From the
Caveolin, an integral membrane protein, is a principal component
of caveolae membranes in vivo. Two isoforms of caveolin have
been identified: a slower migrating 24-kDa species (
Caveolae are small flask-shaped invaginations located at or near
the plasma membrane(1, 2) . They are thought to exist in
most cell types, although they are most abundant in endothelial cells,
type I pneumocytes, adipocytes, fibroblasts, and smooth muscle cells
(reviewed in Refs. 3 and 4).
Functionally, caveolae are involved in
the uptake of small molecules such as folate (5) and the
transport of macromolecules across capillary endothelial cells,
including modified atherogenic low density
lipoproteins(6, 7) . Recently, we and others have
proposed that caveolae may also participate in a subset of
transmembrane signaling events, such as G-protein-coupled
signaling(3, 8, 9, 10, 11) .
Caveolin, a 21-24-kDa integral membrane protein, has been
identified as a principal component of caveolae membranes in
vivo(12) . However, caveolin was first identified as a
major v-src substrate in Rous sarcoma virus-transformed chick
embryo fibroblasts (13). Both cell transformation and tyrosine
phosphorylation of caveolin are dependent on membrane attachment of
v-src(14) , suggesting that caveolin may represent a
critical substrate for cellular transformation. In support of this
view, we have recently observed that both caveolin expression and
caveolae are lost during cell transformation by activated oncogenes
other than v-src (v-abl, bcr-abl,
middle T antigen, and activated ras)(15) . These
results support the hypothesis that caveolin may represent a candidate
tumor suppressor protein(15) . Indeed, Krev-1, a Ras-related
transformation suppressor protein, is concentrated in purified
caveolin-rich membrane domains (9) and purified
caveolae(10) .
Two major isoforms of caveolin are known to
exist: a slower migrating 24-kDa species and a faster migrating 21-kDa
species. For simplicity, we will designate them as
Caveolin
is the product of a single gene(16) . Furthermore, as caveolin
mRNA is a single species, it is unlikely that these two isoforms arise
from differential mRNA splicing(9, 16, 17) . As
both isoforms are present immediately after caveolin synthesis, it does
not appear that there is a precursor-product relationship between
them(18, 19) .
Caveolin is constitutively
phosphorylated on serine residues(8, 14, 20) .
Interestingly, only the
Here,
we have begun to study the differences between these two caveolin
isoforms. Over 20 different monoclonal antibodies have been generated
against native chicken caveolin by Glenney and
co-workers(13, 14) ; only a few of these are reactive
with canine and human caveolin, despite the fact that caveolin is
highly conserved from chicken to man (over 86% identical(16) ).
Among these cross-reactive antibodies we have fortuitously identified
an
Another possibility is alternate
initiation of translation. Caveolin contains a second methionine
residue at position 32 that is conserved in every caveolin cDNA
sequenced to date (see Ref. 32 for an alignment). This methionine might
function as an internal start site during initiation of translation.
This would yield two caveolin isoforms immediately after synthesis that
differ by
The use of a given methionine as a translational start site is
determined in part by surrounding mRNA sequences, known as the Kozak
sequence(35) . Kozak analysis of sequences surrounding
methionine 32 reveals that this residue could act as an internal start
site. The methionine at position 32 in caveolin matches the Kozak
consensus sequence for translation initiation even more closely than
methionine at position 1 (Fig. 5), especially at the two most
critical nucleotide positions.
To
examine this possibility, we performed double labeling with mAb 2234
(
As both caveolin isoforms
contain a complete C-terminal domain (Fig. 4), immunostaining
with anti-caveolin C-terminal-specific antibodies should reflect the
overall distribution of both isoforms. Alternatively, the anti-caveolin
C-terminal-specific antibody may preferentially recognize the
In addition, it should be noted that when
caveolin-expressing FRT cells were used to biochemically purify
caveolae-like structures using an established protocol(8) ,
greater than 95% of caveolin (both isoforms) was recovered within
caveolar membrane domains, an indication that caveolin is correctly
targeted when exogenously expressed in FRT cells (Fig. 8). This
result is in accordance with our observation that FRT cells contain
caveolae-like structures but fail to endogenously express caveolin (8) and recent evidence that caveolin is not required to
maintain the structure or integrity of caveolae(38) .
Here, we have determined that caveolin isoforms differ in
their extreme N-terminal protein sequence, but both contain a complete
C terminus. This conclusion is based on the identification of an
Immunofluorescent localization of caveolin
reveals punctate fluorescent dots or
micropatches(8, 12, 19, 36, 37) .
