Identification, Sequence, and Expression of an Invertebrate Caveolin Gene Family from the Nematode Caenorhabditis elegans
IMPLICATIONS FOR THE MOLECULAR EVOLUTION OF MAMMALIAN CAVEOLIN GENES*

(Received for publication, August 20, 1996, and in revised form, October 23, 1996)

ZhaoLan Tang Dagger , Takashi Okamoto §, Pratumtip Boontrakulpoontawee , Toshiaki Katada par , Anthony J. Otsuka and Michael P. Lisanti Dagger **

From Dagger  The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, § Shriners Hospitals for Crippled Children, Massachusetts General Hospital, Department of Anesthesia, Harvard Medical School, Boston, Massachusetts 02114, the  Department of Biological Sciences, Illinois State University, Normal, Illinois 61790, and the par  Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University, Hongo, Bunkyo, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Caveolae are vesicular organelles that represent an appendage of the plasma membrane. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Caveolin has been proposed to function as a plasma membrane scaffolding protein to organize and concentrate signaling molecules within caveolae, including heterotrimeric G proteins (alpha  and beta gamma subunits). In this regard, caveolin interacts directly with Galpha subunits and can functionally regulate their activity. To date, three cDNAs encoding four subtypes of caveolin have been described in vertebrates. However, evidence for the existence of caveolin proteins in less complex organisms has been lacking.

Here, we report the identification, cDNA sequence and genomic organization of the first invertebrate caveolin gene, Cavce (for caveolin from Caenorhabditis elegans). The Cavce gene, located on chromosome IV, consists of two exons interrupted by a 125-nucleotide intron sequence. The region of Cavce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cavce. This suggests that mammalian caveolins may have evolved from the second exon of Cavce. Cavce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. Remarkably, the invertebrate Cavce protein behaves like mammalian caveolins: (i) Cavce forms a high molecular mass oligomer, (ii) assumes a cytoplasmic membrane orientation, and (iii) interacts with G proteins. A 20-residue peptide encoding the predicted G protein binding region of Cavce possesses "GDP dissociation inhibitor-like activity" with the same potency as described earlier for mammalian caveolin-1. Thus, caveolin appears to be structurally and functionally conserved from worms to man. In addition, we find that there are at least two caveolin-related genes expressed in C. elegans, defining an invertebrate caveolin gene family. These results establish the nematode C. elegans as an invertebrate model system to study caveolae and caveolin in vivo.


INTRODUCTION

Caveolae are vesicular organelles that represent a subdivision of the plasma membrane (1, 2). Although they are found in most cell types, caveolae are most numerous in adipocytes, endothelial cells, fibroblasts, and muscle cells (smooth, skeletal, and cardiac). In transmission electron micrographs they can be distinguished by their characteristic shape: ~50-100-nm vesicles located at or near the plasma membrane (3, 4). Functionally, caveolae have been implicated in a variety of signal transduction related events, including signaling from G protein-coupled receptors and growth factor receptors (5-7).

Caveolin, a 21-24-kDa protein, is an integral membrane component of caveolae (8-12). Caveolin co-purifies with lipid modified signaling molecules, such as G proteins (alpha  and beta gamma subunits), Src-family tyrosine kinases, and H-Ras (see Refs. 6, 9, and 13-18 and references cited within). These molecules appear to be tightly associated as a discrete complex with caveolin as shown using a polyhistidine-tagged form of caveolin for detergent-free affinity purification of caveolae membranes (18).

Caveolin is thought to play an important structural role in the formation of caveolae membranes. In this regard, caveolin may act as a scaffolding protein to organize and concentrate specific caveolin-interacting lipids and proteins within caveolae microdomains (19). In support of this notion, caveolin forms a high molecular mass homo-oligomer (19, 20), interacts specifically with cholesterol and glycolipids (21-23), and interacts directly with signaling molecules, including heterotrimeric G proteins and H-Ras (18, 21, 24). In addition, caveolin interacts only with the GDP-liganded conformation of these molecules and caveolin can function as a GDP dissociation inhibitor (GDI)1 for heterotrimeric G proteins, holding the G protein in the inactive conformation (24).

Caveolin expression appears to drive caveolae formation. For example, caveolae organelles and caveolin protein are induced ~10-25 fold during adipocyte differentiation (25-27). Conversely, caveolin protein expression is down-regulated in response to cell transformation by activated oncogenes (such as H-ras (G12V) and v-abl); down-regulation of caveolin protein coincides with the disappearance of caveolae from these transformed cells (28). Also, recombinant overexpression of caveolin in caveolin-deficient cells can lead to the formation of caveolae in lymphocytes (29) and insect Sf21 cells (30).

However, there are certain cell lines which morphologically contain caveolae, but fail to express caveolin (31). This finding has suggested that other caveolin-related proteins may exist that are immunologically distinct from caveolin. In support of this notion, two novel caveolin-related proteins have recently been identified and cloned. These proteins, termed caveolin-2 and caveolin-3, are the products of separate caveolin genes (27, 32, 33). Thus, caveolin (retermed caveolin-1) is the first member of a multigene family (27).

