Expression of the rat homologue of the Drosophila fat tumour suppressor gene

We have sequenced and defined the expression during rat embryogenesis of the protocadherin fat, the murine homologue of a Drosophila tumour suppressor gene. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A–G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the β-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat.     

Identification and characterization of three members of a novel subclass of protocadherins

Protocadherins are members of a nonclassic subfamily of calcium-dependent cell-cell adhesion molecules in the cadherin superfamily. Although the extracellular domains have several common structural features, there is no extensive homology between the cytoplasmic domains of protocadherin subfamily members. We have identified a new subclass of protocadherins based on a shared and highly conserved 17-amino-acid cytoplasmic motif. The subclass currently consists of 18 protocadherin members. Two of these, PCDH18 and PCDH19, are novel protocadherins and a third is the human orthologue of mouse Pcdh10. All three genes encode six ectodomain repeats with cadherin-like attributes and, consistent with the structural characteristics of protocadherins, a large first exon encodes the extracellular domain of each gene.     

Recent progress in protocadherin research

Protocadherins constitute a large family belonging to the cadherin superfamily and function in different tissues of a wide variety of multicellular organisms. Protocadherins have unique features that are not found in classic cadherins. Expression of protocadherins is spatiotemporally regulated and they are localized at synapses in the CNS. Although protocadherins have Ca(2+)-dependent homophilic interaction activity, the activities are relatively weak. Some protocadherins have heterophilic interaction activity and the cytoplasmic domains associate with the unique cytoplasmic proteins, which are essential for their biological functions. Given the characteristic properties, the large size, and the diversity of members of the protocadherin family, protocadherins may participate in various biological processes. In particular, protocadherins seem to play a central role(s) in the CNS as related to synaptic function.     

DDR1 regulates the stabilization of cell surface E‐cadherin and E‐cadherin‐mediated cell aggregation

The stabilization of cell surface E‐cadherin is important for the maintenance of apical junction complexes and epithelial polarity. Previously, we reported that discoidin domain receptor 1 (DDR1) forms a complex with E‐cadherin at adhesive contacts; however, the regulatory role of DDR1 in the stabilization of cell surface E‐cadherin and E‐cadherin‐mediated cell behaviors remained undefined. To gain insight into these questions, we utilized two stable clones depleted for DDR1 via the small interfering RNA (siRNA) technique, and we over‐expressed DDR1 in MDCK cells. We performed Western blotting, immunofluorescence analysis, flow cytometry, and cell aggregation studies to investigate the effect of DDR1 on cell surface E‐cadherin. The results showed that both DDR1/2 and E‐cadherin use their extracellular domains to form DDR/E‐cadherin complexes. Neither the depletion nor the over‐expression of DDR1 changed the expression level of E‐cadherin in MDCK cells. Collagen disrupted the formation of E‐cadherin complexes and caused E‐cadherin to accumulate in the cytoplasm; however, over‐expression of DDR1 stabilized E‐cadherin on the cell surface and decreased its cytoplasmic accumulation. Furthermore, independently of collagen stimulation, the depletion of DDR1 resulted in a decrease in the level of cell surface E‐cadherin, which consequently caused its cytoplasmic accumulation and decreased E‐cadherin‐mediated cell aggregation. These results indicate that DDR1 can increase the stability of cell surface E‐cadherin and promote MDCK cell aggregation, which may be mediated through the formation of DDR1/E‐cadherin complexes. Overall, these findings have implications for the physiological roles of DDR1 in association with the maintenance of both the adhesion junction and epithelial polarity. J. Cell. Physiol. 224: 387–397, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

Structural and functional characterization of the 5'-flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene.

Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at -1.6 to -2.0 kb and -2.5 to -2. 8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5'-flanking regions of the human (-3712 bp to +10 bp) and rat (-3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.     

A common protocadherin tail: Multiple protocadherins share the same sequence in their cytoplasmic domains and are expressed in different regions of brain

To study the diversity of protocadherins, a rat brain cDNA library was screened using a cDNA for the cytoplasmic domain of human protocadherin Pcdh2 as a probe. The resultant clones contained three different types. One type corresponds to rat Pcdh2; the other two types are distinct from Pcdh2 but contain the same sequence in their cytoplasmic domains and part of the 3′ flanking sequence. To clarify the structure of the proteins defined by the new clones, a putative entire coding sequence corresponding to one of the clones was determined. The overall structure is essentially the same as Pcdh2, indicating that the proteins defined by this clone, and probably by other clones, belong to the protocadherin family. Two PCR experiments and an RNase protection assay showed the existence of the corresponding mRNAs in rat brain preparations. Human and mouse cDNA clones with the same sequence properties were also isolated. Taken together, these results indicate that the clones are not cloning artifacts and that corresponding mRNAs are actually expressed in brains of various species. The results of in situ hybridization showed that the mRNAs corresponding to these clones were expressed in different regions in brain. Since protocadherins encoded by these mRNAs are likely to have different specificity in their interaction and share a common activity at their cytoplasmic domains, these protocadherins may provide a molecular basis, in part, to support the complex cell-cell interaction in brain.     

