From the
University of Nebraska Medical Center,
Department of Oral Biology, College of Dentistry and Eppley Cancer Center,
Omaha, Nebraska 68198 and
Thromgen, Inc., Ann
Arbor, Michigan 48104
Received for publication, April 18, 2003
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ABSTRACT |
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INTRODUCTION |
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Cadherins are comprised of five extracellular repeat domains with conserved
calcium-binding motifs, a single pass transmembrane domain, and a highly
conserved cytoplasmic tail. The first extracellular repeat of type I cadherins
mediates homotypic interactions and contains a conserved
histidine-alanine-valine (HAV) sequence
(13). The conserved calcium
ion-binding sequences LDRE, DXNDN, and DXD coordinate the
calcium ions that bridge the extracellular repeats
(1). Binding of calcium to
these motifs provides rigidity and confers the proper adhesive strength
necessary for cell-cell adhesion
(14). A conserved stretch of
hydrophobic peptides constitutes the transmembrane domain, and the cytoplasmic
domain binds directly to -catenin and p120 catenin with the former
indirectly linking the cadherin to the actin cytoskeleton via interactions
with
-catenin
(1517).
Like type I cadherins, type II cadherins are comprised of five extracellular repeats with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic tail (1). Unlike type I cadherins, the first extracellular repeat of type-II cadherins does not contain the HAV sequence although some contain QAV (18). Type II cadherins are not as well characterized as type I cadherins, although their aberrant expression in cancer has also been described (1922)
In this study, we report the characterization of a novel human type II
cadherin, which has been designated cadherin-24. The shorter isoform of
cadherin-24 encodes a typical type II cadherin, complete with five
extracellular repeats containing conserved calcium-binding motifs, a
transmembrane domain, and a conserved cytoplasmic tail. Homology studies
predict that cadherin-24 is 57% identical to human cadherin-11, a type II
cadherin expressed by osteoblasts and some breast cancer cell lines
(2123).
Like cadherin-11, cadherin-24 exists as two alternatively spliced forms.
However, unlike cadherin-11, the alternative splice site in cadherin-24 is in
the extracellular domain, and the longer, alternatively spliced form binds
-catenin. Cadherin-24 is the first cadherin shown to exist as two
full-length alternatively spliced functional cadherins capable of binding
catenins. Peptide sequence analysis suggests the presence of
-catenin
and p120 catenin-binding sequences in the cytoplasmic domain of cadherin-24,
and immunoprecipitation experiments confirm the ability of cadherin-24 to
associate with
-catenin and p120 catenin.
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EXPERIMENTAL PROCEDURES |
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cDNA CloningSingle strand cDNA was synthesized using a Gene-Amp RNA PCR kit (PerkinElmer Life Sciences). Marathon-ready human mammary gland cDNA was purchased from Clontech Laboratories, Inc. cDNAs were amplified using PCR and degenerate primers as described for amplification of cadherins and proto-cadherins (24). Larger cDNAs were generated using 5' and 3' rapid amplification of cDNA ends (RACE)1 using a kit from Clontech and gene-specific primers. Standard sequencing of PCR products and full-length constructs was done at the Genomics Core Research Facility (University of Nebraska-Lincoln; Lincoln, NE). Oligonucleotides were synthesized by the Molecular Biology Core Facility at the Eppley Institute for Cancer Research (Omaha, NE).
The Genscan algorithm (25) was used to predict the open reading frame and amino acid sequence of cadherin-24 based on partial 5' and 3' RACE products. The gene-specific primers NC40 5'-GCCCCTCCACCCCAGCCAGCTCAT-3' and NC43 5'-AGCTGGCCAGGAGCTGCAGAGTCACACAC-3' were designed based on the nucleotide sequence and the predicted open reading frame. PCR fragments were amplified using the FailSafeTM PCR system (Epicentre Technologies, Madison, WI), subcloned, and sequenced. An expressed sequence tag (EST) (GenBankTM accession number AL137477 [GenBank] ) was obtained from RZPD Deutsches fur Genomforschung GmbH (Berlin, Germany) to assemble the cDNA encoding the predicted open reading frame of cadherin-24. A 2X-birch profilin tag (26) was added to the C terminus of cadherin-24 by PCR and subcloning into a modified version of pSPUTK (27).