Caveolins 1, 2, and 3 are structurally homologous proteins but are immunologically distinct molecules; they have different but overlapping tissue distributions (27, 32-34). For example, the expression of caveolin-3 is absolutely muscle-specific (skeletal and cardiac muscle cells) (32, 34). Caveolin-1 is not expressed in these striated muscle tissues, but smooth muscle cells co-express caveolins 1 and 3 (34). Furthermore, caveolin-1 and -2 are co-expressed in adipocytes and share the same overlapping tissue distribution (27). Thus, a given mammalian cell, such as smooth muscle cells or fibroblasts, may co-express up to three or four immunologically distinct caveolin protein products.

Due to the complexity inherent in studying mammalian systems, we have chosen to search for caveolin genes in model invertebrate organisms. Here, we report the identification of the first invertebrate caveolin gene, Cavce, in the nematode Caenorhabditis elegans. Cavce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. The identification and sequencing of Cavce represents a starting point for reverse genetics experiments designed to isolate animals mutated in a caveolin gene.


EXPERIMENTAL PROCEDURES

Materials

The cDNAs for mammalian caveolins 1, 2, and 3 were as we described previously (27, 31, 32). The C. elegans EST clone yk74b2 was obtained from Dr. Yuji Kohara (Gene Library Laboratory, National Institute of Genetics, Japan). Rabbit polyclonal IgG and mouse monoclonal IgG (mAb 2297) directed against caveolin-1 were the generous gift of Dr. John R. Glenney (Transduction Laboratories, Lexington, KY). Anti-Myc IgG (9E10) and anti-FLAG (M2) IgG were from Santa Cruz Biotechnologies and IBI, respectively. Other reagents were from the following commercial suppliers: prestained protein markers, fetal bovine serum, and other cell culture reagents (Life Technologies, Inc.); and Slow-Fade anti-fade reagent (Molecular Probes, Inc.). Peptide synthesis was performed by the Biopolymers Facility at the Massachusetts Institute of Technology.

Recombinant Expression of Cavce

Epitope-tagged forms of the Cavce cDNA were subcloned into the MCS (KpnI/XbaI) of the vector pCB7 for expression in COS-7 cells. A Myc epitope was incorporated into the N terminus (M<UNL>EQKLISEEDLN</UNL>GG-Cavce) and a FLAG epitope tag was incorporated into the C terminus (Cavce-GG<UNL>DYKDDDDK</UNL>); GG was placed as a spacer between the epitopes and the Cavce coding sequence, as performed previously for caveolins 1, 2, and 3 (11, 27, 32, 33, 35, 36). Using this scheme, both singly tagged and doubly tagged versions of Cavce were produced. Constructs were transiently transfected into COS-7 cells, as described previously. Expression was detected using mAb 9E10 that recognizes the Myc epitope or mAb M2 that recognizes the FLAG epitope.

Velocity Gradient Centrifugation

The molecular mass of Cavce was estimated as described previously for mammalian caveolins 1, 2, and 3 (19, 27, 32). Briefly, samples were dissociated in Mes-buffered saline containing 60 mM octyl glucoside. Solubilized material was loaded atop a 5-40% linear sucrose gradient and centrifuged at 50,000 rpm (340,000 × g) for 10 h in an SW60 rotor (Beckman Instruments). Gradient fractions were collected from above and subjected to immunoblot analysis. Molecular mass standards for velocity gradient centrifugation were as described previously (19, 27, 32). Note that caveolin-2 is a dimer of ~40 kDa, and it correctly migrates between the 29-kDa and 66-kDa molecular mass standards using this velocity gradient system (27).

Immunofluorescence

All reactions were performed at room temperature. Transfected COS-7 cells were briefly washed three times with PBS and fixed for 20 min in PBS containing 4% paraformaldehyde. Fixed cells were rinsed with PBS and treated with 25 mM NH4Cl in PBS for 10 min to quench free aldehyde groups. Cells were then permeabilized with 0.1% Triton X-100 for 10 min and washed with PBS (three times, 10 min each). For double-labeling, the cells were then successively incubated with PBS, 3% bovine serum albumin containing: (i) 50 µg/ml each of normal goat and donkey IgGs, (ii) a 1:400 dilution of mAb 9E10 and 40 µg/ml anti-caveolin-1 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). Alternatively, a 1:400 dilution of anti-FLAG IgG (mAb M2) was utilized in step (ii). The first incubation was 30 min, while primary and secondary antibody reactions were 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

