THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 49, Issue of December 6, pp. 47679 –47685, 2002
Printed in U.S.A.

Identification and Characterization of a Soluble Cadherin-7 Isoform
Produced by Alternative Splicing*
Received for publication, May 30, 2002, and in revised form, September 24, 2002
Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.M205328200

Rie Kawano‡§¶, Noritaka Matsuo‡, Hideaki Tanaka储, Masaru Nasu§, Hidekatsu Yoshioka‡,
and Komei Shirabe‡
From the ‡Department of Anatomy, Biology, and Medicine and the §Department of Infectious Diseases, Oita Medical
University, Hasama-machi, Oita 879-5593, Japan, and the 储Division of Developmental Neurobiology, Kumamoto
University Graduate School of Medical Sciences, Honjo 2-2-1, Kumamoto 860-0811, Japan

Cell adhesion molecules are usually transmembrane glycoproteins that mediate binding interactions at the extracellularsurface and determine the specificity of cell-cell recognition.
The cadherin, integrin, immunoglobulin, and selectin families
are major cell adhesion molecules (1). The molecules of thecadherin family mediate calcium-dependent cell-to-cell adhesion, and they play roles in cell recognition and cell sorting
during development (2, 3). They can be divided into six major
subfamilies: 1) classic (type I) cadherins, 2) atypical (type II)
cadherins, 3) desmocollins, 4) desmogleins, 5) protocadherins,
and 6) Flamingo cadherins, and several cadherins clearly occupy isolated positions in the cadherin superfamily (4).
The type I (classic) and type II (atypical) cadherins have acharacteristic structure, with NH2-terminal domain, four CRs1
(CR1–CR4),1 a pretransmembrane domain (CR5), a transmem-

brane domain, and a highly conserved cytoplasmic domain (2,5), except for T-cadherin (6, 7). CR1 provides the binding specificity of cadherins (8, 9). The type I cadherins have an HAV
motif in the CR1 domain, and this motif is important for calciumdependent adhesion (10). The type II cadherins, on the other
hand, have no HAV site. This difference may be responsible forthe weaker intercellular adhesion shown by type II comparedwith type I cadherin (11). The highly conserved cytoplasmic
domain, which associates with ␣- and ␤-catenin, plakoglobin,and p120, is responsible for the stabilization of cell-to-cell adhesion (12–16).
Cadherin-7, a type II cadherin, is expressed in some populations of migrating neural crest cells, proximal dorsal and ventral roots, and dorsal root ganglia in chicken embryos. It is also
expressed in non-neural crest cells, such as motor neurons, andcells in the floor plate and in myotomes (5). Overexpression of
cadherin-7 blocks neural crest segregation from neuroepithelial cells (17). The regulated expression of cadherins, including
cadherin-7, in the neuroepithelium is essential to maintain its
integrity and to send out neural crest cells in the correct direction (17). Cadherin-7 shows a partial affinity to cadherin-6B,
but the heterophilic interaction is less stable than the homophilic (5). Cadherin-7 is involved in intercellular signaling
between homotypic cells and in the sorting of heterotypiccells, but the precise function of cadherin-7 has not yet been
determined (17, 18).
Splicing variants of type II cadherin have been reported for
cadherin-11 (19) and PB-cadherin (20). These variant forms aretruncated transmembrane protein with a cytoplasmic domainthat differs from full-length forms. In this report, we identified
a soluble splicing variant form of chicken cadherin-7. Competitive PCR analysis and immunostaining showed differential
expression patterns between the variant form and full-length
cadherin-7. The variant protein interacted with and inhibitedcell-to-cell adhesion mediated by full-length cadherin-7 in vitro
and in vivo.

* This study was supported by Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science, Sports and Culture of Japan
11470312 (to H. Y.) and 13680874 (to K. S.). The costs of publication ofthis article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s) AY115555.
¶ To whom correspondence should be addressed. Tel.: 81-97-5865672; Fax: 81-97-549-6302; E-mail: riekawa@oita-med.ac.jp.
1
The abbreviations used are: CR, cadherin repeat; Ab, antibody; HRP,
horseradish peroxidase; PB-cadherin, pituitary gland and brain-cadherin;
P-cadherin, placental-cadherin; T-cadherin, truncated-cadherin; E-cadherin, epithelial-cadherin; E, embryonic day; PBS, phosphate-buffered saline; HCMF, hepes-buffered calcium and magnesium-free Hank’s solution.