Analysis of this distribution by quantitative immunoelectron microscopy
reveals that micropatches correspond to collections of
caveolae(10, 12, 19, 30, 38) .
Based on these ultrastructural studies, it was estimated that greater
than 90% of caveolin is localized within
caveolae(12, 30, 38) . Similarly, greater than
90% of caveolin is recovered within purified caveolae (also known as
caveolin-rich membrane domains), while Golgi-associated caveolin is
excluded from these preparations(9, 21, 38) .
These morphological and biochemical studies have also demonstrated that
both caveolin isoforms are present within
caveolae(9, 10) . However, we show here by confocal
immunofluorescence microscopy that these two isoforms may assume
distinct but overlapping subcellular distributions. This is consistent
with independent morphological evidence suggesting the existence of at
least two distinct subpopulations of caveolae(39, 40) .
Alternate initiation of translation from a single mRNA transcript is
used to generate the two isoforms of other proteins, the progesterone
receptor and the yeast gene MOD5(33, 34) . In the case
of MOD5, these two isoforms are functionally identical, but are
segregated to different subcellular compartments: one is imported into
mitochondria, while the other remains entirely cytoplasmic(34) .
Thus, there is a precedent for using alternate initiation of
translation to segregate isoforms of a given protein to different
regions within the same cell.
As both isoforms of caveolin are
localized within caveolae(9, 10) , this N-terminal
sequence is not required for caveolar localization. As only
The identity of
this serine kinase remains unknown. Caveolin contains consensus sites
for phosphorylation by serine-threonine kinases, protein kinase C
(Ser-37) and casein kinase II (Ser-88)(20, 32) . In
addition, both protein kinase C
In support of our current findings, another
group reported differential immunoreactivity of rabbit anti-peptide
antibodies with the two isoforms of caveolin as an incidental
finding(30) . These investigators concluded that differential
reactivity represented the addition of a post-translational
modification near the N terminus of caveolin without an absence of
specific protein sequences. However, they did not present any evidence
to support this assertion, nor did they examine the completeness of the
N or C terminus of caveolin. Here we have systematically studied these
differences by performing microsequencing, epitope-mapping,
epitope-tagging, and mutational analysis. Taken together, our results
directly demonstrate that
We thank Petra Knaus and Eric Kübler for helpful
discussions, Richard F. Cook for microsequence analysis, John R.
Glenney for generously donating monoclonal antibodies (2234 and 2297),
and Anthony J. Koleske for help in constructing and expressing
C-terminally myc-tagged caveolin.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-isoform) and
a faster migrating 21-kDa species (
-isoform). Little is known
about how these isoforms differ, either structurally or functionally.
Here we have begun to study the differences between these two isoforms.
Microsequencing of caveolin reveals that both isoforms contain internal
caveolin residues 47-77. In a second independent approach, we
recombinantly expressed caveolin in a caveolin-negative cell line (FRT
cells). Stable transfection of FRT cells with the full-length caveolin
cDNA resulted in the expression of both caveolin isoforms, indicating
that they can be derived from a single cDNA. Using extracts from
caveolin-expressing FRT cells, we fortuitously identified a monoclonal
antibody that recognizes only the
-isoform of caveolin. Epitope
mapping of this monoclonal antibody reveals that it recognizes an
epitope within the extreme N terminus of caveolin, specifically
residues 1-21. These results suggest that
- and
-isoforms of caveolin differ in their N-terminal protein
sequences. To independently evaluate this possibility, we placed an
epitope tag at either the extreme N or C terminus of full-length
caveolin. Results of these ``tagging'' experiments clearly
demonstrate that (i) both isoforms of caveolin contain a complete C
terminus and (ii) that the
-isoform contains a complete N terminus
while the
-isoform lacks N-terminal-specific protein sequences.
Mutational analysis reveals that these two isoforms apparently derive
from the use of two alternate start sites: methionine at position 1 and
an internal methionine at position 32. This would explain the
3-kDa difference in their apparent migration in SDS-polyacrylamide
electrophoresis gels. In addition, using isoform-specific antibody
probes we show that caveolin isoforms may assume a distinct but
overlapping subcellular distribution by confocal immunofluorescence
microscopy. We discuss the possible implications of these differences
between
- and
-caveolin.
- and
-isoforms of caveolin, respectively. Little is known about how
these isoforms differ. This may be important for understanding the role
of caveolin and caveolae in normal and transformed cells.