Transfected COS-7 cells grown to confluence in 100-mm dishes were used to prepare caveolin-enriched membrane fractions, essentially as we have described previously (24, 26, 31, 36-38). However, two specific modifications were introduced to allow the purification of caveolin-rich domains without the use of detergent (18, 34). Triton X-100 was replaced with sodium carbonate buffer, and a sonication step was introduced to more finely disrupt cellular membranes (18, 34). After two washes with ice-cold PBS, cells (two confluent 100-mm dishes) were scraped into 2 ml of 500 mM sodium carbonate, pH 11.0. Homogenization was carried out sequentially in the following order using: (i) a loose fitting Dounce homogenizer (10 strokes); (ii) a Polytron tissue grinder (three 10 s bursts; Kinematica GmbH, Brinkmann Instruments, Westbury, NY); and (iii) a sonicator (three 20-s bursts; Branson Sonifier 250, Branson Ultrasonic Corp., Danbury, CT). The homogenate was then adjusted to 45% sucrose by addition of 2 ml of 90% sucrose prepared in MBS (25 mM Mes, pH 6.5, 0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. A 5-35% discontinuous sucrose gradient was formed above (4 ml of 5% sucrose, 4 ml of 35% sucrose; both in MBS containing 250 mM sodium carbonate) and centrifuged at 39,000 rpm for 16-20 h in an SW41 rotor (Beckman Instruments). A light-scattering band confined to the 5-35% sucrose interface was observed that contained caveolin, but excluded most other cellular proteins.

Immunoblotting of Gradient Fractions

Gradient fractions were separated by SDS-PAGE (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 (mAb 2297; 1:400) or with 9E10 ascites (1:500) to visualize epitope-tagged Cavce. For immunoblotting, incubation conditions were as described by the manufacturer (Promega; 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 Cavce that remains associated with caveolin-enriched fractions was estimated by immunoblotting, as described elsewhere (37). 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 previously (39).

GTP Hydrolysis and GTPgamma S Binding Assays

Trimeric Gi2 purified from bovine spleen was provided by T. Asano (40). Trimeric Go purified from bovine brain was provided by T. Katada (41). Steady-state GTP hydrolysis activity was examined as we described previously (42). Briefly, the assay was performed for 20 min at 37 °C in the presence of 20 µM Mg2+ with 10 nM G protein. The GTPgamma S binding assay was performed as we described elsewhere (42) by incubating 10 nM Go with GTP in the presence of 20 µM Mg2+ for 2 min at 37 °C. The Cavce-derived polypeptide contained the sequence DCVWRLNHTVFTAVRLFIYR, corresponding to amino acids 132-151 of Cavce.

Cloning and Analysis of the Genomic Copies of Murine Caveolins 2 and 3

Probes corresponding to the cDNAs of both caveolin-2 and caveolin-3 were labeled with digoxigenin, according to the manufacter's instructions (Boehringer Mannheim). These labeled probes were used to screen a previously described lambda -2 murine genomic library (43, 44) (generous gift of H. Wu and R. Jaenisch, Whitehead Institute). A total of 100,000 plaque-forming units were screened; three independent genomic clones were isolated for caveolins 2 and 3.

To determine possible intron-exon boundaries, we performed PCR with the following primers based on the known cDNA sequences of caveolin-2 and caveolin-3. The caveolin-2 primers were: 2-1, 5'-ATGGACGACGACTCCTACAGC-3'; nucleotides 1-21; 2-2, 5'-ATCGTCCTACGCTCGTACACAATGGAG-3', anti-parallel to nucleotides 386-412; and 2-3, 5'-TCAATCCTGGCTCAGTTGCA-3', anti-parallel to nucleotides 431-450. The caveolin-3 primers were: 3-1, 5'-ATGATGACCGAAGAGC-3', nucleotides 1-16; 3-2, 5'-GATCCAGTGCATCA-3', nucleotides 339-352; 3-3, 5'-AGTGCCCTCGGGCTCCGC-3', anti-parallel to nucleotides 136-153; 3-4, 5'-GGCCCAGATGTGGCAGA-3', anti-parallel to nucleotides 290-306; and 3-5, 5'-TTAGCCTTCCCTTCGCAGCAC-3', anti-parallel to nucleotides 436-456. In all cases, the cDNAs for caveolins 2 and 3 were used as a positive control.

Molecular Cloning of the Cavce-2 cDNA

To obtain the Cavce-2 cDNA, a C. elegans cDNA library was used as the substrate for PCR. Primers were designed based on genomic sequences obtained by the C. elegans Genome project (accession no. Z77655[GenBank]; cosmid C56A3). The primers were as follows (restriction sites are underlined): 5'-CCGGCC<UNL>GAATTC</UNL>ATGACTCGTCAGAATACTTCC-3' and 5'-CCGGCC<UNL>GGATCC</UNL>TTAAACATGATGAATGTGTTT-3'. As expected an ~1-kb band was observed. After gel purification, this product was subjected to direct double-stranded DNA sequencing in both directions. This product was also subcloned into the MCS (EcoRI/BamHI) of the vector pBS II KS+ for additional DNA sequencing and further analysis.

Northern Analysis

C. elegans (N2 strain) were used as the source to purify poly(A)+ RNA. mRNA isolation as well as agarose gel electrophoresis and transfer to nylon membranes was performed as described previously (45, 46); hybridizations were performed in 50% formamide, 5 × SSC, 25 mM sodium phosphate, pH 7.0, 10 × Denhardt's solution, 5 mM EDTA, 1% SDS, 0.1 mg/ml poly(A)+ at 42 °C overnight and subsequently washed in 2 × SSC, 0.1% SDS and 0.5 × SSC, 0.1% SDS at 50 °C; radiolabelded DNA concentrations were at 2 × 106 cpm/ml.