Animals—Fertilized eggs of White Leghorn chickens were purchasedfrom commercial sources. They were incubated at 38 °C, and staging of
the embryos was performed according to Hamburger and Hamilton(21). Rabbits were purchased from commercial sources for the production of polyclonal antibodies (Abs).
Isolation of Variant Form of Chicken Cadherin-7 cDNA—Total RNA
was prepared from embryonic day (E) 12 chicken brains using Isogen(Nippon Gene Co. Ltd., Tokyo, Japan), and cDNA was synthesized from1 ␮g of total RNA using SuperScript II reverse transcriptase (Invitrogen). PCR primers were designed based on the chicken full-length
cadherin-7 cDNA sequence (GenBankTM accession number D42150) as
follows: U1847 (see Fig. 1, B and C), 5⬘-TGGCTATGGAGAGTCAGAATCC-3⬘; L2158 (see Fig. 1, B and C), 5⬘-CAGATTGTTCCTGTCTTCGG-3⬘. The cDNA was amplified with these primers by PCR. The

We identified an alternative mRNA encoding a novel cadherin-7 isoform by reverse transcriptase-PCR of RNA fromday 12 chicken embryos. The alternative mRNA contains 49
bases of insertion in the premembrane region, leading to
the substitution of 14 amino acids and the introduction of apremature stop codon. Identification of a 49-bp insertion
sequence in the genomic DNA corresponding to the intronof the cadherin-7 gene suggests that alternative splicing is
the cause of the alternative mRNA. Transient expression of
the variant form in COS-7 or 293 cells produced a soluble
protein. Aggregation assays and immunoprecipitation
showed that the variant protein interacts with full-length
cadherin-7 in vitro and in vivo and inhibits full-length cadherin-7-mediated cell adhesion. Immunohistochemistry revealed that the variant form was strongly expressed in dermomyotomes rather than in migrating neural crest cells, incontrast to the full-length cadherin-7, suggesting differential regulation of splicing and possible roles of variant cadherin-7 in the development of dermomyotomes and othertissues.

EXPERIMENTAL PROCEDURES

This paper is available on line at http://www.jbc.org

47679

47680The Variant Form of Cadherin-7

PCR products were separated using 4.0% agarose gel electrophoresis.
The amplified fragment (360 bp) was cloned into the pGEM-T Easy
Vector (Promega) and sequenced on an ABI 310 sequencer(PerkinElmer Life Sciences and Applied Biosystems).
Cloning of the Partial Chicken Cadherin-7 Gene—Chick genomic
DNA was extracted from whole E6 embryos according to the standard
protocol (22). Modified nested PCR of the genomic DNA was performed
as follows. The chicken genomic DNA was first amplified using primers
U1593 (see Fig. 3A), 5⬘-AGAAGATGTTGATGAGCCACC-3⬘, and L2158,
and the PCR product was further amplified with primers U1861 (seeFig. 2), 5⬘-CAGAATCCAGCACAGATTGG-3⬘, and L2158, using Ex Taq
polymerase (Takara, Tokyo, Japan). The PCR cycle conditions were 30
cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 10 min, followed by
72 °C for 10 min. The PCR product of about 6 kbp was cloned into thepGEM-T Easy Vector and sequenced.
Competitive PCR for Quantitative Analysis—To develop a competitive PCR assay for variant and full-length chicken cadherin-7, a competitor DNA was prepared. The variant form cDNA was amplified by
PCR using two primers, U1593 and L2158. The amplified 634-bp fragment was cloned into the pGEM-T Easy Vector. The DNA was cut with
NcoI (Takara) and re-ligated to delete a 249-bp NcoI fragment (Fig. 3A).
The copy number of competitor molecules was determined, and then
10-fold serially diluted competitor DNAs were added to PCR reactions.
Total RNAs were prepared from each stage of the chicken embryos
using Isogen, and first-strand cDNAs were synthesized from 1 ␮g oftotal RNAs in 20 ␮l of reaction mixture using SuperScript II reverse
transcriptase. The combinations of PCR primers are shown in Fig. 3A.
The nucleotide sequence of the primer L variant was 5⬘-TTGACCGCTTCTGAGAGGTAGG-3⬘, and the sequences of the other primers aredescribed above. Competitive PCR was performed in 20 ␮l of reaction
mixture containing 0.2 mM each of the four deoxynucleotide triphosphates, 1.5 mM MgCl2, 0.5 ␮M of each primer, 1 ␮l of each 10-foldserially diluted competitor, and 1 unit of rTaq polymerase (Takara)with Taq start antibody (Clontech). The PCR cycle conditions were
94 °C for 2 min, 30 cycles of 94 °C for 40 s, 58 °C for 1 min, 72 °C for 1
min, followed by 72 °C for 5 min. The PCR products were separated by
2% agarose gel electrophoresis, stained with ethidium bromide, andobserved under UV illumination.
Expression of Chicken Full-length Cadherin-7 in L Cell Lines—To
obtain a cDNA clone coding for full-length cadherin-7, a cDNA phagelibrary from E9 chicken brain in the vector lambda ZapII (Stratagene)
was screened with a radiolabeled probe for the cadherin-7 fragment(350 bp). The phage clone containing the cDNA for full-length cadherin-7 was subcloned into the plasmid vector, Bluescript II SK⫹ (pBScad7) (Toyobo, Tokyo, Japan). After digestion with EcoRI, a 3-kb fragment containing the complete coding sequence was recloned into aeukaryotic expression vector, pCAGGS (23), to obtain pCA-cad7. L cells
were transfected with pCA-cad7 using LipofectAMINE Plus reagent(Invitrogen). Transfectants were screened by immunoblotting and immunostaining using CCD7–1, and a stable cell line expressing cadherin-7 (L-cad7) was thus obtained.
Expression of the Variant Form of Chicken Cadherin-7 in COS-7 or
293 Cells—The expression vector for cadherin-7 variant was constructed as follows. cDNA from E12 chicken brain was amplified withtwo primers, U427 (see Fig. 1C) designed in the 5⬘-untranslated region,
5⬘-AGAGACAACTGGAATTGTGCC-3⬘, and L2158 (see Fig. 1, B and C).
Amplified fragments contained both the full-length and variant cDNAs
and were cloned in pGEM-T Easy Vector. A single-clone plasmid containing variant cDNA was selected by PCR using two primers, U1847(see Fig. 1, B and C) and L variant (see Fig. 3A), and verified bysequencing. The selected plasmid, pPCR-v, was amplified with two
primers, U427, and Lxba I, 5⬘-GCTCTAGAATACAGGTTGACCGCTTC3⬘. The primer of Lxba I was designed to remove the stop codon andintroduce an XbaI site at the 3⬘-terminal end. The amplified U427-Lxba
I fragment was cloned in pGEM-T Easy Vector and recloned into pEF1/Myc-His A (Invitrogen) to generate the expression construct pEF-v.
COS-7 or 293 cells were transfected with pEF-v (COS variant cells or
293 variant cells), and 293 cells were transfected with pEF-1/Myc-His A(293 mock cells) as control by LipofectAMINE Plus reagent. Following
incubation with serum-free RPMI 1640 medium (Sigma) for 2 days,
culture media were concentrated using Centricon YM30 tubes (Amicon)
and replaced with 50 mM Tris-HCl (pH 7.5). Media were finally concentrated up to about 30-fold. We confirmed that the expression level ofvariant protein was almost equal with the COS variant and 293 variant
cells (data not shown).
Aggregation Assays—Monolayer cultures of L-cad7 were treated with
0.01% Trypsin (Sigma) and 10 mM CaCl2 in Ca2⫹- and Mg2⫹-free saline
buffered with 10 mM HEPES (HCMF, pH 7.4) for 30 min at 37 °C. The

