(Received for publication, September 27, 1995; and in revised form, November 10, 1995)
From the
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.
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 (- and
-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
-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) (
)(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:
-caveolin contains residues 1-178, while
-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
-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
-caveolin
is phosphorylated on serine residues in vivo(21) .
Thus, coexpression of
- and
-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.
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
10
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-1
encoding residues 1-178
and caveolin-1
encoding residues 32-178 (23) . Thus,
caveolin-1
(147 amino acids) is approximately the same length as
caveolin-2 (149 amino acids) and caveolin-3 (151 amino acids).
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.
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 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 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
and G
with
IC
values of 3 and 5 µM, respectively. A 10
µM concentration of this peptide yielded 80% inhibition
for G
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
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
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; lower,
trimeric G
. The activity is expressed as a percentage of
the basal activity, which was 0.17 ± 0.01 min
for G
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.
Caveolin-3 joins caveolin-1 and caveolin-2 as the third
member of a growing caveolin gene family. As two forms of caveolin-1
(- and
-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 (-
and
-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-1
(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, ()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 -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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31968[GenBank].