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Cloning, Expression Analysis, and Chromosomal Localizationof BH-Protocadherin (PCDH7), a Novel Member

of the Cadherin Superfamily

Kenichi Yoshida,* Kiyomi Yoshitomo-Nakagawa,* Naohiko Seki,†Masahide Sasaki,‡ and Sumio Sugano*,1

*Department of Virology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan;†Laboratory of Gene Structure I, Kazusa DNA Research Institute, Kisarazu 292, Japan; and ‡Department of Biological Science

and Technology, Science University of Tokyo, Chiba 278, Japan

Received October 21, 1997; accepted February 11, 1998

structure, cadherin superfamily molecules are roughlyWe have identified a novel member of the cadherin classified into four classes: classic cadherins, desmoso-

superfamily. Among the members of the superfamily, mal cadherins, protocadherins (Pcdhs), and other typesthis protein exhibited the highest overall homology of cadherin-related proteins (18).with protocadherin-1 (46–49% identity). Its mRNA was Pcdhs and other types of cadherin-related proteinspredominantly expressed in the brain and heart. are found in various tissues in various multicellularHence, we named the gene BH-protocadherin (BH- organisms (1, 3, 9–11, 16, 17, 20). They seem to func-Pcdh) (HGMW-approved symbol PCDH7). BH-Pcdh has tion as cell–cell recognition molecules and as a compo-an extracellular domain consisting of seven repeats of

nent in a signal transduction pathway (18) and to in-the cadherin motif (EC 1 to 7). EC2 of BH-Pcdh isfluence such processes as development and tumorunique in having a 55-amino-acid insertion in the mid-growth (3, 9). Recently, several mammalian Pcdhs havedle of the motif. There are three isoforms of BH-Pcdh,been isolated. Pcdh1 and Pcdh2 are exclusively ex-denoted -a, -b, and -c, which have different cytoplasmicpressed in the brain and thought to play an importanttails and a 47-amino-acid deletion in the EC2–3 regionrole in the central nervous system (17). Here we de-of BH-Pcdh-c. While only a 9.0-kb message was de-scribe the cloning and characterization of a novel mem-tected in normal tissues, 4.5- and 9.0-kb mRNA speciesber of the cadherin superfamily, PCDH7 (BH-Pcdh),2were seen in the human lung carcinoma cell line A549.

Furthermore, only the 4.5-kb mRNA was detected in which shows high similarity with Pcdh1.HeLa cell S3 and human gastric cancer cell lines We developed a new method, which we call trans-MKN28 and KATO-III. Southern blot analysis indi- membrane-domain trapping strategy, for isolatingcated that the BH-Pcdh gene is likely to be conserved cDNAs of putative membrane proteins (manuscript inamong various vertebrates. The BH-Pcdh gene was lo- preparation). Using this method, we isolated fivecalized to human chromosome 4p15. Interestingly, cDNAs of putative membrane proteins from a human4p15 is a region of loss of heterozygosity in some head gastric adenocarcinoma cell line, MKN28. One of theand neck squamous cell carcinomas. q 1998 Academic Press clones, 6-33, consists of a 5* part from the cDNA of the

new membrane protein and a 3 * part from the cDNAof hsp90 (15). To isolate the full-length cDNA of the 5*Cadherins and their related proteins constitute a part of the 6-33 clone, we screened the human gastriccadherin superfamily that is a large and diverse group adenocarcinoma cell line MKN28 cDNA library usingof membrane-associated glycoproteins. This family of 6-33-47 cDNA as a probe (632-bp fragment that corre-proteins shares common structural features: a large sponds to BH-Pcdh-a from nucleotide 3647 to 4278;extracellular domain, which consists of repeats of a so- GenBank Accession No. AB006755).called cadherin motif, and a relatively small cyto-

We obtained three full-length cDNAs, denoted BH-plasmic domain. These domains are connected to eachPcdh-a, -b, and -c. They each contain a single openother by a transmembrane domain. The cadherin motifreading frame that encodes a polypeptide of 1069, 1072,is approximately 110 amino acids long and seems toor 1200 amino acids, respectively. The predicted aminoplay an important role in Ca2/-dependent homophilicacid sequences begin with a 28-amino-acid hydrophobiccell–cell adhesion (6, 7, 19). Based on their molecularstretch, suggesting this is the signal peptide of the pro-tein. The extracellular domain at the N-terminal side

Sequence data from this article have been deposited with theEMBL/GenBank Data Libraries under Accession Nos. AB006755,AB006756, and AB006757. 2 The HGMW-approved symbol for the gene described in this paper

is PCDH7. We used BH-protocadherin (BH-Pcdh) as an alternative1 To whom correspondence should be addressed. Telephone: /81-3-5449-5286. Fax: /81-3-5449-5416. E-mail: ssugano@ims.u-tokyo.ac.jp. name (alias) for PCDH7.