-isoform is phosphorylated in
vivo, while both forms are capable of undergoing serine
phosphorylation in vitro(17) . These observations point
to a functional difference that appears to be recognized by a specific
serine kinase in vivo. Importantly, these observations
demonstrate that the
-isoform is not an artifactual degradation
product of the
-isoform, as only the faster migrating
-isoform is selectively phosphorylated in vivo.
-isoform-specific monoclonal antibody. Identification and
epitope mapping of this monoclonal antibody has provided both
structural and functional information regarding the differences between
- and
-caveolin. In addition, using this isoform-specific
mAb, we show by confocal fluorescence microscopy that
-caveolin
may assume a distinct subcellular distribution. This antibody, which is
commercially available, should prove to be a powerful molecular probe
for studying the function of caveolin isoforms.
Materials
Monoclonal antibodies directed against
full-length caveolin were the generous gift J. R. Glenney (Transduction
Laboratories, Lexington, KY). The monoclonal antibody 9E10 was provided
by The Harvard Monoclonal Antibody Facility (Cambridge, MA). A variety
of other reagents were purchased commercially: fetal bovine serum (JRH
Biosciences), prestained protein markers (Life Technologies, Inc.),
Lab-Tek chamber slides (Nunc Inc., Naperville, IL), normal goat and
donkey IgGs, fluorescein isothiocyanate-conjugated goat anti-mouse
antibody, and lissamine rhodamine B sulfonyl chloride-conjugated donkey
anti-rabbit antibody (Jackson Immunoresearch Laboratory, West Grove,
PA); SlowFade anti-fade reagent (Molecular
Probes).
Purification of Caveolin-enriched
Fractions
Caveolin-rich domains were purified from murine lung
tissue, as described(9, 21) . To further enrich for
caveolin, these domains were resuspended on ice in
Mes(
)-buffered saline (25 mM Mes, pH
6.5, 0.15 M NaCl) containing 60 mM octyl glucoside,
adjusted to 40% sucrose, and refloated using the same type of
bottom-loaded sucrose density gradients (a 5-30% linear sucrose
gradient) used for initial purification. The single light-scattering
band floating at the 15-20% sucrose region was collected, diluted
1:3 with Mes-buffered saline alone and recovered by centrifugation in
the Microfuge (14,000
g for 15 min at 4 °C). This
additional purification step eliminated other 21-24-kDa proteins
that were previously found to co-purify with caveolin. Caveolin
isoforms and proteins co-purifying with caveolin under these conditions
were identified by microsequence analysis. Microsequencing was
performed as we described previously(9) .
Cell Culture
FRT and MDCK cells were propagated as
described previously(8, 22) . Cells were subjected to
steady-state metabolic labeling with S-labeled amino acids
(methionine and cysteine) as described(23) . The expression
levels of a given transfected antigen was increased by an overnight
incubation with normal medium containing 10 mM sodium butyrate
(21, 24).
Transfection and Selection of Stable Cell
Lines
Wild type full-length caveolin and epitope-tagged forms of
caveolin were subcloned into the multiple cloning site (HindIII/BamHI) of the vector pCB7 (containing the
hygro marker; gift of J. Casanova, MGH) for expression in
FRT or MDCK cells, respectively. In order to recombinantly express
epitope-tagged forms of caveolin in MDCK cells, we incorporated the myc
epitope tag into the N terminus (MEQKLISEEDLNGG-caveolin) or the C
terminus (caveolin-GGEQKLISEEDLN) of the cloned canine caveolin cDNA
using PCR primers. We placed GG as a spacer between the epitope and the
caveolin coding sequences, as has been suggested
previously(19, 25) . Correct placement of the epitope
tag and caveolin coding sequences were verified by double-stranded DNA
sequencing. FRT or MDCK cells were stably transfected using a
modification of the calcium-phosphate precipitation
procedure(21, 24) . After selection in media
supplemented with 400 µg/ml hygromycin B, resistant colonies were
picked by trypsinization using cloning rings. Individual clones were
screened by immunofluorescence for recombinant expression of caveolin.
Wild type full-length caveolin expressed in FRT cells was detected
using anti-caveolin IgG (mAb 2297 or 2234). Epitope-tagged forms of
caveolin expressed in MDCK cells were detected using monoclonal
antibody, 9E10, that recognizes the myc epitope (EQKLISEEDLN).