Transfection and Retroviral InfectionTo express human cadherin-24 in mammalian cells, the 2X-Cbirch-tagged construct was ligated into the shuttle vector pMS and subcloned into the retroviral expression vector pLZRS-MS-IRES-neomycin (28, 29). Phoenix packaging cells (29) were transfected with pLZRS-MS-neomycin-cad24-2X-Cbirch using a calcium phosphate kit (Stratagene, La Jolla, CA). Transfectants were selected with 1 µg/ml puromycin (Sigma). Phoenix transfectants were passaged twice in antibiotic-free medium prior to viral harvest. For virus production, cells at 50% confluence were incubated in a 100-mm dish with 5 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 32 °C for 24 h. Medium was collected, filtered through a 0.45-µm filter (Nalgene, Rochester, NY), supplemented with 4 mg/ml polybrene (Sigma), and used immediately. For infection, cells were plated at 105 cells/100-mm dishes and infected 1216 h later. Following 68 h of infection, virus was removed and replaced with fresh medium. Stable cell populations were selected in 1 µg/ml G418 (Invitrogen) for 710 days.
Cell Culture and Cell AggregationMDA-MB-231 cells were obtained from Dr. Mary J. C. Hendrix (University of Iowa, Iowa City, IA). A431D cells have been described previously (30). Phoenix cells (ATCC) have been described (29). All cell lines were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 units/ml penicillin (Sigma), and 100 mg/µl streptomycin (Invitrogen).
For aggregation assays, cells were trypsinized to generate single cell suspensions and resuspended at a density of 2 x 105 cells/ml. 5,000 cells (20 µl) were placed on the inside cover of a 100-mm dish and allowed to aggregate at 37 °C for 18 h. The cells were triturated, remaining aggregates were observed using a Zeiss Axiovert 200M equipped with an ORCA-ER (Hamamatsu) digital camera, and images were collected using OpenLab software (Improvision Inc., Boston, MA).
Detergent Extraction of CellsConfluent monolayers were rinsed three times with phosphate-buffered saline and extracted in TNE (10 mM Tris-acetate, pH 8.0, 0.5% Nonidet P-40, 1 mM EDTA) containing 2 mM phenylmethylsulfonyl fluoride and 2 mM sodium orthovanadate. The cells were placed on ice, scraped, and triturated vigorously for 10 min. Insoluble material was pelleted by centrifugation at 14,000 x g for 15 min at 4 °C, and the supernatant was used immediately or stored at 80 °C.
Immunoprecipitation and ImmunoblotAll polypropylene tubes were rinsed with 0.1% Nonidet P-40 and dried prior to use in immunoprecipitations. 300 µl of cell extract was added to 300 µl of hybridoma-conditioned medium and gently mixed at 4 °C for 30 min. 50 µl of packed anti-mouse IgG affinity gel (ICN Biochemical Co., Costa Mesa, CA) was added, and mixing was continued for 30 min. Immune complexes were washed five times with TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 2 mM sodium orthovanadate, resuspended in 50 µl of 2x Laemmli sample buffer, and boiled for 5 min, and the proteins were resolved by SDS-PAGE (31). Proteins were electrophoretically transferred overnight to nitrocellulose membranes and immunoblotted as described previously (32).
ImmunofluorescenceCells were grown on glass coverslips to 80% confluence, fixed in 1% paraformaldehyde for 30 min, and permeabilized in methanol at 20 °C for 5 min. After three 5-min washes in serum-free culture medium, the coverslips were blocked in 10% goat serum in culture medium for 30 min and processed as described previously (32).
AntibodiesThe 4A6 mouse monoclonal antibody against the
birch epitope was a kind gift from Dr. Manfred Rudiger (Zoological Institute,
Braunschweig, Germany). Mouse monoclonal antibodies to -catenin (1G5)
and
-catenin (5H10) have been described previously
(33). The mouse monoclonal
antibody to p120 catenin (pp120) was obtained from BD Transduction
Laboratories.
Tissue Expression ScreeningRelative expression levels of cadherin-24 mRNA were estimated by reverse transcription PCR using a human multiple tissue cDNA panel (Clontech Laboratories, Inc.) and the FailSafeTM PCR System (Epicentre) with gene-specific primers NC42 5'-CCCCTGCCAGCCCAATCAGATACTCCATCC-3' and NC43, described above. PCR products were resolved on a 1% agarose gel.
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RESULTS |
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We employed PCR to more carefully examine the region of alternative splicing using cDNA from MDA-MB-231 and primers NC42 and NC43 (see "Experimental Procedures"). Three bands (Fig. 2) were excised and sequenced. The slowest migrating band was not related to cadherin-24 and was presumed to be an artifact. Sequence analysis confirmed that the other two bands were cadherin-24. The longer form contained an insertion of 114 bp in the fourth extracellular repeat of the predicted amino acid sequence (Fig. 1). Data from the Human Genome Project confirmed that the 114-bp insert is an authentic exon flanked by well defined intron boundary sequences.
