Shintaro T. Suzuki

Protocadherins: a large family of cadherin-related molecules in central nervous system.

Using the polymerase chain reaction, we have isolated numerous rat and human cDNAs of which the deduced amino acid sequences are highly homologous to the sequences of the extracellular domain of cadherins. The entire putative coding sequences for two human proteins defined by two of these cDNAs have been determined. The overall structure of these molecules is very similar to that of classic cadherins, but they have some unique features. The extracellular domains are composed of six or seven subdomains that are very similar to those of cadherins, but have characteristic properties. The cytoplasmic domains, on the other hand, have no significant homology with those of classic cadherins. Since various cDNAs with almost identical features were obtained also from Xenopus, Drosophila and Caenorhabditis elegans, it appears that similar molecules are expressed in a variety of organisms. We have tentatively named these proteins protocadherins. They are highly expressed in brain and their expression appears to be developmentally regulated. The proteins expressed from the two full‐length cDNAs in L cells were approximately 170 or 150 kDa in size, and were localized mainly at cell‐cell contact sites. Moreover, the transfectants showed cell adhesion activity.

Cloning of five human cadherins clarifies characteristic features of cadherin extracellular domain and provides further evidence for two structurally different types of cadherin

The entire coding sequences for five possible human cadherins, named cadherin-4, -8, -11, -12 and -13, were determined. The deduced amino acid sequences of cadherin-4 and cadherin-13 showed high homology with those of chicken R-cadherin or chicken T-cadherin, suggesting that cadherin-4 and cadherin-13 are mammalian homologues of the chicken R-cadherin or T-cadherin. Comparison of the extracellular domain of these proteins with those of other cadherins and cadherin-related proteins clarifies characteristic structural features of this domain. The domain is subdivided into five subdomains, each of which contains a cadherin-specific motif characterized by well-conserved amino acid residues and short amino acid sequences. Moreover, each subdomain has unique features of its own. The comparison also provides additional evidence for two structurally different types of cadherins: the first type includes B-, E-, EP-, M, N-, P- and R-cadherins and cadherin-4; the second type includes cadherin-5 through cadherin-12. Cadherin-13 lacks the sequence corresponding to the cytoplasmic domain of typical cadherins, but the extracellular domain shares most of the features common to the extracellular domain of cadherins, especially those of the first type of cadherins, suggesting that cadherin-13 is a special type of cadherin. These results, and those of other recent cloning studies, indicate that many cadherins with different properties are expressed in various tissues of different organisms.

Cadherin 11 Expression Marks the Mesenchymal Phenotype: Towards New Functions for Cadherins?

Cadherin-11 (cad-11) belongs to the cell adhesion type II cadherin family, which seems to have different functions from the classic cadherin family. This study shows the overall pattern of cad-11 gene expression during rat embryonic development, from the pregastrula to very late embryonic stage. Cad-11 is the first cadherin found to be highly expressed in the dispersed and migrating mesenchymal cells that originate from the neuroectodermal neural crest cells and from the pre-chordal and paraxial mesoderm. A burst of cad-11 expression appears during the epithelial to mesenchymal transition, as observed by sclerotome formation. Cad-11 mRNAs were present in all mesenchymal cells throughout the embryo, regardless of their embryonic origin. A proximo-distal and antero-posterior gradient of cad-11 expression is seen in the limb buds, genitalia, and tail. As development proceeds, while all epithelium are negative, cad-11 is present in all mesenchymal cells involved in various morphogenetic events, such as the mesenchyme condensations during chondrogenesis and in the formation of sclera, cornea, naris, palate and meninges. Cad-11 was strongly expressed in mesenchyme during lung or kidney branching morphogenesis or the many epithelium to mesenchyme inductions that operate in the nasal septum, skin, vibrissae, teeth and various glands. High levels of cad-11 transcripts were also found in the dispersed cells of the hyaloid plexus in the vitreous body and in the invading mesenchyme within the trabeculae of the outflow tract of the heart. Cad-11 is thus specific to the mesenchymal phenotype whatever the stage of embryonic development.

Human Osteoblasts Express a Repertoire of Cadherins, Which Are Critical for BMP‐2–Induced Osteogenic Differentiation

Direct cell–cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription‐polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS‐2 and MG‐63, expressed mRNA for cadherin‐11 (C11) and N‐cadherin (N‐cad). HOBs and BMCs also expressed low levels of cadherin‐4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein‐2 (BMP‐2) treatment of either BMCs or HOBs. Likewise, N‐cad mRNA did not change during BMP‐2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down‐regulated by BMP‐2 treatment of normal cells. Both C11 and C4 were localized to sites of cell–cell contact in both HOBs and BMCs, colocalized with β‐catenin, and bands corresponding to cadherins were coimmunoprecipitated by a β‐catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N‐cad partially inhibited Ca2+‐dependent cell–cell adhesion and completely prevented BMP‐2–induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin‐mediated cell‐to‐cell adhesion is critical for normal human osteoblast differentiation.

Structural and functional diversity of cadherin superfamily : are new members of cadherin superfamily involved in signal transduction pathway?