Mutational Analysis of Caveolin
Isoforms
Methionine residues at either position 1 or at position
32 were mutated to valine by changing ATG to GTG. Single base mutations
(A to G) were introduced into the carboxyl-terminally myc-tagged canine
caveolin cDNA by PCR site-directed mutagenesis(26) . After
subcloning into the pCB7 vector, the corresponding constructs were
transiently transfected into Cos7 cells by the DEAE-dextran
method(27) . 48 h post-transfection, cells were scraped into
lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1%
Triton X-100, 60 mM octyl glucoside, 5 mM EDTA, 1
mM phenylmethylsulfonyl fluoride). Insoluble material was
removed by centrifugation at 15,000 g for 10 min, and
the supernatant was diluted with an equal volume of SDS-PAGE sample
buffer containing 100 mM NaOH and boiled for 5 min. The
equivalent of 5% of a 10-cm dish of Cos7 cells was analyzed by SDS-PAGE
(14% acrylamide) followed by Western blotting with mAb 9E10.
Construction and Expression of GST-fusion
Proteins
Full-length caveolin (residues 1-178), each
caveolin subdomain (N terminus (residues 1-101), the
transmembrane domain (residues 102-134), and the C terminus
(residues 135-178)), and deletion mutants of the N-terminal
domain (residues 1-21, 1-41, 1-61, 1-81,
25-58, 61-101, 71-101, 76-101, 81-101)
were amplified by PCR and separately subcloned into the multiple
cloning site of the vector pGEX-4T-1 to obtain GST-fusion proteins when
expressed in a suitable Escherichia coli strain (BL21, lacking
lon and ompT proteases; Novagen, Inc.). The exact reading frame and
caveolin coding sequences were verified by double-stranded DNA
sequencing. GST-fusion proteins were purified by affinity
chromatography using glutathione agarose, as described
previously(28) .
Preparation of C-terminal Specific Anti-caveolin
IgG
A peptide encoding the entire canine caveolin C-terminal
domain (residues 135-178) was synthesized, purified, and used to
immunize rabbits with RIBI's adjuvant following a standard
protocol(29) . Test bleeds were taken at 0, 5, 7, 9, and 16
weeks and monitored for antibody titer by immunoprecipitation of
metabolically labeled MDCK lysates. Anti-caveolin IgGs were purified
using protein A-Sepharose as described by the manufacturer.
Immunofluorescence
FRT cells (grown in Lab-Tek
chamber slides at a subconfluent density) were washed three times with
PBS and fixed for 45 min in PBS containing 3% paraformaldehyde, 10
mM NaIO, and 70 mM lysine-HCl. Fixed
cells were rinsed with PBS and treated with 100 mM NH
Cl in PBS for 10 min to quench free aldehyde groups.
Cells were then permeabilized with 0.1% Triton X-100 for 10 min, either
at room temperature or on ice, and washed with PBS, four times, 10 min
each. The cells were then successively incubated with PBS, 2% bovine
serum albumin containing: (i) 50 µg/ml each of normal goat and
donkey IgGs, (ii) a 1:300 dilution of anti-caveolin mAb 2234 and 40
µg/ml anti-caveolin C-terminal-specific polyclonal IgG, and (iii)
fluorescein isothiocyanate-conjugated goat anti-mouse antibody (5
µg/ml) and lissamine rhodamine B sulfonyl chloride-conjugated
donkey anti-rabbit antibody (5 µg/ml). 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 SlowFade anti-fade reagent and observed under a
Bio-Rad MR600 confocal fluorescence microscope.
Microsequence Analysis of Caveolin Isoforms
Two
major isoforms of caveolin have been identified by one- or
two-dimensional gel electrophoresis of cell extracts, including MDCK
cells (Fig. 1A). In order to investigate the difference
between these two isoforms, we partially purified caveolin from murine
lung tissue, a caveolin-rich tissue source, and subjected these
isoforms to microsequence analysis (Fig. 1B). N-terminal
microsequencing revealed that the N terminus of each isoform is
blocked. We next performed internal microsequencing with the
endoproteinase Lys-C. Sequencing of these peptides, detailed in , reveals that both caveolin isoforms contain internal
caveolin residues 47-77. In addition, -caveolin appears to
contain a complete N-terminal domain (caveolin residues 5-26; ), and
-caveolin appears to contain a complete
C-terminal domain (caveolin residues 165-176; ).
Thus, the faster migrating
-isoform might still lack a complete
N-terminal domain.
Figure 1:
Caveolin
isoforms and caveolin-associated proteins. A, caveolin
isoforms in MDCK cells. Cells were directly lysed in sample buffer and
subjected to SDS-PAGE. After transfer to nitrocellulose, caveolin
isoforms were detected with a specific monoclonal antibody (mAb 2297).