RESULTS AND DISCUSSION

A Caveolin Homologue from the Nematode C. elegans: Identification, Sequence, and Genomic Organization

In order to identify an invertebrate caveolin gene, we searched existing data bases of model invertebrate organisms using the protein sequences of mammalian caveolins 1, 2, and 3. Using this approach, we identified a short EST sequence potentially encoding a caveolin homologue in the nematode C. elegans.

This EST clone (yk74b2, accession no. D71360[GenBank]) was obtained from Dr. Yuji Kohara (C. elegans Genome Project) for further study. Analysis and sequencing of clone yk74b2 revealed that it contains an open reading frame encoding a caveolin-related protein of 235 amino acids (708 bases). Fig. 1 shows the deduced protein sequence. For simplicity, we have termed this protein caveolince (or Cavce; for caveolin from C. elegans). Cavce contains a 151-amino acid N-terminal domain, a 32-amino acid membrane spanning segment, and a 52-amino acid C-terminal domain, with a predicted molecular mass of 26,293 Da. Cavce is substantially longer than the known mammalian caveolins, which are ~147-178-amino acid in length.


Fig. 1. cDNA sequence and deduced protein sequence of an invertebrate caveolin, Cavce, from the nematode C. elegans. Numbers at left indicate amino acid positions. Note that Cavce contains three potential sites for phosphorylation by casein kinase II ((S/T)-X2-(D/E)) at amino acid positions 3, 27, and 58. It also contains a consensus site for amidation (X-G-(R/K)-(R/K)) at amino acid position 47 and a single consensus site for SH-3 domain binding (P-X2-P) at amino acid position 35.
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Using the C. elegans Blast server, we compared the deduced protein sequence of Cavce with the sequences of catalogued genomic clones prepared by the C. elegans Genome Project. Our searches indicate that the Cavce gene is included within the ~40-kb insert of cosmid T13F2 which maps to chromosome IV, more specifically within the major sperm protein region. This direct nucleotide sequence comparison between the Cavce cDNA and the corresponding genomic sequence of cosmid T13F2 allowed us to deduce the intron-exon organization of the Cavce gene. As illustrated in Fig. 2, the Cavce gene consists of two exons interrupted by a 125-base intron sequence. The 5' and 3' DNA sequences flanking the intron correspond to the accepted consensus sequences for donor and acceptor splice sites (Fig. 2B).


Fig. 2. Genomic organization of the Cavce gene. A, intron-exon boundaries are indicated and were deduced by comparision of the cDNA sequence with the sequence of genomic clone T13F2. The Cavce gene consists of two exons with a 125-base pair intron. B, the sequences flanking the intron are shown and conform to accepted consensus sequences for donor and acceptor splice sites.
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Fig. 3 shows an alignment of the Cavce protein sequence with the known protein sequences of mammalian caveolins. The region of highest homology is restricted to Cavce residues 100-235. This 135-amino acid region is 67% similar and 37% identical to the mammalian caveolins 1 and 3; 65% similarity and 32% identity were obtained by comparision with caveolin-2. Thus, Cavce is roughly equally related to all three known mammalian caveolins and could represent a common ancestor. Interestingly, Cavce residues 100-235 are restricted to the second exon of Cavce that encodes residues 93-235. Thus, the region of Cavce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cavce. This suggests that mammalian caveolins may have evolved from this second exon of Cavce.


Fig. 3. Alignment of the invertebrate Cavce protein with the protein sequences of known mammalian caveolin genes. Residues identical to Cavce are boxed. The position of the G protein binding region in mammalian caveolins is overlined; the position of the hydrophobic membrane spanning region is indicated by a boldface overline. A conserved tyrosine residue that is the site for v-Src phosphorylation in caveolin-1 (30) is indicated by the pound sign (#); a C-terminal cysteine residue that is a potential site for palmitoylation is indicated by an asterisk.
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This Cavce region (residues 93-235) is 142 amino acids in length, approximating the size of mammalian caveolins. Also, this Cavce region is predicted to contain the most important features of mammalian caveolins, the N-terminal oligomerization domain and G protein-interacting sequences, the membrane-spanning segment, and the C terminus.

Perhaps the first exon encoded by Cavce evolved into a separate mammalian gene product. Interestingly, searches with the deduced protein sequence of the first coding exon revealed that this region of the Cavce protein is most closely related to a region of the mammalian MARCKS protein (myristoylated alanine-rich C-kinase substrate) and a MARCKS-related protein termed MAC-MARCKS.

Recombinant Expression and Characterization of the Cavce Protein Product

To study the properties of the Cavce protein, we constructed an epitope-tagged form of Cavce for expression in mammalian cells. A Myc epitope tag was placed at its extreme N terminus and a FLAG epitope tag was placed at its extreme C terminus. Note that epitope tagging in this fashion does not affect the behavior of mammalian caveolins 1, 2, and 3, or their subcellular localization within caveolae membranes (11, 27, 32, 33, 35, 36).