458GENOMICS 49, 458–461 (1998)ARTICLE NO. GE9852710888-7543/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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FIG. 1. Alignment of the deduced amino acid sequences of Pcdh1 and BH-Pcdh. Vertical lines indicate identity, plus signs indicatesimilarity, and gaps are introduced to optimize the alignment. (A) The extracellular domain sequences of Pcdh1 and BH-Pcdh are aligned.Indicated is the 47-amino-acid deletion of BH-Pcdh-c (underline). (B) The cytoplasmic domain sequences of Pcdh1 and BH-Pcdh are aligned.Alternatively spliced Pcdh1 and BH-Pcdh-a, -b, and -c are also indicated.

consists of seven cadherin repeats. A 24-amino-acid hy- acids, respectively. Thus, isoforms have characteristiccytoplasmic tails.drophobic stretch corresponds to the transmembrane

domain (8). The sizes of the cytoplasmic domains of The overall structure of deduced BH-Pcdh polypep-tide showed the highest homology with Pcdh1 amongBH-Pcdh-a, -b, and -c are 168, 171, and 346 amino

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overall, BH-Pcdh and Pcdh1 proteins share 46–49%identity.

The EC1 of BH-Pcdh lacks the HAV adhesion recog-nition sequences that are involved in the homophiliccell–cell binding function of the type I classic cadherins(2). These HAV tripeptides are also absent in Pcdhs.BH-Pcdh has an exceptionally long EC2 (165 aminoacids compared to the usual 110 amino acids), and themiddle region of EC2 does not show any homology withother repeats or proteins in the database. The EC2 ofPcdhs is relatively variable when compared with thesame region of other cadherin repeats (16, 17). Twocadherin-related proteins, LI-cadherin (1) and Ksp-cadherin (20), are also known to have two additionalsubdomains around EC2. Furthermore, BH-Pcdh-c hasa 47-amino-acid deletion (underlined in Fig. 1A) fromthe middle of EC2 to the N-terminal of EC3 including(L)DRE and DXNDNXPXF, which consist of possibleCa2/-binding sites of the cadherin motif (12).

The size of the cytoplasmic domain of BH-Pcdh-c istwice as long as those of BH-Pcdh-a and -b and is simi-lar to an alternatively spliced product of Pcdh1 (Fig.1B). This finding shows that Pcdh1, BH-Pcdh, and theirrelated proteins constitute a novel Pcdh subfamily.This well-conserved C-terminal region between BH-Pcdh-c and Pcdh1 suggests the functional importanceof this region. The cytoplasmic domain of BH-Pcdhlacks the homologous sequence corresponding to thecatenin-binding site described for the classic cadherins(13). It is possible that the cytoplasmic domain of BH-Pcdh associates with proteins other than catenins.

To examine BH-Pcdh mRNA size and its expressionin various tissues, Northern blot analysis was performed(Human Multiple Tissue Northern Blot, Clontech, 7760-1 and 7767-1). As shown in Fig. 2A, BH-Pcdh was ex-pressed as a major transcript of about 9.0 kb. Amongthe human tissues examined, the expression of BH-PcdhFIG. 2. Northern blot analysis of the expression of BH-Pcdh inmRNA was predominant in the brain and heart and lessvarious human tissues (A) and in human cancer cell lines (B). Theso in the stomach, thyroid, spinal cord, and placenta.nylon membrane containing 2 mg of poly(A)/ RNA extracted from

the tissues and cancer cell lines, indicated across the top, was hybrid- The expression pattern is different with Pcdhs whoseized with the 32P-labeled 6-33-47 cDNA (632 bp, positions 3647– expression is restricted in the brain (17).4278; GenBank Accession No. AB006755). The origins of the poly(A)/ Northern blot analysis of various human cancer cellRNA are as follows: HL-60, promyelocytic leukemia cell line; HeLa,

lines was also performed (Fig. 2B). Interestingly, wecervical carcinoma cell line (S3); K-562, chronic myelogenous leuke-mia cell line; MOLT-4, lymphoblastic leukemia cell line; Raji, Bur- found a 4.5-kb message expressed in various tumor cellkitt’s lymphoma cell line; SW480, colorectal adenocarcinoma cell line; lines. Human lung carcinoma cell line A549 expressesA549, lung carcinoma cell line; G361, melanoma cell line (Human both 4.5- and 9.0-kb messages. Other cell lines, HeLa,Cancer Cell Line Multiple Tissue Northern Blot, Clontech, 7757-1); MKN28, or KATO-III, expressed only a 4.5-kb message.MKN28, gastric adenocarcinoma cell line; KATO-III, gastric adeno-