Slower migrating and faster migrating isoforms were designated as
- and
-caveolin, respectively. Similar results were obtained
by two-dimensional gel electrophoresis (not shown). B,
microsequence analysis of caveolin isoforms. Caveolin-rich membrane
domains were purified from murine lung tissue and used to further
purify caveolin (see ``Experimental Procedures''). After
SDS-PAGE and transfer to nitrocellulose, caveolin isoforms and proteins
co-purifying with caveolin were subjected to microsequence analysis.
Note that caveolin stains negatively with either Ponceau S or Coomassie
Blue. The identity of each band is summarized here. These proteins
included a transmembrane protein (CD36), four
glycophosphoinositide-linked proteins (membrane dipeptidase,
5`-nucleotidase, ceruloplasmin, carbonic anhydrase IV), actin, a
calcium-binding protein (calsequestrin), cytoplasmically oriented
signaling molecules (Lyn tyrosine kinase; G
), a novel
45-kDa protein, and caveolin. See Table I for the specific caveolin
sequences obtained.
Expression of the Full-length Caveolin cDNA in a
Caveolin-negative Cell Line Yields Both Caveolin Isoforms
FRT
cells fail to express detectable levels of caveolin mRNA or protein,
although they contain caveolae-like structures(8, 22) .
As a consequence, we stably expressed full-length caveolin in FRT cells
to study its properties. Our results indicate that both caveolin
isoforms derive from a single cDNA. This conclusion is consistent with
studies demonstrating a single species of caveolin mRNA (9, 16, 17) and that in vitro translation produces both isoforms(19) . However, these
authors did not rule out the possibility that this is an artifact of
the in vitro translation system. To examine this possibility,
we derived stable cell lines. Fig. 2shows FRT cells stably
transfected with the full-length canine caveolin cDNA. Immunoblotting
with monoclonal antibody 2297 reveals both - and
-caveolin in
FRT transfectants (Fig. 2A). However, we noticed that
immunoblotting of the same cell lysate with another monoclonal
antibody, 2234, only revealed
-caveolin (Fig. 2B).
These results suggest that monoclonal 2234 is
-specific and
recognizes an epitope that is absent from
-caveolin. Thus,
monoclonal antibody 2234 could be used as a molecular probe to
structurally and functionally distinguish between
- and
-caveolin.
Figure 2:
Recombinant expression of full-length
caveolin in FRT cells. FRT cells fail to express caveolin mRNA or
protein. As such, we used FRT cells as an expression system to study
caveolin. FRT cells were stably transfected with a plasmid encoding the
full-length canine caveolin cDNA. After selection, stable cell lines
were obtained that express caveolin. U, untransfected control
FRT cells; T, FRT cell transfectants recombinantly expressing
caveolin. A, note that both caveolin isoforms are present in
FRT cells transfected with the full-length caveolin cDNA and are
recognized by monoclonal antibody 2297. B, in contrast,
monoclonal 2234 selectively recognizes only the slower migrating
-isoform of caveolin.
Epitope Mapping of an
We next used epitope mapping to understand why
monoclonal 2234 might only recognize -Isoform-specific Monoclonal
Antibody
-caveolin. As the substrate
for antibody binding, we expressed full-length caveolin and portions of
caveolin as GST-fusion proteins. After purification, fusion proteins
were subjected to immunoblotting with either monoclonal antibody (2234
or 2297) (Fig. 3A). Based on their differential
immunoreactivity, mAb 2234 recognizes an epitope within caveolin
residues 1-21, while mAb 2297 recognizes an epitope within
caveolin residues 61-71 (summarized schematically in Fig. 3B). As mAb 2297 identifies both caveolin isoforms,
these results are consistent with the above microsequence analysis
demonstrating that both isoforms contain caveolin residues 47-77.
In addition, these results directly show that these two isoforms differ
by a discrete N-terminal epitope. This might reflect the addition of a
post-translational modification that renders
-caveolin nonreactive
to mAb 2234 or, more likely, the absence of N-terminal protein
sequence.
Figure 3:
Epitope mapping of mAbs 2234 and 2297. A, full-length caveolin and portions of caveolin were
recombinantly expressed as GST-fusion proteins and purified by affinity
chromatography on glutathione agarose. Based on their location within
an intact caveolin molecule, they received the following designation: FL, full-length caveolin; C, caveolin C-terminal
domain; T, caveolin transmembrane domain; and N,
caveolin N-terminal domain. In addition, caveolin residues contained
within a given fusion protein are indicated in parentheses. Upper panel, Coomassie Blue staining of each purified fusion
protein. Lower panels, immunoblotting of fusion proteins with
a given monoclonal antibody (2234 or 2297). B, schematic
diagram summarizing the immunoreactivity of mAbs 2234 and 2297 with
caveolin fusion proteins. Note that mAb 2234 recognizes an epitope
within caveolin residues 1-21, while mAb 2297 recognizes a
different epitope within caveolin residues
61-71.