Fig. 4A shows the recombinant expression of the epitope-tagged Cavce protein in COS-7 cells. Expression yielded a protein product of ~32-33 kDa. Similar results were obtained using singly tagged versions of Cavce containing exclusively either the Myc or the FLAG epitope. Thus, as with mammalian caveolins (27, 32), Cavce migrates several kilodaltons higher than is predicted from its amino acid sequence.


Fig. 4. Recombinant expression of Cavce in mammalian cells. A, expression of epitope-tagged Cavce in COS-7 cells yielded a protein product of ~32-33 kDa. B, velocity gradient analysis of Cavce. COS-7 cells expressing epitoped-tagged Cavce were solubilized and loaded atop a 5-40% sucrose gradient, as described previously for mammalian caveolin proteins. After centrifugation, fractions were collected and subjected to SDS-PAGE/Western blot analysis. Note that Cavce migrates mainly in fractions 7 and 8. The migration of caveolin-1 is shown for comparison; caveolin-1 migrates mainly in fraction 6. Arrows mark the positions of molecular mass standards. In A and B, expression of Cavce was detected with the mAb 9E10 that recognizes the Myc epitope. Similar results were obtained using mAb M2 that recognizes the FLAG epitope (not shown). Caveolin-1 was detected with mAb 2297.
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Caveolin-1 forms a ~350-kDa homo-oligomer containing ~14-16 caveolin monomers per oligomer (19, 20). These homo-oligomers are thought to function as building blocks in the construction of caveolae membranes. Similarly, caveolin-3 forms homo-oligomers of the same size as caveolin-1 (32). In contrast, caveolin-2 exists as a homodimeric complex (27). In caveolin-1, the oligomerization domain has been localized to a 41-amino acid membrane proximal region of the cytoplasmic N-terminal domain using a battery of glutathione S-transferase-caveolin fusion proteins expressed in Escherichia coli (19). This region is highly conserved in all mammalian caveolin subtypes and invertebrate Cavce.

Thus, we next investigated the oligomeric state of Cavce. For this pupose, we employed an established velocity gradient system developed previously to study the oligomeric state of mammalian caveolins 1, 2, and 3 (19, 27, 32). Fig. 4B shows that Cavce behaved as a high molecular mass complex, migrating between the 200- and 443-kDa molecular mass standards (peak fractions 7 and 8). The migration of caveolin-1 is shown for comparison (peak fraction 6). As expected, Cavce migrated with a slightly higher molecular mass than caveolin-1, as Cavce has a higher monomeric molecular mass than caveolin-1.

Subcellular Distribution and Membrane Topology of Cavce

To determine if caveolin-1 and Cavce co-fractionate when Cavce is expressed in mammalian cells, we transiently expressed Cavce in COS-7 cells and subjected them to subcellular fractionation. A protocol involving homogenization in sodium carbonate followed by equilibrium sucrose density centrifugation was used to separate membranes enriched in caveolin-1 from the bulk of cellular membranes and cytosolic proteins (18, 34). In this fractionation scheme, immunoblotting with anti-caveolin-1 IgG can be used to track the position of caveolae-derived membrane domains (24, 26, 31, 36-38). Fig. 5 shows that ~90-95% of caveolin-1 and Cavce co-fractionate and are localized to the same low density fractions (gradient fractions 5 and 6).


Fig. 5. Detergent-free subcellular fractionation of mammalian cells expressing Cavce. COS-7 cells expressing epitoped-tagged Cavce were subjected to subcellular fractionation after homogenization in a buffer containing sodium carbonate (see "Experimental Procedures"). In this fractionation scheme, note that (i) Cavce (fractions 5 and 6) is separated from most cellular proteins (fractions 9-13) and (ii) Cavce and mammalian caveolin-1 co-fractionate (fractions 5 and 6).
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Immunostaining of COS-7 cells expressing epitope-tagged Cavce revealed punctate fluorescence or micropatches along the surface of the cell and within the perinuclear region (Fig. 6, left). A similar staining pattern has been observed for the distribution of mammalian caveolins (9, 11, 12, 27, 31-33, 35, 36, 47). In addition, double-labeling of cells co-transfected with Cavce and caveolin-1 revealed significant co-localization of these distinct caveolin proteins (Fig. 6). This is consistent with results demonstrating their co-fractionation during subcellular fractionation (Fig. 5).


