(Received for publication, August 20, 1996, and in revised form, October 23, 1996)
From 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
Department of Physiological
Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University,
Hongo, Bunkyo, Tokyo 113, Japan
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 ( and
subunits). In this regard, caveolin interacts directly with
G
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.
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 ( and
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.
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 CavceEpitope-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
(MGG-Cavce) and a FLAG epitope tag was
incorporated into the C terminus (Cavce-GG
); 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.
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).
ImmunofluorescenceAll 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 FractionationTransfected 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 FractionsGradient 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 GTPTrimeric
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 GTPS 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.
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 -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.
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
ATGACTCGTCAGAATACTTCC-3
and
5
-CCGGCC
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.
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.
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.
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. 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.
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 ProductTo 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.
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 CavceTo 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).
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).
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.
A Cav ce-derived Peptide Suppresses the GTPase Activity and GTP
Mammalian caveolins functionally interact directly with
G protein -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 GTPS
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 GTP
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).
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.
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. 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.
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.
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].
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.