treated cells were washed three times in HCMF with 1 mM EDTA anddissociated into single cells (24). The dispersed cells were suspended in
HCMF with 1 mM CaCl2 or 1 mM EDTA at a density of 2 ⫻ 105 cells/ml.
24-well plates (Iwaki, Tokyo, Japan) were coated with 1% bovine serum
albumin (Sigma) for 1 h at room temperature and washed twice with
HCMF. The cell suspension (0.5 ml) was dispensed into each well with
30-fold-concentrated culture medium from COS variant cells or COS-7
cells. The plates were incubated at 37 °C on a gyratory shaker rotating
at 40 rpm.
Antibodies—Antibodies specific to the variant form of cadherin-7,
anti-variant Ab, were generated in rabbits by immunizing with the
COOH-terminal
peptideofthevariant
form,
NH2-GDVSPYLSEAVNLY-COOH (Fig. 1B), conjugated with Keyhole Limpet
hemocyanin as described before (25). The antiserum was affinity-purified with an epoxy-activated Sepharose 6B column (Amersham Biosciences) that was modified with the variant peptide. The other antibodies used in the experiment were as follows: mouse IgG monoclonal
antibody against cadherin-7; CCD7–1 (a generous gift from Drs. S.
Nakagawa and M. Takeichi of Kyoto University); HRP-conjugated antimouse IgG (Wako, Osaka, Japan); biotinylated anti-mouse IgG (Wako);
HRP-conjugated anti-rabbit IgG (Wako); and Texas Red-conjugatedstreptavidin (Caltag).
Immunoblotting Analysis—The concentrated culture media of
L-cad7, 293 variant and 293 mock cells were mixed with 2⫻ sample
buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 2% SDS, 20% sucrose,
1.2% mercaptoethanol, 0.01% BPB). These cells were lysed with 1⫻
sample buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS, 10%
sucrose, 0.6% mercaptoethanol, 0.005% BPB). The samples were boiled
for 5 min and run in 10% SDS-polyacrylamide gels and transferred topolyvinylidene difluoride membranes. The blots were incubated withappropriate antibodies, and the signals were detected using the ECL or
ECL Plus systems (Amersham Biosciences).
Immunoprecipitation—The L-cad7 cells were dissociated into single
cells as described in the methodology for the aggregation assay andre-aggregated in culture medium of COS variant cells in the presence of
CaCl2 for 15 min. The re-aggregated cells were lysed in lysis buffer (150mM NaCl, 50 mM HEPES, pH 7.5, 5 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 0.1% trypsin
inhibitor) with 1% Triton X-100.
E5 chicken embryos were homogenized in lysis buffer without Triton
X-100. The tissue lysate was centrifuged at 70 ⫻ g for 5 min at 4 °C. The
supernatant (whole homogenate) was centrifuged at 65,000 ⫻ g for 1 h
at 4 °C, and fractionated into the supernatant (soluble fraction) and
precipitate. The precipitate was suspended in lysis buffer with 1%
Triton X-100 and centrifuged at 14,000 ⫻ g for 5 min at 4 °C. The
supernatant (membranous fraction) was then collected.
For immunoprecipitation, the cell lysate and whole homogenate with
1% Triton X-100 was incubated with a 1/10 volume of purified antivariant Ab at 4 °C for 5 h, then protein G beads (Amersham Biosciences) were added, and the mixture was incubated at 4 °C for 1 h.
The beads were washed three times with lysis buffer with 1% Triton
X-100 and dissolved in sample buffer. Bound proteins were eluted fromthe beads by boiling for 5 min.
Immunostaining—Cultured cells were fixed and permeabilized in
100% methanol with 3% H2O2 for 1 h at room temperature. They wereincubated with CCD7–1 antibody or anti-variant Ab, followed by incubation with HRP-conjugated second antibody. For the detection of HRP,
Tyramid reagent (PerkinElmer Life Sciences) was used. For doublelabel immunostaining, they were incubated with CCD7–1 antibody,followed by biotinylated anti-mouse IgG and Streptavidin-Texas Red,and then with anti-variant Ab, followed by anti-rabbit IgG-HRP and
Tyramid reagent.
E5 chicken embryos were fixed in 4% paraformaldehyde at 4 °C for
2 h and washed in PBS at 4 °C overnight. They were permeabilized ina graded series of methanol in PBS (25, 50, 75, and 100% at 4 °C for 30
min each), placed in 6% H2O2 in methanol at room temperature for 2 h,and re-hydrated in PBS. Nonspecific binding of antibodies was blocked
by immersion in 10% skim milk with 0.01% Triton X-100 in PBS(PBSMT) at room temperature for 2 h. Embryos were incubated with
CCD7–1 antibody or anti-variant antibody at 4 °C overnight. The embryos were washed in 0.01% Triton X-100 in PBS (PBST) for 6 h at 4 °C,
immersed in PBSMT for blocking of nonspecific binding of antibody,and then incubated with an appropriate HRP-conjugated second antibody at 4 °C overnight. The embryos were washed in PBST for 6 h at
4 °C. For the detection of HRP activity, they were immersed in PBS
containing 166 ␮g/ml 3,3⬘-diaminobenzidine at room temperature for 30
min, and H2O2 was added to a final concentration of 0.003%. Embryos
were dehydrated in a graded series of ethanol, followed by 100% chlo-

