at high stringency with this novel genomic sequence. This mRNA is present only in skeletal muscle, diaphragm, and heart, suggesting muscle-specific expression of this novel gene product. The distribution of caveolin-1 mRNA is shown for comparison. In contrast, caveolin-1 mRNA is 2.5 kilobases and is most highly expressed (in descending order) in white adipose tissue, lung, and muscle tissues. Thus, this presumptive caveolin-related protein appeared distinct from caveolin-1 in its mRNA size and tissue distribution. Also, it is important to note that detection of caveolin-1 mRNA within skeletal muscle tissue reflects its expression within the endothelial cells of the muscle tissue, but not in the muscle cells themselves (see ``Discussion'' for a more detailed explanation).


Figure 1: Northern blot analysis of the tissue distribution of caveolin-3. Each lane contains 1 µg of poly(A) RNA prepared from a given tissue. Blots were probed with the caveolin-1 cDNA, stripped, and reprobed with caveolin-3. Caveolin-1 and caveolin-3 mRNAs have different transcript sizes: 2.5 kilobases for caveolin-1 and 1.2 kilobases for caveolin-3. After the initial hybridizations, the blot was stripped and reprobed with a cytosolic hsp70 cDNA as a control for equal loading (as we published previously(21) ).



To obtain the full-length cDNA, this polymerase chain reaction-generated genomic probe was used to screen a rat heart cDNA library (gt11). Five independent positive clones were obtained from screening 6 times 10^5 recombinant phage. Fig. 2shows the predicted translation product of one of these positive cDNA clones. This full-length clone contained the same DNA sequence that was present within the probe used for screening. The open reading frame encodes a protein of 151 amino acids with a predicted molecular mass of 17,403.9 Da. It is smaller than rat caveolin-1, which is 178 amino acids and has a predicted molecular mass of 20,408.2 Da. We termed this protein caveolin-3. Data base searches with the deduced protein sequence of caveolin-3 revealed significant identity only to caveolin-1 and caveolin-2, but not to any other known proteins.


Figure 2: Deduced protein sequence of rat caveolin-3. The protein sequence of rat caveolin-3 is compared with the known sequences of rat, chick, mouse, dog, and human caveolin-1 and with human caveolin-2. Residues identical to caveolin-3 are boxed. The position of the G-protein-binding region of caveolin-1 is overlined; the position of the hydrophobic membrane-spanning region is indicated by a boldface overline. A conserved methionine residue (an alternate start site in caveolin-1) and three conserved cysteine residues (sites of palmitoylation in caveolin-1) are indicated by the pound sign and asterisks, respectively.



An alignment of sequences of rat caveolin-3 with caveolin-1 and caveolin-2 shows that caveolin-3 is more similar to caveolin-1 than to caveolin-2 (Fig. 2). Specifically, caveolin-1 and caveolin-3 are 65% identical and 85% similar based on conservative amino acid substitutions. A single stretch of eight amino acids (FEDVIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a ``caveolin signature sequence'' that is characteristic of members of the caveolin gene family. In addition, the three cysteine residues that undergo palmitoylation in caveolin-1 (Fig. 2, see asterisks) (32) are absolutely conserved in caveolin-3, but not in caveolin-2. This suggests that caveolin-3 may be palmitoylated at these residues as well. Like caveolin-1, caveolin-3 contains a 33-amino acid membrane-spanning segment and a 44-amino acid C-terminal domain. However, the N-terminal domain of caveolin-3 is shorter than that of caveolin-1 by 27 amino acids. This fits well with the observation that two isoforms of caveolin-1 are generated by alternate translation initiation sites (methionines 1 and 32), yielding two protein products that differ in their extreme N terminus: caveolin-1alpha encoding residues 1-178 and caveolin-1beta encoding residues 32-178 (23) . Thus, caveolin-1beta (147 amino acids) is approximately the same length as caveolin-2 (149 amino acids) and caveolin-3 (151 amino acids).

Caveolin-3 Is Induced during Differentiation of Skeletal Myoblasts in Culture

As caveolin-3 mRNA is most highly expressed in muscle tissue types (Fig. 1), we used C2C12 cells to examine if caveolin-3 expression is regulated during skeletal muscle differentiation. 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. As a positive control for differentiation, the expression of troponin I mRNA (fast isoform) was also monitored. The fast isoform of troponin I is expressed only in skeletal muscle cells, but not in cardiac muscle or other tissues(40) . The constitutive expression of ribophorin I mRNA was monitored as a control for equal loading.