Fig. 6. Localization of Cavce and caveolin-1 within a single cell. COS-7 cells were co-transfected with epitope-tagged Cavce and untagged mammalian caveolin-1. Cavce expression was detected with the mouse mAb M2 that recognizes the FLAG tag; caveolin-1 expression was detected using rabbit polyclonal IgG directed against caveolin-1. Control experiments using singly transfected populations of cells confirmed the specificity of these antibodies; no cross-reaction was observed (not shown). Transfected cells expressing both Cavce and caveolin-1 were selected for imaging by laser confocal fluorescence microscopy. Primary antibodies were detected using distinctly tagged fluorescent secondary antibodies (rhodamine-conjugated for Cavce (left) and fluorescein-conjugated for caveolin-1 (right)). Virtually identical results were obtained when mAb 9E10 that recognizes the Myc epitope was used to detect epitope-tagged Cavce (not shown).
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The transmembrane domain of mammalian caveolins is thought to form a hairpin loop within the membrane, allowing both the N-terminal domain and the C-terminal domain to remain entirely cytosolic (35, 36, 48). In accordance with this topology, caveolin is inaccessible to biotinylation probes that efficiently label other plasma membrane proteins within caveolae (19). This unusual membrane topology has been confirmed using several independent approaches, including epitope tagging (35, 36). In this regard, immunolocalization of an N-terminal epitope tag or a C-terminal epitope tag attached to caveolin-1 requires detergent permeabilization to allow specific IgG access to the cytoplasm (35, 36).

Here, we have performed the analogous experiment with Cavce. As illustrated in Fig. 7, immunolocalization of Cavce using the N-terminal Myc tag or the C-terminal FLAG tag required detergent permeabilization; no immunostaining was observed if the permeabilization step was omitted. Thus, it appears that Cavce assumes the same unusual cytoplasmic membrane topology as mammalian caveolins, with both N-terminal and C-terminal domains facing the cytoplasm.


Fig. 7. Membrane orientation of Cavce. After fixation with paraformaldehyde, COS-7 cells expressing epitope-tagged Cavce were immunostained with anti-Myc IgG or anti-FLAG IgG in the presence or absence of detergent (0.1% Triton X-100), as indicated. Bound primary antibodies were visualized with a rhodamine-conjugated donkey anti-mouse IgG; see "Experimental Procedures." Note that visualization of either epitope (Myc or FLAG) is strictly dependent on membrane permeabilization.
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A Cav ce-derived Peptide Suppresses the GTPase Activity and GTPgamma S Binding of Purified Heterotrimeric G Proteins

Mammalian caveolins functionally interact directly with G protein alpha -subunits (18, 21, 24, 27, 32). This binding activity is encoded by a 20-amino acid membrane proximal region within caveolins 1, 2, and 3. Peptides encoding this region differentially affect the GTPase activity of G proteins, depending on the caveolin subtype. For example, the caveolin-1-derived peptide acts as a GDI, while the same region of caveolin-2 acts as a GTPase-activating protein (GAP) (24, 27). The caveolin-3-derived peptide expresses both GDI and GAP activities: (i) at nanomolar concentrations, the caveolin-3-derived peptide stimulates their GTPase activity; and (ii) at micromolar concentrations, it suppresses their GTPase activity (32). We have suggested such GAP and GDI activities could function in concert to recruit and sequester G proteins in the GDP-liganded conformation within caveolae (27, 32). In this two-step mechanism, GAP activity would first actively place the G protein in the inactive GDP-bound state, and GDI activity would then hold the G protein in the inactive GDP-bound state by preventing GDP/GTP exchange (27, 32).

The predicted G protein binding region of Cavce is indicated in Fig. 1. We synthesized a peptide encoding this 20-amino acid region of Cavce to evaluate its effect on the functional properties of heterotrimeric G proteins. Fig. 8 shows the effect of this Cavce-derived peptide on the GTPase activity and GTPgamma S binding of the purified trimeric Go protein. Note that the steady-state GTP hydrolysis activity of Go was dose-dependently inhibited by this peptide with an IC50 value of 4 µM. A similar inhibitory effect was also observed for trimeric Gi2, yielding an IC50 value of 2 µM. For both Go and Gi2, complete inhibition was observed at 10 µM. Thus, the Cavce-derived peptide negatively affects both Go and Gi2 GTPase activity in a similar fashion. In addition, at a concentration of 10 µM, the Cavce-derived peptide completely abolished GDP/GTP exchange as measured by GTPgamma S binding of purified Go. Thus, it appears that this Cavce-derived peptide possesses "GDI-like activity" with the same potency as described earlier for mammalian caveolin-1 (24). A homologue of the mammalian Go protein has recently been identified in C. elegans (49).


Fig. 8. Effect of the Cavce-derived polypeptide on the basal GTPase activity and GTPgamma S binding of purified heterotrimeric G proteins. Upper and middle panels, effect of the Cavce peptide on the GTPase activity of purified trimeric Go and Gi2. The activity is expressed as a percentage of the basal activity, which was 0.17 ± 0.01 min-1 for Go and 0.05 ± 0.005 min-1 for Gi2 (mean ± S.E., n = 3). Lower panel, effect of the Cavce peptide on the GTPgamma S binding activity of purified trimeric Go protein. Activity is expressed as a percentage of basal activity. 100% represents the basal binding of GTPgamma S to Go, of which Kapp was 0.20 ± 0.02 min-1 (mean ± S.E., n = 3). All experiments were performed at least three times independently, and values indicate the mean ± S.E.
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Genomic Organization of Murine Caveolin-2 and Caveolin-3 Genes