The Variant Form of Cadherin-7

FIG. 1. Identification of the variant form of chicken cadherin-7. A, RT-PCR analysis on total RNA from day 12 embryonicchicken brains. Lane 1, 100-bp ladder maker; lane 2, the RT-PCR
products. An arrow and an arrowhead indicate the amplified DNA
fragments of variant and full-length chicken cadherin-7, respectively.
In the reaction, primers U1847 and L2158 were used for the amplification of cDNA encoding a partial CR4 domain and pretransmembrane
fragment of cadherin-7. B, partial nucleotide sequences and predicted
amino acid sequences of chicken full-length cadherin-7 and the variant
form. The large and small boxes indicate a 49-bp insertion and termination codon, respectively. The horizontal arrows show the bindingsites for the two primers, U1847 and L2158, used in the PCR reaction.
C, schematic representation of chicken full-length cadherin-7 (top) andthe variant form (bottom). The boxes and horizontal lines indicate thecoding and untranslated regions, respectively. Each box is as follows:
hatched box, precursor region; dotted box, NH2-terminal domain;
CR1– 4, cadherin repeats 1– 4; preTM, pretransmembrane domain; TM,
transmembrane domain; CP, cytoplasmic domain; closed box, the
COOH-terminal segment of the variant form of cadherin-7. ATG, TAG,
TGA, and arrows indicate first methionine of translation, stop codons,
and the binding sites of primers used for PCR, respectively.
roform, 50% paraffin in chloroform, and then embedded in paraffin.
Serial 10-␮m sections were cut on a microtome, de-waxed, and mounted
on glass slides using Permount (Fisher).
RESULTS