A large number of cadherins and cadherin‐related proteins are expressed in different tissues of a variety of multicellular organisms. These proteins share one property: their extracellular domains consist of multiple repeats of a cadherin‐specific motif. A recent structure study has shown that the cadherin repeats roughly corresponding to the folding unit of the extracellular domains. The members of the cadherin superfamily are roughly classified into two groups, classical type cadherins proteins and protocadherin type according to their structural properties. These proteins appear to be derived from a common ancestor that might have cadherin repeats similar to those of the current protocadherins, and to have common functional properties. Among various cadherins, E‐cadherin was the first to be identified as a Ca2+‐dependent homophilic adhesion protein. Recent knockout mice experiments have proven its biological role, but there are still several puzzling unsolved properties of the cell adhesion activity. Other members of cadherin superfamily show divergent properties and many lack some of the expected properties of cell adhesion protein. Since recent studies of various adhesion proteins reveal that they are involved in different signal transduction pathways, the idea that the new members of cadherin superfamily may participate in more general cell‐cell interaction processes including signal transduction is an intriguing hypothesis. The cadherin superfamily is structurally divergent and possibly functionally divergent as well.

The extracellular domains of E- and N-cadherin determine the scattered punctate localization in epithelial cells and the cytoplasmic domains modulate the localization

The accumulation of classical cadherins is essential for their function, but the mechanism is poorly understood. Hence, we investigated the accumulation of E- and N-cadherin and the formation of cell junctions in epithelial cells. Immunostaining revealed a scattered dot-like accumulation of E- and N-cadherin throughout the lateral membrane in MDCK II and other epithelial cells. Mutant E-cadherin lacking the beta-catenin binding site accumulated granularly at cell-cell contact sites and showed weak cell aggregation activity in cadherin-deficient epithelial cells, MIA PaCa2 cells. Mutant E-cadherin lacking the p120-catenin binding site exhibited scattered punctate accumulation and strong cell adhesion activity in MIA PaCa2 cells. Electron microscopy demonstrated that MIA PaCa2 transfectants of E-cadherin containing beta-catenin binding site formed adherens junction, whereas E-cadherin lacking the binding site did not. Mutant N-cadherins showed accumulation properties similar to those of corresponding mutant E-cadherins. Moreover, wild type and mutant N-cadherin lacking the p120-catenin binding site showed subapical accumulation in polarized DLD-1 cells, whereas mutant N-cadherin lacking beta-catenin binding site did not. These results indicate that the extracellular domains of E- and N-cadherin determines the basic localization pattern, whereas the cytoplasmic domains modulate it thereby affects the cell adhesion activity, subapical accumulation, and the formation of adherens junction.

Restricted expression of protocadherin 2A in the developing mouse brain.

Protocadherins are cell-cell adhesion molecules that are thought to be involved in neural development. Here, we report the expression pattern of protocadherin 2A (Pc2A) in the developing mouse brain as determined by the in situ hybridization technique. In the postnatal day 2 brain, various regions expressed Pc2A including the cerebellar cortex, ventral posterior thalamic nucleus, dorsal lateral geniculate nucleus, hippocampus and cerebellum (Purkinje cells). In particular, some ependymal cells that form the lining of the lateral ventricle and the third ventricle and floor plate cells lining the fourth ventricle showed prominent expression. In the adult brain, strong expression was restricted to the Purkinje cells. Expression in other areas of the adult brain was down-regulated to a faint level, and only a weak signal was detected in regions such as the retina, olfactory bulb, dentate gyrus of the hippocampus, and in some parts of the medial eminence. These observations suggest that Pc2A is expressed in various regions of the brain in a developmentally regulated manner.

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization.

Abstract. By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24→q25, 18p12→p11, 8q32.1 and 2q22.1→q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues for rat Smad2 (Madh2) and Smad4 (Madh4) genes were assigned to Chinese hamster chromosomes (CGR) 5q28, 2q17, 4q26, 2p29→p27, 2q112→q113 and 2q112→q113, respectively. The chromosome assignments of homologues of Ctse and Cspg5 reinforced well-known homologous relationships among mouse chromosome (MMU) 1, RNO 13 and CGR 5q, and among MMU 9, RNO 8 and CGR 4q, respectively. The chromosome locations of homologues for Madh2, Madh4 and Pcdh13 genes suggested that inversion events were involved in chromosomal rearrangements in the differentiation of MMU 18 and RNO 18, whereas most of MMU 18 is conserved as a continuous segment in CGR 2q. Furthermore, the mapping result of Myo10 and homologues suggested an orthologous segment of MMU 15, RNO 2 and CGR 2.

Sequence and Distribution of Xenopus Laevis E-cadherin Transcripts

Cadherins are calcium-dependent adhesive glycoproteins implicated in histogenetic processes. In Xenopus laevis, the distribution of classical cadherins N, E, EP, XB and U has been determined by immunofluorescence labeling or by in situ hybridization. In this study, we report the full-length sequence of the E-cadherin cDNA. Comparison with the other cadherin sequences available indicates that Xenopus E-cadherin is as homologous to Xenopus EP-cadherin as to the chicken L-CAM and to the mammalian E-cadherin. Although Xenopus E-cadherin protein sequence exhibits many short conserved motifs present in other E-cadherins, it differs remarkably from the chicken L-CAM and the mammalian E-cadherin in its appearance after gastrulation. In situ hybridization data showed that E-cadherin transcripts are homogenously distributed in all differentiating epithelia from early tailbud to post-metamorphic stage. In contrast to mouse E-cadherin, Xenopus E-cadherin was not detected transiently in the nervous system during embryogenesis and in the post-metamorphic stages.