Independently of our work on Cavce, we cloned the genomic copies of the murine caveolin-2 and caveolin-3 genes. To our surprise, caveolins 2 and 3 appear to be intronless single exon genes. Using PCR, we attempted to determine their intron-exon organization. In both cases, the cDNAs for caveolins 2 and 3 were used as positive controls. Fig. 9 shows that the same size PCR products were obtained when either of the cDNAs or the genomic clones were used as the template. These results indicate that no introns are present within the caveolin-2 and caveolin-3 genes. These result provide further support for the hypothesis that mammalian caveolins evolved from a single exon, related to the second exon of the Cavce gene. As the caveolin-1 gene contains three exons (9), it is possible that caveolin-1 evolved from caveolin-3. Caveolins 1 and 3 are the most closely related; the protein sequences of caveolins 1 and 3 are ~85% similar and ~65% identical. These results have interesting implications for understanding the molecular evolution of the mammalian caveolin gene family.


Fig. 9. Genomic organization of murine caveolin-2 and caveolin-3 genes. The cDNAs and genomic clones for caveolins 2 and 3 were subjected to PCR with specific primers to deduce potential intron-exon boundaries. Primer sequences are detailed under "Experimental Procedures." Note that no PCR products were observed if either the cDNAs or genomic templates were selectively omitted from the reaction; this critical negative control was run in parallel with the positive samples shown in panels A and B (not shown). A, caveolin-2: lane 1, primers 2-1 and 2-2; and lane 2, primers 2-1 and 2-3. A product of ~450 bp is observed in all cases, as expected based on the caveolin-2 cDNA sequence. Identical results were obtained with the caveolin-2 cDNA and genomic clone. B, caveolin-3: lane 1, primers 3-1 and 3-3; lane 2, primers 3-1 and 3-4; lane 3, primers 3-1 and 3-5; and lane 4, primers 3-2 and 3-5. Based on the cDNA sequence of caveolin-3, the expected size of PCR products for lanes 1-4 is 153, 306, 456, and 118 bp, respectively. Identical results were obtained with the caveolin-3 cDNA and a genomic clone. C, proposed genomic organization of Cavce and known mammalian caveolin genes. The structure of the chicken caveolin-1 gene was published previously by Glenney and Soppet (9); we confirmed the same genomic organization after cloning the murine caveolin-1 gene (data not shown).
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Are There Multiple Caveolin Genes in C. elegans?

Given the existence of multiple mammalian caveolin genes, we continued to search for other caveolin-related genes in C. elegans. Additional data base searches revealed a genomic sequence (accession no. Z77655[GenBank]; cosmid C56A3) that appeared similar to mammalian caveolins and Cavce. This gene is located on chromosome V and is, thus, distinct from the Cavce gene.

To determine if this gene is expressed, we used a PCR-based approach to clone the cDNA for this gene from a C. elegans cDNA library. This approach yielded an ~1-kb product of the appropriate size. DNA sequencing of multiple clones and direct sequencing of PCR products revealed an open reading frame encoding a protein of 351 amino acids (Fig. 10A), with a calculated molecular mass of 40,822.1 Da. For simplicity, we have termed this second novel caveolin-related protein Cavce-2 and retermed Cavce as Cavce-1. Cavce-2 contains a predicted 261-amino acid N-terminal domain, a 29-amino acid membrane spanning segment, and a 61-amino acid C-terminal domain.


Fig. 10. Sequence and analysis of Cavce-2. A, cDNA sequence and deduced protein sequence of Cavce-2. Numbers at left indicate amino acid positions. Note that Cavce-2 contains many consensus sites for potential post-translational modifications/interactions, including: 12 sites for phosphorylation by protein kinase C ((ST)-X-(RK)) at amino acid positions 12, 109, 116, 122, 143, 147, 148, 184, 148, 259, 329, and 334; 7 sites for phosphorylation by casein kinase II ((ST)-X (2)-(DE)) at amino acid positions 7, 22, 59, 103, 137, 226, and 335; 1 site for tyrosine phosphorylation ((R/K)-X2,3-(D/E)-X2,3-Y) at amino acid position 214; and 3 sites for SH-3 domain binding (P-X2-P) at amino acid positions 15, 159, and 187. B, alignment of the Cavce-2 protein with the protein sequences of Cavce-1 and known mammalian caveolin genes. Only the homologous region of Cavce-2 is shown; this represents a 138-amino acid stretch from residues 214-351. Residues identical to Cavce-2 are boxed. Note that in many cases Cavce-2 is more similar to mammalian caveolin-2.
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Fig. 10B shows an alignment of the Cavce-2 protein with the protein sequences of Cavce-1 and known mammalian caveolin genes. The most homologous region of Cavce-2 is a 138-amino acid stretch from residues 214-351. This homologous region includes the putative N-terminal oligomerization and G protein binding domains, the membrane spanning region and the entire C-terminal domain. This 138-amino acid region of Cavce-2 is ~44% similar and ~24% identical to the mammalian caveolins 1, 2, and 3. Perhaps surprisingly, Cavce-2 is only ~28% identical to Cavce-1. Thus, Cavce-2 is roughly equally related to all three known mammalian caveolins and Cavce-1. Also, analysis of the Cavce-2 protein sequence using the Prosite data base reveals potential sites for phosphorylation and SH-3 domain binding (Fig. 10A; see legend).