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cDNA and Amino Acid Sequence of Cadherin-24 The nucleotide sequences and the deduced amino acid sequences of the two distinct cDNA clones are shown in Fig. 1. The short form contains a 2,346-bp open reading frame that encodes a putative 781-amino-acid protein, whereas the long form includes an open reading frame of 2,460 bp, which encodes a putative 819-amino-acid protein. We thus termed the cDNA clones cadherin-24 short form and cadherin-24 long form. The predicted start codon has a purine in the 3 position in accordance with the Kozak criteria (34). Cadherin-24 contains a hydrophobic signal sequence and the postulated furin cleavage site of cadherin precursor polypeptides (35, 36). The deduced amino acid sequence of mature cadherin-24 displays homology with the cadherin family as it includes: 1) an extracellular domain comprised of five cadherin-specific repeats (Fig. 1, arrows); 2) a transmembrane domain (Fig. 1, underlined); and 3) a cytoplasmic tail complete with amino acid sequences previously reported to bind catenins (37, 38). There are three potential N-linked glycosylation sites in the extracellular domain that are pointed out by asterisks.
Comparison of Cadherin-24 with Other CadherinsFig. 3 compares the amino acid sequences of cadherin-24, cadherin-11, cadherin-8, N-cadherin, E-cadherin, and P-cadherin. Cadherin-24 shows a total identity of 57% with cadherin-11, 55% with cadherin-8, 36% with N-cadherin, 33% with E-cadherin, and 31% with P-cadherin. The first two extracellular repeats of cadherin-24 show the highest homology among the compared cadherins (Table I). The extracellular domain of cadherin-24 includes the characteristic cadherin consensus sequences DXD, DRE, and DXNDN that are believed to be involved in Ca2+ binding (39). The N-terminal WV in EC1 is conserved, and the classical HAV sequence is replaced with QAV. All 4 cadherin conserved cysteine residues are present in EC5.
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Expression of Cadherin-24 We analyzed tissue-specific expression of cadherin-24 mRNA in human tissues by reverse transcription-PCR using gene-specific primers flanking the alternative exon insertion point. Fig. 4 shows that cadherin-24 short form is highly expressed in all tissues examined, whereas cadherin-24 long form was expressed in brain, kidney, lung, pancreas, and placental tissue. This is in contrast to MDA-MB-231 human mammary tumor cells, where there was approximately equal expression of the long and short forms (Fig. 2).
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Functional Analysis of Cadherin-24 To determine whether
cadherin-24 is a functional cadherin that mediates cell-cell adhesion,
birch-tagged constructions of either the long form or the short form were
transduced into the cadherin-negative A431D cell line. The expression level
was examined by immunoblot and immunofluorescence
(Fig. 5). Cadherin-24-2X-birch
short form and cadherin-24-2X-birch long form each were detected as a doublet
with molecular weights of 110,00 and 105,000
(Fig. 5A). The upper
band is probably not fully processed and likely retains the prosequence. This
is commonly seen when cadherins are exogenously expressed in A431D cells
(32). The transfectants
exhibited elevated levels of
-catenin and
-catenin, suggesting
that both cadherin-24 short form and cadherin-24 long form interact with and
stabilize the catenins. Immunofluorescence analysis showed that both the long
form and the short form of cadherin-24 were localized to cell-cell borders
(Fig. 5B). The diffuse
signal seen in the perinuclear region probably reflects partially processed
intracellular forms of cadherin-24 corresponding to the higher molecular
weight bands seen in Fig.
5A, lanes 2 and 3.
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To confirm that both forms of cadherin-24 interact with catenins, the
cadherin was immunoprecipitated from extracts of transfected cells using an
antibody against the birch tag. Fig.
6A shows that -catenin,
-catenin, and p120 catenin
all co-immunoprecipitate with both the long form and the short form of
cadherin-24. A431D cells express two isoforms of p120, and both forms
co-immunoprecipitate with both cadherin-24 proteins. To determine whether the
cadherin-24 isoforms could mediate cell-cell interactions, we performed
aggregation assays using the control A431D cells and the cells transduced with
each form of cadherin-24. Fig.