Epitope Tagging Reveals That Both Caveolin Isoforms
Contain a Complete C-terminal Domain, but Differ at Their Extreme N
Termini
To monitor the completeness of the N- or C-terminal ends
of caveolin independently of mAb 2234, we next expressed epitope-tagged
forms of caveolin in MDCK cells. Using PCR, we placed a myc epitope tag
(EQKLISEEDLN) at either the extreme N or C terminus of full-length
caveolin (Fig. 4). Caveolin isoforms can then be visualized after
recombinant expression using a monoclonal antibody (9E10) that is
directed against the myc epitope.
Figure 4:
Recombinant expression of epitope-tagged
forms of caveolin. A, expression of caveolin containing an
epitope tag placed at its extreme N terminus. Left, note that
immunoblotting with mAb 9E10, which recognizes the myc epitope tag,
only reveals the slower migrating -isoform. Right,
immunoblotting with anti-caveolin IgG reveals that both isoforms are
synthesized in transfected MDCK cells. U, untransfected
control cells; T, cell transfectants expressing recombinant
caveolin. B, expression of caveolin containing an epitope tag
placed at its extreme C terminus. Immunoblotting with mAb 9E10, which
recognizes the myc epitope tag, reveals both the
- and
-isoforms of caveolin in transfected MDCK cells. U,
untransfected control cells; T, cell transfectants expressing
recombinant caveolin. C, to evaluate the relative importance
of methionine 32 in generating caveolin isoforms, we mutated the
methionine residue at position 1 (
Met-1) or at position 32
(
Met-32) to valine by changing ATG to GTG by PCR. For these
experiments, we used C-terminally myc-tagged caveolin and recombinant
expression in COS cells. Immunoblotting with mAb 9E10 reveals the
myc-tagged protein products. Note that caveolin
Met-1 only gives
rise to the
-isoform, while caveolin
Met-32 only yields the
-isoform. U, untransfected control cells; T,
cell transfectants expressing recombinant
caveolin.
Consistent with previous topology
studies demonstrating that both the N- and C-terminal domains of
caveolin face the cytoplasm(30, 31) , immunofluorescent
detection of either N- or C-terminally tagged caveolin with mAb 9E10
required detergent permeabilization (not shown). Expression of caveolin
containing a C-terminal tag yielded both caveolin isoforms when
visualized with an antibody that recognizes the myc epitope (Fig. 4, A and B). These results clearly
demonstrate that both isoforms contain a complete C terminus. In
contrast, N-terminally tagged caveolin yielded only the -isoform
when visualized using an antibody that recognizes the myc epitope.
However, both isoforms were expressed in these cells transfected with
N-terminally tagged caveolin, as visualized with mAb 2297 (Fig. 4, A and B). These results independently
demonstrate that (i) caveolin isoforms derive from a single cDNA and
(ii) that the
-isoform contains a complete N terminus while the
-isoform lacks N-terminal-specific protein sequences, as it fails
to contain the myc epitope. These studies directly support our results
from epitope mapping of mAb 2234.
Mutational Analysis of the Generation of Caveolin
Isoforms
Although we have determined that caveolin isoforms
differ in N-terminal protein sequence, it is unclear how these two
isoforms are generated. One possibility is that -caveolin
undergoes rapid co-translational or post-translational proteolytic
processing by a specific aminopeptidase or an endopeptidase to generate
-caveolin. This would be supported if a precursor-product
relationship existed between
- and
-caveolin. Although we
cannot completely exclude this possibility, it appears unlikely as both
caveolin isoforms are present immediately after caveolin
synthesis(8, 19) .
3 kDa, and the smaller isoform would lack specific
N-terminal protein sequences such as residues 1-21, as we
observe. Alternate initiation of translation from a single mRNA
transcript is used to generate two isoforms of other proteins, the
progesterone receptor and the yeast gene MOD5(33, 34) .
Figure 5:
Kozak nucleotide consensus for initiation
of translation. A, above is shown the Kozak consensus
sequence. The A of the AUG is designated as +1, as described
previously (35). Nucleotide positions -3 and +4 are most
critical for selection of an AUG for initiation of translation (35).