To ensure that both Cavce-1 and Cavce-2 are expressed, we performed Northern analysis using purified poly(A)+ RNA (Fig. 11). Hybridization with the Cavce-1 cDNA probe revealed an mRNA species of ~1.4 kb, while hybridization with the Cavce-2 cDNA probe demonstrated a 2.4-kb species. Little or no cross-hybridization was observed even at lower stringency. It is also interesting to note that the message for Cavce-1 was more abundant in eggs than mixed stages, suggesting developmental regulation of its expression.


Fig. 11. Northern blot analysis of the expression of Cavce-1 and Cavce-2. Each lane contains ~5 µg of poly(A)+ RNA prepared from eggs or mixed stages of C. elegans. Blots were first probed with the Cavce-1 cDNA, stripped, and reprobed with the Cavce-2 cDNA. Cavce-1 and Cavce-2 have different transcript sizes: ~1.4 kilobases for Cavce-1 and ~2.4 kilobases for Cavce-2. Note that the message for Cavce-1 is more abundant in eggs, while the message for Cavce-2 is roughly equal in eggs and mixed stages.
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Conclusions

The small soil nematode, C. elegans, has become an established model organism for studying developmental processes and signal transduction using genetic approaches. Its genome is relatively small, roughly (null)/1;30 that of the human (50). During development, 131 of the 1090 total somatic cells undergo apoptosis (51). The mature adult contains only 302 neurons, which represent ~(null)/1;3 of the total cells of the organism. C. elegans expresses many signaling molecules that have known mammalian counterparts, such as heterotrimeric G proteins, Ras proteins, and receptor-tyrosine kinases (49, 52). Many of these signaling molecules have been shown to function as key regulators in C. elegans development. Moreover, the C. elegans system provides an opportunity to perform reverse-genetic analysis in a moderately complex organism through (i) transposon insertion and excision mutagenesis (53) and (ii) the construction of transgenic animals (54).

Here, we have described the identification and characterization of two C. elegans homologues of mammalian caveolins, termed Cavce-1 and Cav ce-2 (see Fig. 12 for a comparison). This critical first step will allow us to take advantage of the C. elegans system to study the proposed functions of caveolae and caveolin using the established in vivo genetic approaches outlined above. Thus, it is likely that multiple caveolin genes exist in other simple model organisms such as Drosophila melanogaster and Saccharomyces cerevisiae.


Fig. 12. Summary of known caveolin family members. The overall structure of the four known mammalian caveolin gene products (caveolins 1alpha , 1beta , 2, and 3) is shown and compared with the structure of invertebrate Cavce-1 and -2. Caveolin-1 exists as two isoforms (alpha  and beta ) differing in their translation initiation sites; the beta -isoform lacks the first 31 amino acids (36). All four mammalian caveolin products contain the invariant sequence FEDVIAEP within their hydrophillic N-terminal domains (27, 32); this peptide sequence is FEDIFGEA in Cavce-1 and FFEVFNEP in Cavce-2. Both mammalian caveolins and invertebrate caveolins contain a characteristic and unusually long membrane spanning segment (TM) of ~29-33 amino acids. Overall amino acid length, percent similarity, and identity to caveolin-1 and GDI or GAP activities are summarized at the right. *From Cavce-1 residues 100-235; @from Cavce-2 residues 214-351.
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FOOTNOTES

*   This work was supported in part by National Institutes of Health FIRST Award GM-50443 (to M. P. L.), a grant from the Elsa U. Pardee Foundation (to M. P. L.), and a grant from the W. M. Keck Foundation to the Whitehead Fellows Program (M. P. L). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U66405[GenBank] and U75587[GenBank].


**   To whom correspondence should be addressed: The Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142-1479. Tel.: 617-258-5225; Fax: 617-258-9872; E- mail: lisanti{at}wi.mit.edu.
1    The abbreviations used are: GDI, GDP dissociation inhibitor; GAP, GTPase-activating protein; mAb, monoclonal antibody; Mes, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PCR, polymerase chain reaction; kb, kilobase(s); EST, expressed sequence tag.

Acknowledgments

We thank Dr. Harvey F. Lodish for enthusiasm and encouragement; Dr. Philipp Scherer for critical discussions; Dr. John R. Glenney for antibodies directed against caveolin-1; Drs. Charles S. Rubin, Mary-Ann Land, and Natalie Rebeiz for advice on the C. elegans system; Dr. Miriam Hasson for helpful discussions; Dr. Barbara Conradt for C. elegans genomic DNA. Dr. T. Asano for providing purified trimeric Gi2 proteins; members of the Lisanti laboratory for helpful discussions; Ya-Huei Tu for help with immunofluorescence microscopy; and Dr. Yuji Kohara (National Institute of Genetics, Japan) for providing the C. elegans EST clone.


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