6B shows that both the long form and the short form cause
the cells to form large aggregates, whereas the control A431D cells do not
aggregate. Thus cadherin-24 is expressed as two alternatively spliced forms,
and each form is a fully functional cadherin that mediates cell-cell
interactions.
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Genomic Organization of Cadherin-24
Fig. 7 shows the
genomic structure of cadherin-24. The gene encoding human cadherin-24 lies on
the long arm of chromosome 14 in a generich segment near the centromere and is
transcribed toward the centromere. The closest neighbor of cadherin-24 on the
5' side is acinus (GenBankTM accession code NM_014977
[GenBank]
). Acinus and
cadherin-24 are transcribed in the same direction with the 5' end of the
cadherin-24 transcript only 1 kb from the 3'end of acinus. The
3' end of the cadherin-24 transcript lies only
12 kb from the
5' end of PSMB5 encoding the proteosomal
5 subunit (NM_002797
[GenBank]
).
The cadherin-24 gene is predicted to consist of 14 exons. The most 5'
and the most 3' exons are non-coding. All the introns start with GT and
end with AG. Although our longest 5' RACE product extended into exon 1,
the EST BX248750
[GenBank]
adds additional sequence to the 5' end and includes the
most 5' sequence in the databases. A number of ESTs, including
GenBankTM accession number AL137477
[GenBank]
, end in a poly(A) tail, which is
located just 3' of an AATAAA consensus poly adenylation signal
(40), suggesting that these
ESTs define the 3' end of the mRNA.
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DISCUSSION |
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Two transcripts encoding different protein isoforms of human cadherin-11
have been described that are predicted to contain identical extracellular
domains but completely different cytoplasmic tails
(23). The cytoplasmic tail of
the longer isoform contains typical p120 catenin and -catenin-binding
regions (GenBankTM accession code NM_001797
[GenBank]
), whereas that of the shorter
isoform does not (NM_033664
[GenBank]
). The transcript encoding the shorter protein
contains an extra exon relative to the transcript encoding the more typical
cadherin. The extra exon interrupts codon 632, which is within the predicted
transmembrane domain of each isoform. Although a number of other cadherins,
including human cadherin-24, contain an exon-intron boundary in a similar
position within the transmembrane domain, no other cadherins have been
reported to be alternatively spliced in this position. Recently, the shorter
isoform of cadherin-11 has been reported to affect cell behavior
(21).
Two transcripts for rat cadherin-22, also known as rat PB-cadherin, have
also been reported (GenBankTM accession numbers D83348
[GenBank]
and D83349
[GenBank]
). The
transcripts encode proteins that diverge completely in sequence after 23
residues of their putative cytoplasmic tails. Thus, as with human cadherin-11,
the longer isoform has binding sites for both p120 catenin and -catenin,
whereas the shorter isoform does not. We used each transcript to search the
November 2002 freeze of the rat genome
(genome.ucsc.edu/)
and found that the entire sequence of the more typical transcript maps to rat
chromosome 3. However, the sequence unique to the variant transcript maps to
rat chromosome 6. In addition, the point where the two transcripts diverge
does not correspond to a normal exon-intron boundary. These data raise the
possibility that the variant transcript may be a chimeric cDNA rather than an
alternatively spliced transcript.
In addition to cadherin-24, there have been a few other reports of
cadherins that are alternatively spliced in the extracellular domain.
Recently, a secreted form of chicken cadherin-7 was described that was due to
alternative splicing at an exon-intron boundary 55 amino acids into
extracellular repeat 5 (43).
The variant transcript contained an exon that is normally skipped. After 14
codons, the variant exon contained a termination codon; thus, the resulting
polypeptide was predicted to be secreted. The authors found that the truncated
protein inhibited cell-cell adhesion mediated by cadherin-7. Since type II
cadherins can interact heterophilically
(44), the secreted protein has
the potential to affect the adhesive interactions between a variety of type II
cadherins.
Similar to the case of chicken cadherin-7, two transcripts encoding rat
cadherin-8 that diverge from one another in extracellular repeat 5 have been
described (45). One transcript
encodes a cadherin with a typical structure (GenBankTM accession number
AB010436
[GenBank]
), and the other encodes a protein that diverges from the first after
20 amino acids into extracellular repeat 5 (AB010437
[GenBank]
). The variant
protein terminates after a further 19 amino acids, suggesting that it may be
secreted. When we analyzed the sequences of the two transcripts using the
November 2002 freeze of the rat genome, we found that the variant transcript
diverged precisely at an exon-intron boundary, but the variant transcript
continued into the adjacent intron throughout the remainder of its sequence.