Identification of a Variant Form of Chicken Cadherin-7—
Two DNA fragments were detected by PCR reaction on cDNAfrom E12 chicken brain using primers that amplified the cDNA
encoding for partial CR4 and the pretransmembrane domain of
cadherin-7 (Fig. 1A). Nucleotide sequence data showed that thelonger fragment had the partial cDNA sequence of full-length
cadherin-7 and an insert sequence of 49 bp, which caused
frameshift and a stop codon within the insert, and the shorter
fragment corresponded to the partial cDNA sequence of fulllength cadherin-7 (Fig. 1B). Because the stop codon existed
before the transmembrane domain, the variant transcript
coded for the soluble form of cadherin-7, possessing only the
extracellular domains of full-length cadherins (Fig. 1C).
To examine the origin of the 49-bp insert and exon-intron
junction in the chicken genome, a genomic DNA fragment of ⬃6kbp, coding for the CR4 and pretransmembrane region, wascloned by PCR. The DNA clone was sequenced, and four exons
were detected (Fig. 2B). Consensus GT and AG sequences,including the adjacent polypyrimidine tract were found in the

47681

FIG. 2. Partial structure and nucleotide sequence of thechicken cadherin-7 gene. A, schematic representation of the cadherin-7 genomic organization leading to two alternative splicings: thevariant form and full-length cadherin-7. About 6 kbp of genomic DNA
fragment was cloned from chicken embryo by the PCR method. The
arrows indicate the primers used, U1861 and L2158. B, the partial
nucleotide sequence of genomic DNA of chicken cadherin-7. The nucleotide sequences representing the exons are boxed. The mRNA of variant
form only includes the additional exon (n⬘) sequence. The horizontal
bars and underlines indicate the GT/AG sequences in the splicing sites,
and the polypyrimidine tract adjacent to AG dinucleotides, respectively.

49-bp insertion (26). All data suggested that the variant form of
cadherin-7 had been generated by alternative splicing (Fig.
2A).
Expression Level of mRNA of the Variant Form in Chicken
Embryos—We carried out competitive PCR to compare thetranscription levels of the variant form and full-length cadherin-7 at different developmental stages (Fig. 3). The expression level of the variant form was very low in whole embryos atstage 10, compared with that of the full-length cadherin-7.
Because neural crest migration, somitogenesis, and myogenesis in the rostral region precede those in the caudal region, we
examined the expression level of the variant form and fulllength cadherin-7 in rostral and caudal halves of chick embryos
at stage 17 separately. A significant expression of variant
mRNA was detected in the rostral half of the trunk, although
expression in the caudal half was still low. The amounts ofvariant transcripts in the rostral half were in the range fromone-tenth to one-fifth of that of full-length cadherin-7. After
this stage, the expression level of variant form in the trunk atwing level was almost constant until E9, maintaining the sameratio to that of full-length cadherin-7 (Fig. 3C).
Expression of the Variant and Full-length Forms of Cadherin-7 in Vitro—To obtain information on the variant protein,
we generated anti-variant Abs that specifically recognized thevariant form of cadherin-7 by immunizing rabbits with theshort COOH-terminal peptide. We introduced the cDNA of thevariant form into COS-7 or 293 cells (COS or 293 variants)
transiently and the cDNA of full-length cadherin-7 into L cells(L-cad7) stably. The expressions of full-length cadherin-7 andthe variant form were confirmed by immunostaining with
CCD7–1 and anti-variant Ab (Fig. 4A). It is noteworthy thatfull-length cadherin-7 signal was detected at the cell boundary,indicating that it was functioning in cell-to-cell interaction,

47682The Variant Form of Cadherin-7

FIG. 4. Immunostaining and immunoblotting of the variant
and full-length cadherin-7-expressing cells. A, immunofluorescent
staining of L-cad7 (1 and 2) and COS variant (3 and 4). Cells were
stained with CCD7–1 (1 and 3) and anti-variant Ab (2 and 4). The
signals in 2 were nonspecific, and in 3 and 4 the same cells weredouble-stained. Bars, 50 ␮m. B, immunoblotting analysis using
CCD7–1 (lanes 1– 6) and anti-variant Ab (lanes 7–12). The concentrated
conditioning medium of 293 variant (lanes 1 and 7), 293 mock (lanes 3
and 9), L-cad (lanes 5 and 11), and whole cell lysate of the 293 variant(lanes 2 and 8), 293 mock (lanes 4 and 10), L-cad7 (lanes 6 and 12) wereapplied. The arrow, arrowhead, and asterisk indicate full-length cadherin-7 (105 kDa), the variant protein with myc-His tag (73 kDa), anddegradation products of the variant protein, respectively. The protein
marker represents a pre-stained protein marker broad range (NewEngland BioLabs).