Since we isolated BH-Pcdh cDNA from MKN28 cells,carcinoma cell line; MKN45, gastric adenocarcinoma cell line;HepG2, hepatocellular carcinoma cell line; and SK-N-MC, neuro- the clone corresponds to the 4.5-kb message. To under-blastoma cell line. Rehybridization of the same filters with b-actin stand the relationship between the 4.5- and 9.0-kb mes-probe showed that a comparable amount of RNA was loaded in each sages, we performed RT-PCR using human brainlane (data not shown). Sizes in kilobases are indicated along the left.

poly(A)/ RNA (Clontech) and primers covering variousparts of the BH-Pcdh-a cDNA. The sequence analysisrevealed that the 9.0-kb message includes the 4.5-kbthe members of the cadherin superfamily. The homol-

ogy between BH-Pcdh and Pcdh1 was 47% at the extra- message and that at least the open reading frame wasidentical.cellular domain (Fig. 1A), while homology between

Pcdh1 and Pcdh2 is 40% (17). The cytoplasmic domain We examined the conservation of the BH-Pcdh genein different eukaryotic species. Southern blotting of thealso showed significant homology (39–51% identity)

with that of Pcdh1 (Fig. 1B), but not with Pcdh2, Pcdh3, genomic DNAs (Zoo Blot, Clontech) so far tested gaveone band, except for yeast genomic DNA, which showedor any other cadherin superfamily molecules. Thus,

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cadherin superfamily that controls imaginal disc morphogene-no band, suggesting the presence of a homologous sin-sis in Drosophila. Genes Dev. 9: 1530–1542.gle-copy gene in various vertebrate genomes (data not

4. Cox, R. D., Burmeister, M., Price, E. R., Kim, S., and Myers,shown).R. M. (1990). Radiation hybrid mapping: A somatic cell genetic

Finally, chromosomal assignment of the novel BH- method for constructing high-resolution maps of mammalianPcdh gene was done by PCR analysis of NIGMS Human/ chromosomes. Science 250: 245–250.Rodent Somatic Cell Hybrid Mapping Panel 2 (National 5. Drwinga, H. L., Toji, L. H., Kim, C. H., Greene, A. E., and Muli-

vor, R. A. (1993). NIGMS human/rodent somatic cell hybridInstitute of General Medicine Service, Coriell Cell Reposi-mapping panel 1 and 2. Genomics 16: 311–314.tories) (5). The PCR primer set was designed for the 3*

6. Geiger, B., and Ayalon, O. (1992). Cadherins. Annu. Rev. Celluntranslated region of the BH-Pcdh-a cDNA (5*-CTA-Biol. 8: 307–332.

ATGGATGTCTGAGTCACC-3* for the forward direction 7. Grunwald, G. B. (1993). The structural and functional analysisand 5*-AGACAAAGACATAAGAGGCTG-3* for the re- of cadherin calcium-dependent cell adhesion molecules. Curr.verse direction). PCR products (162 bp) specifically ampli- Opin. Cell Biol. 5: 797–805.

8. Kyte, J., and Doolittle, R. F. (1982). A simple method for dis-fied with the oligonucleotide set were detected only fromplaying the hydropathic character of a protein. J. Mol. Biol.the hybrid containing human chromosome 4. We per-157: 105–132.formed further mapping analysis using PCR-based radia-

9. Mahoney, P. A., Weber, U., Onofrechuk, P., Biessmann, H., Bry-tion hybrid panels (Genebridge4; Research Genetics, Inc.) ant, P. J., and Goodman, C. S. (1991). The fat tumor suppressor(4). The radiation hybrid data were submitted to the gene in Drosophila encodes a novel member of the cadherinWhitehead Institute/MIT Center for Genome Research gene superfamily. Cell 67: 853–868.