Compared with the constitutive expression of ribophorin I mRNA, caveolin-3 mRNA was undetectable in precursor myoblasts and strongly induced during myoblast differentiation (Fig. 3). Troponin I mRNA was dramatically induced to a similar extent as caveolin-3. These results are consistent with the selective expression of caveolin-3 mRNA in skeletal muscle and other muscle tissues (Fig. 1) and suggest that caveolin-3 may function in muscle from the earliest stages of its development.


Figure 3: Induction of caveolin-3 mRNA during the differentiation of C2C12 skeletal myoblasts. Each lane contains 4 µg of poly(A) RNA prepared from proliferating (P) or differentiated (D) C2C12 skeletal myoblasts. After initial hybridization with the cDNA for caveolin-3, the blot was stripped and reprobed with the ribophorin I cDNA as a control for equal loading and the troponin I (fast isoform) cDNA as a positive control for skeletal myoblast differentiation.



Recombinant Expression of Caveolin-3

To study the properties of caveolin-3, we recombinantly expressed a myc epitope-tagged form of the protein. Caveolin-1 tagged with the myc epitope is functionally indistinguishable from endogenous caveolin-1(23, 31, 32) . Fig. 4A illustrates recombinant expression of myc-tagged caveolin-3. Expression of caveolin-3 yielded a protein product of the expected molecular mass (18-20 kDa) that was slightly smaller than caveolin-1.


Figure 4: Recombinant expression of caveolin-3. A, expression of myc-tagged caveolin-3 in COS-7 cells yielded a protein product of the expected molecular mass (18-20 kDa). U, mock-transfected control; T, transfectants expressing caveolin-3. B, velocity gradient analysis of caveolin-3. Cells expressing myc-tagged caveolin-3 were solubilized, loaded atop a 5-40% sucrose gradient, and subjected to centrifugation for 10 h as we described previously for caveolin-1(15) . Note that caveolin-3 migrates mainly in fractions 6 and 7, identical to the migration of caveolin-1 shown for comparison. Arrows mark the positions of molecular mass standards. In A and B, expression of caveolin-3 was detected by immunblot analysis with monoclonal antibody 9E10, which recognizes the myc epitope (EQKLISEEDLN); caveolin-1 was detected using monoclonal antibody 2297.



Caveolin-1 exists as an 350-kDa homo-oligomer containing 14-16 monomers per oligomer as shown using several approaches including velocity gradient centrifugation(15, 16) . Oligomerization activity is localized to residues 61-101 of the cytoplasmic N-terminal domain of caveolin-1(15) ; this 41amino acid sequence is sufficient to confer oligomerization of the same stoichiometry upon a fusion protein (15) . As only seven amino acid changes occur in this 41-amino acid stretch between caveolin-1 and caveolin-3 and six out of seven of these changes are conservative substitutions, caveolin-3 might also form well-defined high molecular mass homo-oligomers. To test this hypothesis, we used an established velocity gradient system developed previously to study oligomers of caveolin-1(15) . Like caveolin-1, caveolin-3 migrated as a high molecular mass complex between the 200- and 443-kDa molecular mass standards (Fig. 4B). In contrast, several other integral membrane proteins migrate at their expected monomeric molecular mass in these same velocity gradients(15) .

The immunostaining pattern obtained with caveolin-3 is shown in Fig. 5and is very similar to immunostaining patterns observed previously for caveolin-1 and caveolin-2(7, 9, 14, 23, 24, 31) . Many small micropatches are present throughout the cell and along the cell surface. In addition, double-labeling experiments employing cells cotransfected with caveolin-1 and caveolin-3 demonstrate significant colocalization of these two distinct gene products (Fig. 5).


Figure 5: Immunolocalization of caveolin-1 and caveolin-3 within a single cell. COS-7 cells were cotransfected with untagged caveolin-1 and myc-tagged caveolin-3. Cells expressing both caveolin gene products were selected for imaging by confocal laser fluorescence microscopy. Caveolin-1 expression was detected with a rabbit polyclonal IgG probe that specifically reacts only with caveolin-1; caveolin-3 expression was detected with monoclonal antibody 9E10, which recognizes the myc epitope. Control experiments employing singly transfected populations of COS-7 cells confirmed the specificity of these antibodies; no cross-reaction was observed (data not shown). Bound primary antibodies were visualized by incubation with distinctly tagged fluorescent secondary antibodies (fluorescein-conjugated for caveolin-1 and rhodamine-conjugated for caveolin-3; see ``Experimental Procedures'').