FIG. 3. Competitive PCR analysis of the variant form and fulllength cadherin-7. A, schematic representation of amplified fragments and the competitor. The letters indicate the following: a and a⬘,amplified fragments of the variant and full-length form using U1593
and L2158; b, an amplified fragment of the variant form using theprimers U1593 and L variant; c1 and c2, amplified fragments of thecompetitor using the primers U1593 and L2158, and U1593 and L
variant, respectively. B, agarose gel analysis of competitive PCR products using RNAs from chicken embryonic tissues. Each gel shows a
representative experiment from five similar results. The sources ofRNAs are as follows: A and B, total embryo at stage 10; C and D, caudal
half trunk at stage 17; E and F, rostral half trunk at stage 17; G and H,
trunk at wing level at stage 23; I and J, trunk at wing level at stage 29;
K and L, trunk at wing level at 9E. The combination of primers U1593
and L2158 was used in A, C, E, G, I, and K, whereas that of U1593 andL variant were used in B, D, F, H, J, and L. The lanes contain the
products amplified from a constant amount of cDNA mixture, and thecompetitor DNA was prepared in successive 10-fold dilutions. a, a⬘, b,
c1, and c2 indicate the amplified DNA fragments. Note that the fragments a and a⬘ showed almost the same mobility on the 2% agarose gel.

whereas the signal for the variant form was found inside thecell body.
Immunoblotting, using CCD7–1 and the anti-variant Ab,
detected the 73-kDa band of the variant form with an Myc-His
tag in the concentrated culture medium of 293 variant cells(Fig. 4B, lanes 1 and 7). In the cell lysate of 293 variant,
CCD7–1 detected this band weakly (Fig. 4B, lane 2), but antivariant Ab did not detect it (Fig. 4B, lane 8), because thesensitivity of anti-variant Ab was lower than that of CCD7–1.
It was thought that the lower bands of lanes 1 and 2 (Fig. 4B)
were degradation products. CCD7–1 detected the 105-kDa
band of full-length cadherin-7 in the lysate of L-cad7 cells (Fig.
4B, lane 6), but anti-variant Ab detected no band (Fig. 4B, lane12). Full-length cadherin-7 expressed by L-cad7 was highly
degradable during lysis procedures as previously reported (17).
The lower band of lane 6 (Fig. 4B) reflects these degradation
products. Because full-length cadherin-7 was a membranous
protein, CCD7–1 did not detect it in the concentrated culture
medium of L-cad7 (Fig. 4B, lane 5). Anti-variant Ab detected noband in the concentrated culture medium of L-cad7 (Fig. 4B,

C, quantification of the results of competitive PCR. The values represent the means ⫾S.D. values of five separate experiments. The black
and white bars indicate the relative expression level of the variant andfull-length forms, respectively. The results were demonstrated as -foldrelativity. glyceraldehyde-3-phosphate dehydrogenase levels of eachsample were not different (data not shown).

The Variant Form of Cadherin-7

FIG. 5. Effects of the variant protein on cell aggregation mediated by full-length cadherin-7. A, time course of cell aggregation
mediated by full-length cadherin-7 with or without the variant protein.
L-cad7 cells were dissociated by trypsin treatment and were allowed toaggregate in conditioning medium from COS variant cells that contained the variant protein (closed circles) or COS-7 cells (closed squares)in the presence of 1 mM CaCl2. The aggregation was represented by theindex Nt/No, where No is the initial cell number before aggregation and
Nt is the total particle number at incubation time t (minutes). Values
represent the means ⫾S.D. of six separate experiments. B, appearanceof L-cad7 cells in aggregation assays with or without the variant protein. The L-cad7 cells were aggregated for 30 min in conditioning medium from COS variant cells (panel 1) or COS-7 cells (panel 2) with 1
mM CaCl2. Insets show mock experiments in the absence of CaCl2. Bars,
50 ␮m. C, co-immunoprecipitation of the variant protein and full-length
cadherin-7. After an aggregation assay was performed with the variant
protein and 1 mM CaCl2 for 15 min, the cell lysate was immunoprecipitated with anti-variant Ab. The precipitates were loaded onto the SDSpolyacrylamide gel for immunoblotting analysis (lanes 2 and 3). The
filter was incubated with CCD7–1 (lane 2) or anti-variant Ab (lane 3).
For comparison, the lysate of L-cad7 cells was loaded, blotted, andincubated with CCD7–1 (lane 1). The signals were detected using an
ECL system. The arrow, arrowhead, and asterisk indicate full-length
cadherin-7, the variant protein with the myc-His tag, and degradation
products of full-length cadherin-7, respectively.

lane 11). CCD7–1 and anti-variant Ab detected no band in thelysate and concentrated medium of 293 mock cells.
Effect of the Variant Form on Cell Adhesion Mediated by
Full-length Cadherin-7 in Vitro—We introduced a cell aggregation assay mediated by full-length cadherin-7 using L-cad7
cells to examine a possible function for the variant protein (5).
The aggregation of L-cad7 cells was significantly slower in thepresence of the conditioned medium from COS variant cells
than in that of COS-7 cells within 30 min. Even after 120 min,the difference was still apparent. These aggregations were
calcium-dependent (Fig. 5, A and B). These data suggest thatthe variant form inhibits cell aggregation mediated by fulllength cadherin-7.
We performed immunoprecipitation to confirm interaction of
the variant protein with full-length cadherin-7. We collected
and lysed the cells after 15-min aggregation, and immunoprecipitation was performed using anti-variant Ab. The full-length
cadherin-7 co-immunoprecipitated with the variant protein as