10. Miller, J., and McClay, D. R. (1994). Expression of cadherin andSTS mapping server (Cambridge, MA), and statisticalcatenin orthologs during sea urchin development. Mol. Biol.analysis was performed using the RHMAPPER softwareCell 5: 106a.package (http://www-genome.wi.mit.edu/cgi-bin/contig/

11. Oda, H., Uemura, T., Harada, Y., Iwai, Y., and Takeichi, M.rhmapper.pl). The data vector for the gene was (1994). A Drosophila homolog of cadherin associated with arma-0100000111 0010000101 0011010011 0000000100 dillo and essential for embryonic cell–cell adhesion. Dev. Biol.1010101100 0100001000 1100011010 1101000011 165: 716–726.1111011000 010. Using an lod threshold ofú17, we were 12. Overduin, M., Harvey, T. S., Bagby, S., Tong, K. I., Yau, P.,

Takeichi, M., and Ikura, M. (1995). Solution structure of theable to establish linkage of the gene to chromosome 4,epithelial cadherin domain responsible for selective cell adhe-and the gene was placed between markers GCT6F03 andsion. Science 267: 386–389.AFMA041WD5. The gene is 3.36 cR distal to the marker

13. Ozawa, M., Ringwald, M., and Kemler, R. (1990). Uvomorulin–GCT6F03 (LOD ú3). The marker, GCT6F03, maps catenin complex formation is regulated by a specific domain in150.37 cR from the top of the chromosome 4 linkage the cytoplasmic region of the adhesion molecule. Proc. Natl.

Acad. Sci. USA 87: 4246–4250.group. Recent studies have shown that the loss of hetero-14. Pershouse, M. A., El-Nagger, A. K., Hurr, K., Lin, H., Yung,zygosity at chromosome 4, including 4p15, is detected

W. K. A., and Steck, P. A. (1997). Deletion mapping of chromo-in some head and neck squamous cell carcinomas (14).some 4 in head and neck squamous cell carcinoma. OncogeneExperiments are currently under way to define the loss 14: 369–373.

of heterozygosity on 4p15 of some of the carcinoma cell 15. Rebbe, N. F., Ware, J., Bertina, R. M., Modrich, P., and Stafford,lines used in this study. D. W. (1987). Nucleotide sequence of a cDNA for a member of

the human 90kDa heat-shock protein family. Gene 53: 235–245.

ACKNOWLEDGMENTS 16. Sago, H., Kitagawa, M., Obata, S., Mori, N., Taketani, S., Ro-chelle, J. M., Seedling, M. F., Davidson, M., St. John, T., and

This work was supported in part by grants-in-aid for scientific Suzuki, S. T. (1995). Cloning, expression, and chromosomal lo-research from the Ministry of Education, Science, Sports, and Cul- calization of a novel cadherin-related protein, protocadherin-3.ture of Japan. Kenichi Yoshida is a research fellow of the Japan Genomics 29: 631–640.Society for the Promotion of Science. 17. Sano, K., Tanihara, H., Heimark, R. L., Obata, S., Davidson,

M., St. John, T., Taketani, S., and Suzuki, S. (1993). Protocad-herins: A large family of cadherin-related molecules in central

REFERENCES nervous system. EMBO J. 12: 2249–2256.18. Suzuki, S. T. (1996). Structural and functional diversity of cad-

1. Berndorff, D., Gessner, R., Kreft, B., Schnoy, N., Lajous-Petter, herin superfamily: Are new members of cadherin superfamilyA., Loch, N., Reutter, W., Hortsch, M., and Tauber, R. (1994). involved in signal transduction pathway? J. Cell. Biochem. 61:Liver–intestine cadherin: Molecular cloning and characteriza- 531–542.tion of a novel Ca2/-dependent cell adhesion molecule expressed 19. Takeichi, M. (1991). Cadherin cell adhesion receptor as a mor-in liver and intestine. J. Cell Biol. 125: 1353–1369. phogenetic regulator. Science 251: 1451–1455.

2. Blaschuk, O. W., Sullivan, R., David, S., and Pouliot, Y. (1990). 20. Thomson, R. B., Igarashi, P., Biemesderfer, D., Kim, R., Abu-Identification of a cadherin cell adhesion recognition sequence. Alfa, A., Soleimani, M., and Aronson, P. S. (1995). Isolation andDev. Biol. 139: 227–229. cDNA cloning of Ksp-cadherin, a novel kidney-specific member

of the cadherin multigene family. J. Biol. Chem. 270: 17594–3. Clark, H. F., Brentrup, D., Schneitz, K., Bieber, A., Goodman,C., and Noll, M. (1995). Dachsous encodes a member of the 17601.

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