To further examine the colocalization of caveolin-1 and caveolin-3, we stably expressed caveolin-3 in MDCK cells, which contain endogenous caveolin-1. To separate membranes enriched in caveolin-1 from the bulk of cellular membranes and cytosolic proteins, an established equilibrium sucrose density gradient system was utilized (7, 8, 12, 13, 17, 18, 21, 23, 34, 35, 41). In this fractionation scheme, immunoblotting with anti-caveolin-1 IgG can be used to track the position of caveola-derived membranes within these bottom-loaded sucrose gradients. Using this procedure, caveolin-1 is purified 2000-fold relative to total cell lysates as 4-6 µg of caveolin-rich domains (containing 90-95% of total cellular caveolin-1) are obtained from 10 mg of total MDCK proteins(13, 17) . We (7, 8) and others (21) have shown that these caveolin-rich fractions exclude >99.95% of total cellular proteins and also markers for the non-caveolar plasma membrane, Golgi apparatus, lysosomes, mitochondria, and endoplasmic reticulum. Fig. 6illustrates that in this fractionation scheme, 90-95% of both caveolin-1 and caveolin-3 cofractionate and are targeted to the same low density fractions, suggesting that they colocalize to similar areas of the plasma membrane. This is consistent with results demonstrating their colocalization by fluorescence microscopy in intact cells (Fig. 5).


Figure 6: Subcellular fractionation of MDCK cells recombinantly expressing caveolin-3. The distribution of total cellular proteins, caveolin-1, and caveolin-3 is shown. One-ml sucrose gradient fractions were collected from the top and analyzed by Ponceau S staining (upper panel) or immunoblotting (lower panels). Fractions 1-8 are the 5-30% sucrose layer, fractions 9-12 are the 40% sucrose layer, and fraction 13 is the insoluble pellet. Fractions 9-12 represent the ``loading zone'' of these bottom-loaded flotation gradients and contain the bulk of cellular membranes and cytosolic proteins (see ``Experimental Procedures''). Note that fractions 4 and 5 retain >95% of caveolin-1 and caveolin-3 and exclude 99.95% of total cellular proteins (based on independent protein determinations) and markers for the endoplasmic reticulum, Golgi apparatus, non-caveolar plasma membrane, mitochondria, and lysosomes as shown previously(7, 8, 21) .



A Caveolin-3-derived Polypeptide Functionally Affects the GTPase Activity of Purified Heterotrimeric G-proteins

Caveolin-1 functionally interacts directly with G-protein alpha-subunits(17) . Residues 82-101 of caveolin-1 are most critical for this interaction, and a caveolin-1-derived polypeptide encoding these residues can functionally suppress the basal GTPase activity of purified heterotrimeric G-proteins, apparently by inhibiting GDP/GTP exchange (17) . Thus, this caveolin-1-derived peptide acts as a GDI for heterotrimeric G-proteins(17) . This activity requires the complete caveolin-1 sequence from residues 82 to 101 as other peptides containing caveolin-1 residues 84-92 or 93-101 do not contain GDI activity(^2); also, a polypeptide derived from a caveolin-1 region that is not required for G-protein binding (residues 53-81) has no effect(17) .

A region that corresponds to caveolin-1 residues 82-101 is present within caveolin-3 and is highly conserved (six amino acid changes occur in a 20-amino acid stretch; all six changes are conservative substitutions) (Fig. 2). Thus, the effect of the corresponding caveolin-3-derived polypeptide on the functional properties of purified trimeric G-proteins (G(o) and G) was next evaluated. Fig. 7shows the effect of the caveolin-3-derived polypeptide on the turnover number of GTPase activity of purified trimeric G-proteins. Like polypeptides derived from caveolin-1, this caveolin-3-derived polypeptide dose-dependently suppressed the GTPase activity of both trimeric G(o) and G with IC values of 3 and 5 µM, respectively. A 10 µM concentration of this peptide yielded 80% inhibition for G(o) and total inhibition for G. This is the same potency reported previously for the caveolin-1-derived polypeptide(17) . However, unlike the caveolin-1-derived polypeptide, lower concentrations of the caveolin-3-derived polypeptide stimulated the GTPase activity. The steady-state GTPase activities of G(o) and G were dose-dependently stimulated by nanomolar concentrations with EC values of 500 and 300 nM, respectively. Maximal stimulation was 1.6- and 1.3-fold over the basal activities of G(o) and G, respectively. These findings are consistent with the recent observation that a polypeptide derived from the corresponding region of caveolin-2 selectively stimulates the GTPase activity of purified trimeric G-proteins, acting as a GTPase-activating protein (GAP)(24) . Thus, this caveolin-3-derived polypeptide has both activities that are found separately in caveolin-1 and caveolin-2.