Below, the corresponding mRNA sequences for canine caveolin are listed
for methionine at amino acid position 1 (M-1) and at amino
acid position 32 (M-32). B, the positions of epitopes
recognized by mAbs 2234 (residues 1-21) and 2297 (residues
61-71) are indicated relative to methionine 1 and methionine
32.
Fig. 4C shows that
methionine 32 acts as an internal start site to generate caveolin
isoforms. For these experiments, we used C-terminally myc-tagged
caveolin and recombinant expression in COS cells. To evaluate the
relative importance of methionine 32 in generating caveolin isoforms,
we mutated the methionine residue at position 1 (Met-1) or at
position 32 (
Met-32) to valine by changing ATG to GTG. Caveolin
Met-1 only gives rise to the
-isoform, indicating that this
isoform can be derived from an internal start site when the normal
start site at position 1 is removed. Conversely, caveolin
Met-32
only yields the
-isoform, demonstrating that an internal
methionine at position 32 is necessary to generate the
-isoform.
Taken together, these studies strongly indicate that methionine 32 can
act as an internal start site to generate caveolin isoforms.
Localization of Caveolin Isoforms
Caveolin appears
predominantly as distinct ``micropatches'' by
immunofluorescent
microscopy(8, 12, 19, 36, 37) .
In cells that contain endogenous caveolin or in cells where caveolin is
recombinantly overexpressed, these micropatches correspond to
collections of caveolae as seen by immunoelectron
microscopy(10, 12, 19, 30, 38) .
Using antibodies that recognize both caveolin isoforms, biochemical and
morphological studies indicate that greater than 90% of both caveolin
isoforms are localized to
caveolae(9, 10, 12, 30, 38) .
However, independent morphological evidence (immunolocalization of the
inositol 1,4,5-trisphosphate receptor and the plasma membrane
Ca-ATPase) has suggested that at least two distinct
populations of caveolae may exist(39, 40) . Thus,
structural differences between
- and
-caveolin might still
convey distinct subcellular localization of these two isoforms.
-isoform-specific) and anti-caveolin C-terminal-specific
polyclonal IgG using subconfluent caveolin-expressing FRT cells which
assume a fibroblastic morphology. These two primary antibodies were
chosen for double-labeling experiments as they were elicited in
different animal species (mouse versus rabbit), minimizing
possible cross-reaction of the individual primary antibodies with
distinctly tagged secondary antibodies. Immunostaining was visualized
by confocal microscopy. Staining with both antibodies was dependent on
caveolin expression, as no staining was observed with untransfected FRT
cells (not shown). Fig. 6shows the characterization of the
anti-caveolin IgG directed against the C terminus of caveolin (residues
135-178) and that this antibody specifically recognizes caveolin.
Figure 6:
Characterization of anti-caveolin
C-terminal specific antibodies. Anti-caveolin IgG were elicited against
the entire C-terminal domain of caveolin (residues 135-178) (see
``Experimental Procedures''). Left, MDCK cells were
metabolically labeled to steady-state with S-labeled amino
acids and subjected to immunoprecipitation with either preimmune IgG (PI) or anti-caveolin IgG (
-C-Cav). Note the
specific immunoprecipitation of both caveolin isoforms. Right, a
purified GST fusion protein encoding the C-terminal domain of caveolin
(residues 135-178; GST-C-Cav) was subjected to
immunoprecipitation with either preimmune IgG (PI) or
anti-caveolin IgG (
-C-Cav). Immunoprecipitation of the
GST-tagged fusion protein was monitored by Western blotting with
anti-GST IgG. HC, rabbit IgG heavy chain products
immunoreactive with the secondary antibody used to visualize bound
rabbit anti-GST antibodies.
The immunostaining pattern observed with mAb 2234 is shown in Fig. 7A. Many small micropatches are present throughout
the cell and along the cell surface. However, there is an intense
accumulation of micropatches within the cell (Fig. 7A, open arrows) and less accumulation along the edge of the cell (Fig. 7A, closed arrows). As shown in Fig. 7B, immunostaining with anti-caveolin
C-terminal-specific IgG reveals a markedly different overall pattern.
As with mAb 2234, many micropatches are present throughout the cell. In
contrast, there is an intense accumulation of micropatches along the
edge of the cell (Fig. 7B, closed arrows),
while less accumulation within the body of the cell (Fig. 7B, open arrows). However, a longer
exposure of panel B demonstrates that there is immunostaining
within the body of the cell (Fig. 7C, open
arrow).