47683

FIG. 6. The variant and full-length cadherin-7 in chicken embryos. A, immunoblotting and co-immunoprecipitation analysis of fractionated tissue homogenate from E5 chicken embryos, showing thewhole homogenate (lane 1), soluble fraction (lane 2), membranous fraction (lane 3), the concentrated conditioning medium of COS variant
cells (lanes 4 and 5), and immunoprecipitation with anti-variant Ab(lanes 6 and 7). The filter was stained with CCD7–1 (lanes 1– 4 and 6)
or anti-variant Ab (lanes 5 and 7). The arrow indicates full-length
cadherin-7; arrowhead, the variant protein; asterisk and sharp sign,degradation products of full-length cadherin-7. B, immunostaining.
Transverse (1, 2) and horizontal (3, 4) sections of the trunk region in E5
embryos. Whole embryos were stained with anti-variant Ab that detects
only the variant form (1, 3), and with CCD7–1 that detects both fulllength and the variant form of cadherin-7 (2, 4). Arrows indicate dermomyotomes; SC, spinal cord; D, dorsal root ganglion. Bars, 100 ␮m.

shown in Fig. 5C. These results indicate that the variant protein interacts with full-length cadherin-7.
The Variant and Full-length Cadherin-7 Proteins in Chicken
Embryos—To localize the variant protein in chicken embryo,
we fractionated the homogenate of whole E5 chicken embryos(Fig. 6A, lane 1) into soluble (Fig. 6A, lane 2) and membranous
fractions (Fig. 6A, lane 3). CCD7–1 detected three bands in thewhole homogenate. The 73-kDa band seemed to be the variant
form, because anti-variant Ab detected the 73-kDa band thatco-migrates with the variant cadherin-7 expressed in COS variant cells (Fig. 6A, lanes 4 and 5). The 105- and 80-kDa bands
seemed to correspond to the full-length cadherin-7 and its
degradation product, respectively (see Fig. 5C, lane 1). The
majority of the variant form was found in the soluble fraction,
but some was in the membranous fraction. Because the variant
protein was soluble and was still found after resuspension andrecentrifugation of the membranous fraction, we believe thatthe variant form in the membranous fraction interacts withfull-length cadherin-7. The full-length and degraded forms of
cadherin-7 were found only in the membranous fraction.
To examine the interaction of the variant protein with fulllength cadherin-7 in vivo, we performed immunoprecipitationfrom the whole homogenate solubilized with 1% Triton X-100
using anti-variant Ab. Full-length cadherin-7 was co-immunoprecipitated with the variant protein (Fig. 6A, lanes 6 and 7).
These results indicate that some of the variant protein interacts with the full-length cadherin-7. The degradation form of
80 kDa appears to have no interaction with the variant form,
because it was not co-immunoprecipitated. The 100-kDa bandthat was co-immunoprecipitated with anti-variant Ab seemed
to be another degradation product of full-length cadherin-7,
because this band was often observed for L-cad7 cells (Fig. 6A,
lane 6).
We examined the expression pattern of the variant form in47684The Variant Form of Cadherin-7

E5 chicken embryo tissues, by comparing it with the staining
generated by the CCD7–1 antibody that detects both fulllength and variant cadherin-7. The variant form was expressedtransiently and strongly in dermomyotomes, then at the dorsal
and ventral lips and in the myotome. Faint signals of thevariant form were detected in proximal dorsal and ventral roots
and in dorsal root ganglia (Fig. 6B, 1 and 3). In contrast, thestronger signals of full-length cadherin-7 were detected in dorsal root ganglia and in proximal dorsal and ventral roots, andfainter signals were detected in dermomyotomes (Fig. 6B, 2
and 4). The full-length cadherin-7 was strongly expressed in
migrating neural crest cells, but signals of the variant form
were faint in these cells (data not shown).
DISCUSSION