Figure 7: Effect of caveolin-3-derived polypeptides on the basal GTPase activity of purified heterotrimeric G-proteins. Upper, trimeric G(o); lower, trimeric G. The activity is expressed as a percentage of the basal activity, which was 0.17 ± 0.01 min for G(o) and 0.05 ± 0.005 min for G (mean ± S.E.; n = 3). All experiments were done independently three times, and the values indicate the means ± S.E.




DISCUSSION

Caveolin-3 joins caveolin-1 and caveolin-2 as the third member of a growing caveolin gene family. As two forms of caveolin-1 (alpha- and beta-isoforms) exist(23) , there are now four distinct caveolin protein products (Fig. 8).


Figure 8: Schematic diagram summarizing known caveolin family members. The overall structures of caveolin-1 (alpha- and beta-isoforms), caveolin-2, and caveolin-3 are shown. All four protein products contain the invariant sequence FEDVIAEP within their hydrophilic N-terminal domains. Note that caveolin-1beta (147 amino acids) is approximately the same length as caveolin-2 (149 amino acids) and caveolin-3 (151 amino acids); percent similarity and identity of caveolin-2 and caveolin-3 to caveolin-1 are shown. GDI and GAP activities are also noted. TM, transmembrane domain.



Caveolin-1, -2, and -3 all have a similar overall structure with a hydrophilic N-terminal domain, a 33-amino acid membrane-spanning segment, and a 43-44-amino acid hydrophilic C-terminal domain, and all three contain the invariant sequence FEDVIAEP within their N-terminal domains. These similarities predict that caveolin-2 and caveolin-3 should assume the same hairpin topology as caveolin-1; the N- and C-terminal domains of caveolin-1 are known to face the cytoplasm(23, 32, 42) . Caveolin-2 and caveolin-3 colocalize with caveolin-1 when recombinantly expressed within a single cell (caveolin-1 and caveolin-2 (24) ; caveolin-1 and caveolin-3 (this report)), indicating that all three caveolins localize to caveolae. Based on sequence homology, caveolin-3 is most closely related to caveolin-1 and is most distant from caveolin-2. In accordance with this homology, both caveolin-1 (15, 16) and caveolin-3 (this report) form high molecular mass oligomers, while only a dimeric form of caveolin-2 has been detected(24) .

Caveolin-1 and caveolin-2 are most highly expressed in white adipose tissue, and both are induced during adipocyte differentiation(21, 24) ; caveolin-3 is most highly expressed in muscle tissues and is induced during the differentiation of skeletal myoblasts in culture. This suggests that caveolin-1 and caveolin-2 may be very important for adipocyte function, while caveolin-3 is important for muscle cell types. The tissue-specific expression of different caveolin genes could help generate the formation of caveolae that are tailored to the function of a given cell type. For example, smooth muscle cell caveolae remain attached to the plasma membrane and are viewed as static structures, while endothelial cell caveolae detach from the plasma membrane and participate in endocytic and transcytotic transport events (1) . The reason for these cell type-specific differences in the behavior of caveolae remains unknown; these differences could reflect tissue-specific expression of functionally distinct caveolin genes.

Prior to the identification of caveolin-3, we looked for expression of caveolin-1 in skeletal muscle fibers by immunofluorescence microscopy using four different caveolin-1-specific antibodies. Caveolin-1 expression was detected in surrounding endothelial cells, but not in skeletal muscle fibers, (^3)explaining the apparent detection of small amounts of caveolin-1 mRNA in skeletal muscle tissue (Fig. 1, upper panel). Also, we were unable to detect caveolin-1 protein in differentiated or undifferentiated C2C12 skeletal myoblasts. As muscle cells contain caveolae, it appeared that caveolin-1 was not required for the formation of skeletal muscle cell caveolae. We also considered the possibility that a muscle-specific form of caveolin might exist that was not recognized by available caveolin-1 antibodies. However, we had no direct evidence to support this prediction. Our current observations that caveolin is a multigene family and that caveolin-3 mRNA is predominantly expressed in muscle tissues and differentiated C2C12 myoblasts may explain our inability to detect caveolin-1 in skeletal muscle fibers and C2C12 cells. Thus, a failure to detect caveolin-1 expression should not be interpreted as an absence of caveolae. For example, based on a lack of caveolin-1 expression, other investigators have concluded that caveolae do not exist in lymphocytes and neurons(43, 44) . However, this predates the observation that caveolin is actually a multigene family of immunologically distinct but homologous molecules. Thus, other as yet undiscovered caveolin genes may exist that are expressed in lymphocytes and neuronal cells.