Figure 7:
Immunolocalization of caveolin isoforms.
Confocal fluorescent microscopic images of subconfluent FRT cells
stably transfected with caveolin. The same field of cells is shown in A and B, but they differ in the nature of the primary
and secondary antibodies. A, immunostaining with mAb 2234
revealed by a fluorescein isothiocyanate-conjugated goat anti-mouse
secondary antibody. B, immunostaining with a rabbit
anti-caveolin C-terminal-specific IgG revealed by a lissamine rhodamine
B sulfonyl chloride-conjugated donkey anti-rabbit secondary antibody.
It should be noted that B was intentionally printed at a
lesser exposure to allow more adequate comparison between A and B. C, same as B, except it was
exposed longer to show micropatches within the perinuclear region that
are not apparent in B (open arrow). Both antibodies
show many micropatches throughout the cell, including the cell surface (closed arrows in A and B) and within the
cell (open arrows in A and B). These
micropatches clearly coincide throughout the cell, demonstrating
significant co-localization. However, strikingly, mAb 2234
preferentially stains accumulations of caveolin micropatches within the
body of the cell (open arrows in A), while the
C-terminally directed antibody primarily stains caveolin micropatches
at the edge of the cell (closed arrows in B). Bar = 10 µm.
The intense immunostaining in Fig. 7B (closed arrows) may represent the leading edge of the
cell, as caveolae are known to be morphologically concentrated at the
leading edge(12) . In addition, as the -isoform is
predominantly excluded from this region (Fig. 7A, closed arrows), this intense immunostaining (Fig. 7B, closed arrows) should correspond to
the localization of the
-isoform.
-isoform by immunostaining, although it appears to recognize both
forms by immunoprecipitation (Fig. 6). Regardless of whether this
C-terminally directed antibody recognizes both isoforms or only the
-isoform, our results directly demonstrate that these isoforms are
differentially localized within a single cell as mAb 2234 is
-isoform-specific.
Figure 8:
Subcellular fractionation of FRT cells
expressing caveolin. Distribution of total cellular proteins and
caveolin. FRT cells expressing caveolin were fractionated using a
well-established protocol that separates caveolae-like structures from
the bulk of cellular membranes and cytosolic proteins (8). One-ml
sucrose gradient fractions were collected from the top and analyzed
after SDS-PAGE and transfer to nitrocellulose. Fractions 1-7 are
the 5-30% sucrose layer, while fractions 8 and 9 are the 40%
sucrose layer. Note that fractions 8 and 9 represent the ``loading
zone'' of these bottom-loaded flotation gradients and contain the
bulk of cellular membrane and cytosolic proteins (see
``Experimental Procedures''). Upper, Ponceau S
staining of total cellular proteins. Lower, immunoblotting
with anti-caveolin mAb 2297. Fractions 3 and 4, representing
caveolae-enriched membranes, retained >95% of caveolin and excluded
>99% of total cellular proteins (based on independent protein
determinations).
-caveolin-specific monoclonal antibody whose epitope maps to
residues 1-21 of caveolin. This antibody fails to recognize
-caveolin. The most straightforward interpretation is that the
-isoform lacks an N-terminal-specific protein sequence found in
the
-isoform. Independent confirmation of this assertion was
obtained by tagging the N or C terminus of caveolin with the myc
epitope tag to monitor the completeness of the N or C terminus.
Mutational analysis reveals that these two isoforms apparently derive
from the use of two alternate start sites: methionine at position 1 and
an internal methionine at position 32. Thus, these observations account
for the
3-kDa difference observed in the migration of the
-
and
-isoforms.
-caveolin undergoes phosphorylation in vivo(17) ,
this N-terminal protein sequence might play a ``negative
regulatory role'' by preventing the recognition of
-caveolin
by an endogenous caveolin-associated serine kinase.
and casein kinase II
are concentrated in purified caveolin-rich membrane domains(9) .
Serine phosphorylation is apparently important for regulating the
function of caveolae as (i) protein kinase C activators cause caveolae
to flatten out and prevent uptake of folate via caveolar potocytosis (41) and (ii) a serine phosphatase inhibitor (okadaic acid)
dramatically alters the subcellular distribution of
caveolae(42) .
-caveolin contains a complete N and C
terminus, while
-caveolin lacks specific N-terminal protein
sequence. In addition, as mAb 2234 is commercially available, this will
greatly facilitate the use of this antibody as a molecular probe for
studying caveolin isoforms.
Table: Internal microsequencing of caveolin isoforms
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.