In the present study, we first report an identification of thesoluble isoform for cadherin-7. Differential expression of theisoform and inhibitory effect on the cell adhesion mediated byfull-length cadherin-7 suggest new type of regulatory mechanism of cell adhesion and cell signaling.
Identification of the Soluble Isoform of Cadherin-7—We identified the variant form of cadherin-7 in chicken embryos. Molecular cloning of the genomic fragment revealed that the variant mRNA originated by alternative splicing. Such a splicing
variant of type II cadherin has been reported for cadherin-11(19) and PB-cadherin (20). These variant proteins are truncated forms with an intact transmembrane and a different
cytoplasmic domain. The variant protein of cadherin-7 consistsof an NH2-terminal domain, four CRs (CR1–CR4), a shorter
pretransmembrane domain, and a distinct COOH-terminal
polypeptide of 14 amino acids but lacks transmembrane andcytoplasmic domains. The variant protein might not be a glycosyl phosphatidylinositol-anchored protein, because it does
not have the hydrophobic COOH-terminal domain essential for
glycosyl phosphatidylinositol-modification (27), in contrast to
T-cadherin (6, 7). To investigate whether the variant protein issoluble, we transfected the cDNA of the variant form of cadherin-7 into COS-7 and 293 cells and detected the variant
protein in the conditioning medium as a soluble molecule.
Furthermore, we fractionated the homogenates of E5 embryos
into soluble and membranous fractions. The majority of thevariant form was detected in the soluble fraction, and the small
amount was found in the membranous fraction. We believe thatthe variant form of the membranous fraction interacts withfull-length cadherin-7. Some soluble forms of the type I and II
cadherins that were generated by proteolysis have been reported previously (28, 29). Thus, this is the first demonstrationof a soluble form of type I and II cadherins generated byalternative splicing.
Inhibition of Cell Adhesion Mediated by Full-length Cadherin-7 by the Variant Form—It is known that each subtype of
cadherin molecules prefers homophilic to heterophilic interactions. Thus, cadherin molecules play a role in intracellularsignaling between homotypic cells and in sorting heterotypiccells (5), and the CR1 domain determines their binding specificity (8, 9). The extracellular domain is able to act as a ligand
for the same subtype of membranous-bound cadherins andinhibits adhesion mediated by the same subtype of membranous-bound cadherins. The soluble fragment of E-cadherin,
generated by proteolysis, is able to disrupt cell-to-cell adhesionin cultured epithelial cells (28) and is involved in carcinoma
invasion in vivo (30 –33). Soluble P-cadherin, generated byproteolysis, is present in human milk and serum of women withor without breast cancer, but the exact function of this protein
is not clear (34, 35). And the soluble recombinant T-cadherin
inhibits neurite extension from selected neuron populations(36). Several mutations in cadherin-23, a member of the cad-herin superfamily, introduce premature stop codons, and thesoluble form of cadherin-23, as the result, is produced. Thesemutations cause hearing loss. The effect of soluble caderin-23
on this symptom is not clear, because it is thought that thisarises from a loss of function of full-length cadherin-23 (37, 38).
Both variant and full-length cadherin-7 have the same EC1domain. It is possible that the variant protein binds to fulllength cadherin-7 and inhibits cell-cell adhesion mediated byfull-length cadherin-7. To examine this possibility, we performed aggregation assays with or without the variant form.
The variant cadherin-7 slowed aggregation, indicating its inhibitory activity against the full-length cadherin-7. Furthermore, to confirm the interaction between the variant protein
and the full-length cadherin-7, we performed immunoprecipitation using anti-variant Ab, and full-length cadherin-7 molecules were co-precipitated. Interaction of the variant proteinwith full-length cadherin-7 suggests that the variant form interrupts trans-interaction of homophilic binding of full-length
cadherin-7 in vitro and in vivo.
Expression and Possible Role of the Variant Form of Cadherin-7 in Chicken Embryos—The expression patterns of cadherin-7 have been well investigated in early chicken embryos(5, 17). The cDNA probe used in these studies for in situ
hybridization encoded the CR3 and CR4 regions of cadherin-7,
and an anti-cadherin-7 monoclonal antibody, CCD7–1, recognized the extracellular domain. Therefore, they detected bothfull-length and variant forms of cadherin-7 at the transcriptional and protein levels. In this report, we performed competitive PCR and immunostaining using an anti-variant-specificantibody. Full-length cadherin-7 was strongly expressed in migrating neural crest cells; the signal of the variant form was
detected strongly on dermomyotomes but only weakly in proximal dorsal and ventral roots and in dorsal root ganglia. The
expression pattern of the variant form on dermomyotomes resembles the expression pattern of MyoD at stage 23 (39). The
dermomyotome is a developmentally transient tissue that displays extensive cell movements and gives rise to both muscle
and dermis.
During development, dynamic cell adhesion, detachment,
and migration are essential processes for morphogenesis, andtherefore the regulation of cadherin activities must be involved(40). E-cadherin-mediated cell-cell adhesions are dynamicallyregulated by internal processes. Growth factor activation ofreceptor tyrosine kinase induces phosphorylation of E-cadherinand ␤-catenin, ubiquitination, and endocytosis, and epithelial
cells then change their morphology (41). Our finding of inhibition against full-length cadherin-7 by a soluble variant form
raises the possibility that an alternative regulation mechanismof cadherin activity from outside the cells occurs during development. To establish the mechanism of regulation of cadherin
activity by the soluble form, it is necessary to study further invivo on the role of the soluble form in dermomyotome
development.
Acknowledgments—We thank Drs. S. Nakagawa and M. Takeichi of
Kyoto University for generous gifts of CCD7–1 antibody and hybridoma, Dr. S. Hirano of Department of Medical Technology of Niigata
University for helpful suggestion, Dr. H. Kikuchi of Department of
Infectious Diseases of Oita Medical University, and Y. Yamashita of theDivision of Developmental Neurobiology of Kumamoto University forthe support of this work.
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