It will be interesting to determine whether the variant cDNA represents an
incompletely processed transcript or a secreted form of the protein since a
secreted form of cadherin-8 would be expected to interfere with cell-cell
adhesion mediated by both cadherin-8 and cadherin-11
(44).
Human cadherin-6 (also known as K-cadherin) has been reported to be alternatively spliced in the extracellular domain (46). These authors reported several variants of human cadherin-6 and characterized a variant called cadherin-6/2 that was missing a portion of extracellular repeats 3 and 4. Using the sequence in GenBankTM accession number NM_004932 [GenBank] to represent the typical human cadherin-6 transcript and the November 2002 freeze of the human genome, we found that cadherin-6/2 is missing exon 7. Since exon 7 contains 254 nucleotides, splicing exon 6 to exon 8 would be expected to change the reading frame, and a stop codon would be encountered 15 codons after the splice junction. Thus, the expected result would be a secreted protein. However, cadherin-6/2 mediated cell adhesion when transfected into L-calls. In addition to missing exon 7, cadherin-6/2 was also missing nucleotide 34 of exon 8, putting the reading frame back into that of cadherin-6, so that cadherin-6/2 also contained an intact cytoplasmic tail. A search of the EST databases did not identify any additional cadherin-6 sequences missing exon 7 or residue 34 of exon 8. It would be interesting to see whether other tissues that express cadherin-6 produce variant transcripts.
The open reading frames of the two transcripts of cadherin-24 contain 781 and 819 codons. The algorithm of Nielsen et al. (47) predicted signal peptidase cleavage between amino acids 16 and 17. The mature proteins are likely to start with Ser-45, which is the residue immediately downstream of a consensus prohormone convertase cleavage site. As with other type II cadherins, the mature extracellular domains are predicted to contain five extracellular repeats. The extracellular portion of mature cadherin-24 contains three potential N-linked glycosylation sites at amino acids 446, 548, 563 of the open reading frame of the shorter splice form.
The alternative sequence in the long form of cadherin-24 is inserted just
C-terminal to Leu-454. Based upon the x-ray structure of the extracellular
portion of Xenopus C-cadherin, this position corresponds to the end
of strand F in EC4
(48). Just upstream of the
site of insertion in cadherin-24 lies Glu-453. This residue aligns with
Asp-414 in mature Xenopus C-cadherin, which is involved in
coordinating calcium ion number 3 between EC3 and EC4. See Boggon et
al. (48) for the
numbering of the resides in C-cadherin. This calcium ion is unique in the
x-ray structure of C-cadherin in that the side chain of Gln-397, rather than
the backbone carbonyl, is involved in coordinating it. The insertion of 38
amino acids at this site in cadherin-24 has the potential to disrupt the
binding of one or more of the calcium ions between EC3 and EC4, and as a
result, the potential to disrupt the structure at the EC3EC4 boundary.
Recent data suggest that multiple cadherin extracellular repeats may be
involved in homophilic binding
(49). If this is the case, the
insertion of 38 residues at the EC3EC4 boundary of cadherin-24 may be
expected to have an impact on how cells adhere to one another. However, the
long splice form of cadherin-24 still localizes to cell-cell borders and
mediates cell aggregation (Figs.
5 and
6).
In this report, we have identified for the first time a cadherin that is alternatively spliced in the extracellular domain, producing a longer splice form that is active in cell adhesion and retains its catenin-binding sites. Interestingly, the insertion in the longer form may alter its ability to bind calcium, which would be predicted to interfere with cadherin function. However, experimental data show that both forms mediate cell aggregation (Fig. 6). We have used PCR to show that cadherin-24 is widely expressed in normal tissues and that the more common transcript is the short form missing the insert in extracellular domain 4. Since we identified cadherin-24 in the MDA-MB-231 human breast cancer cell line, it will be interesting to determine whether the alternatively spliced form is expressed in tumors. We are currently developing antibody reagents to distinguish the two forms for such studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM51188 (to
M. J. W.) and DE12308 (to K. R. J.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: The University of Nebraska Medical Center, Eppley Cancer Center, 987696 University of Nebraska Medical Center, Omaha, NE 68198-7696. Tel.: 402-559-3892; Fax: 402-559-3739; E-mail: mwheelock{at}unmc.edu.
1 The abbreviations used are: RACE, rapid amplification of cDNA ends; EST,
expressed sequence tag.
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ACKNOWLEDGMENTS |
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REFERENCES |
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