What could be the functional significance of caveolin diversity? Cell fractionation studies indicate that caveolin-1 copurifies with signal transducers, including G-proteins(7, 8, 9, 10, 11, 12, 17, 23) . Using an in vitro binding assay, caveolin-1 interacts directly with inactive G-protein alpha-subunits; this G-protein binding activity is located within residues 82-101 of the cytoplasmic N-terminal domain of caveolin-1(17) . A polypeptide containing caveolin-1 residues 82-101 functionally suppresses the basal activity of purified trimeric G-proteins by inhibiting GDP/GTP exchange, acting as a GDI for trimeric G-proteins(17) . Conversely, the corresponding polypeptide from caveolin-2 acts as a GAP(24) . As both caveolin-1 and caveolin-2 are coexpressed within the same cells, we have proposed that they could act in concert to recruit and sequester G-proteins in the GDP-liganded conformation within caveolae for presentation to activated G-protein-coupled receptors(24) . More specifically, we have postulated that caveolin-2 would act first as a GAP to actively place the G-protein in the inactive GDP-bound state, and caveolin-1 would then act as a GDI to hold the G-protein in the inactive conformation by preventing GDP/GTP exchange(24) . Here, we find that the corresponding caveolin-3-derived polypeptide has both activities found separately in caveolin-1 and caveolin-2; at nanomolar concentrations, the caveolin-3-derived polypeptide acts to stimulate the GTPase activity of purified trimeric G-proteins, and at micromolar concentrations, it suppresses their GTPase activity. Thus, caveolin-3 might subsume the functional roles of both caveolin-1 and caveolin-2 in certain muscle cell types.

We have previously evaluated the effect of over 200 polypeptides on the functional activities of purified trimeric G-proteins(39, 45, 46, 47, 48, 49, 50) , and the caveolin-derived peptides ( (17) and (24) and this report) are the only polypeptides that produce GDI and/or GAP activities. However, many receptor-derived polypeptides contain GDP dissociation stimulator activity as expected, stimulating both GTPase activity and GTPS binding at micromolar concentrations(39, 45, 46, 47, 48, 49, 50) .

It has been known for many years that caveolae are morphologically abundant in all muscle cell types (smooth, cardiac, and skeletal) (51, 52, 53, 54, 55, 56, 57, 58) . This appears to be relevant to the pathogenesis of Duchenne's muscular dystrophy. More specifically, (i) dystrophin has been localized to plasma membrane caveolae in smooth muscle cells(59) , and (ii) skeletal muscle caveolae undergo characteristic changes in size and their distribution in patients with Duchenne's muscular dystrophy, but not in other forms of muscular dystrophy examined(60) . This indicates that caveolae may play an important role in muscle biology. In this regard, future studies on caveolin-3 should help to elucidate the role of caveolae in muscle cell membrane biology.


FOOTNOTES

*
This work was supported in part by a grant from the W. M. Keck Foundation to the Whitehead Fellows program (to M. P. L.), National Institutes of Health FIRST Award GM-50443 (to M. P. L.), National Institutes of Health Grants GM-49516 and DK-47618 (to H. F. L.), and a grant from Bristol-Myers-Squibb (to I. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31968[GenBank].

§
Supported by a Swiss National Science Foundation fellowship.

Recipient of fellowships from the Byotai-Taisha Foundation and the Mochida Memorial Foundation.

**
To whom correspondence should be addressed: Whitehead Inst. for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479. Tel.: 617-258-5225; Fax: 617-258-9872; lisanti{at}wi.mit.edu.

(^1)
The abbreviations used are: GDI, GDP dissociation inhibitor; MDCK, Madin-Darby canine kidney; Mes, 4-morpholineethanesulfonic acid; MBS, Mes-buffered saline; PBS, phosphate-buffered saline; GAP, GTPase-activating protein; GTPS, guanosine 5`-O-(3-thiotriphosphate).

(^2)
M. P. Lisanti and T. Okamoto, unpublished observations.

(^3)
M. P. Lisanti et al., unpublished observations.


ACKNOWLEDGEMENTS

We thank the following people: Mark Chafel for confocal immunofluorescence microscopy, Guilia Baldini for help in Northern blot analysis, John R. Glenney for generously donating anti-caveolin-1 monoclonal antibodies, Tomiko Asano for purified G, Tatsuya Haga for purified G(o), and Marcia Glatt and other members of the Whitehead purchasing department for dedicated service.


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