©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Caveolin Isoforms Differ in Their N-terminal Protein Sequence and Subcellular Distribution
IDENTIFICATION AND EPITOPE MAPPING OF AN ISOFORM-SPECIFIC MONOCLONAL ANTIBODY PROBE (*)

Philipp E. Scherer(§) (1), ZhaoLan Tang (1), Miyoung Chun(¶) (1), Massimo Sargiacomo(**) (1), Harvey F. Lodish (1) (2), Michael P. Lisanti (§§) (1)

From the (1)FromThe Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479 and the (2)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (-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.


INTRODUCTION

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 - 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.

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 -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.

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 -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.


EXPERIMENTAL PROCEDURES

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 NHCl 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.


RESULTS

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 -Isoform-specific Monoclonal Antibody

We next used epitope mapping to understand why monoclonal 2234 might only recognize -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) .

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 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) .

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.


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.

To examine this possibility, we performed double labeling with mAb 2234 (-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.

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 -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.

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) .


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).




DISCUSSION

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 -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.

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 -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.

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 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) .

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 -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



FOOTNOTES

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

§
Funded by a Swiss National Science Foundation fellowship.

Supported by a fellowship from the Life Science Research Foundation.

**
Present address: Dept. of Hematology & Oncology, Istituto Superiore di Sanitá, Viale Regina Elena, 299, 00161 Rome, Italy.

§§
To whom correspondence and reprint requests should be addressed: The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479. Tel.: 617-258-5225; Fax: 617-258-9872; E-mail: lisanti@wi.mit.edu.

The abbreviations used are: Mes, 4-morpholineethanesulfonic acid; MDCK, Madin-Darby canine kidney cells; PCR, polymerase chain reaction; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

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.


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