Elastic versus brittle mechanical responses predicted for dimeric cadherin complexes

Cadherins are a superfamily of adhesion proteins involved in a variety of biological processes that include the formation of intercellular contacts, the maintenance of tissue integrity, and the development of neuronal circuits. These transmembrane proteins are characterized by ectodomains composed of a variable number of extracellular cadherin (EC) repeats that are similar but not identical in sequence and fold. E-cadherin, along with desmoglein and desmocollin proteins, are three classical-type cadherins that have slightly curved ectodomains and engage in homophilic and heterophilic interactions through an exchange of conserved tryptophan residues in their N-terminal EC1 repeat. In contrast, clustered protocadherins are straighter than classical cadherins and interact through an antiparallel homophilic binding interface that involves overlapped EC1 to EC4 repeats. Here we present molecular dynamics simulations that model the adhesive domains of these cadherins using available crystal structures, with systems encompassing up to 2.8 million atoms. Simulations of complete classical cadherin ectodomain dimers predict a two-phased elastic response to force in which these complexes first softly unbend and then stiffen to unbind without unfolding. Simulated α, β, and γ clustered protocadherin homodimers lack a two-phased elastic response, are brittle and stiffer than classical cadherins and exhibit complex unbinding pathways that in some cases involve transient intermediates. We propose that these distinct mechanical responses are important for function, with classical cadherin ectodomains acting as molecular shock absorbers and with stiffer clustered protocadherin ectodomains facilitating overlap that favors binding specificity over mechanical resilience. Overall, our simulations provide insights into the molecular mechanics of single cadherin dimers relevant in the formation of cellular junctions essential for tissue function.     

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (ΨR2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5′ upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the D1 D-loop sequence is also due to the 5′ and 3′ regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the D1 D-loop region.     

Modulation of Cell–Cell Adherens Junctions by Surface Clustering of the N-Cadherin Cytoplasmic Tail

Cadherins mediate the formation of cell–cell adherens junctions (AJ) by homophilic interactions through their extracellular domains as well as by interacting with the actin cytoskeleton via their cytoplasmic portions. Cadherin clustering initiates cytoplasmic signaling that results in the assembly of structural components into cell–cell AJ. To elucidate the function of the cytoplasmic tail of cadherins in initiating the assembly signal, we generated and characterized a chimeric cadherin tail fused to an inert transmembrane anchor. The chimera enabled us to cluster the cadherin cytoplasmic tail in the absence of extracellular portions of the molecule. The transfected cadherin tail chimera localized to cell–cell AJ of epithelial cells, indicating that the submembrane junctional plaque has the capacity to recruit additional cadherins, with no involvement of their extracellular domains. Expression of the chimera in cells of mesenchymal origin resulted in dominant negative effects on the formation of cell–cell AJ. Surface clustering of cadherin cytoplasmic tails induced the recruitment of components and structural assembly of cell–cell AJ, thereby reversing the initial dominant–negative effects. We conclude that the cadherin cytoplasmic tail contains information required to direct the molecule to cell–cell AJ. Its function as modulator of cell–cell AJ depends on cell type and on whether the tail is clustered.     

In search of the hair-cell gating spring elastic properties of ankyrin and cadherin repeats

Mechanotransduction in vertebrate hair cells involves a biophysically defined elastic element (the "gating spring") that pulls on the transduction channels. The tip link, a fine filament made of cadherin 23 linking adjacent stereocilia in hair-cell bundles, has been suggested to be the gating spring. However, TRP channels that mediate mechanotransduction in Drosophila, zebrafish, and mice often have cytoplasmic domains containing a large number of ankyrin repeats that are also candidates for the gating spring. We have explored the elastic properties of cadherin and ankyrin repeats through molecular dynamics simulations using crystallographic structures of proteins with one cadherin repeat or 4 and 12 ankyrin repeats, and using models of 17 and 24 ankyrin repeats. The extension and stiffness of large ankyrin-repeat structures were found to match those predicted by the gating-spring model. Our results suggest that ankyrin repeats of TRPA1 and TRPN1 channels serve as the gating spring for mechanotransduction.     

A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52 degrees mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.     

Genomic sequence and organization of the family of CNR/Pcdhalpha genes in rat

CNR/Pcdhalpha family proteins are known as synaptic cadherins and Reelin receptors. Here we report the complete genomic sequence and organization of the rat CNR. The rat CNR cluster encodes 15 variable and 3 constant exons. The genomic organizations of the rat, mouse, and human CNR/Pcdhalpha are orthologous. The percentage identity of the coding regions between the rat and the mouse is 93.6% on average at the nucleic acid level, and between rat and human it is 82.8%. The rat CNRs (v1-v13) also contain an RGD motif in the extracellular cadherin 1 domains and cysteine repeats that are characteristic of the transmembrane and cytoplasmic domains of CNR proteins. The number of variable exons in the rat CNR cluster is identical to that of the human. The rat CNR cluster has one more variable exon than is found in laboratory mouse strains, because in the mouse a variable exon located between v7 and v8 is divided by the insertion of a retrotransposon. This exon is not disrupted in the rat, in which it is transcribed. By in silico analysis, CNR/Pcdhalpha was also mapped to rat chromosome 18, but the orientation was opposite for the mouse CNR/Pcdhalpha gene cluster. The relative expression profiles of the rat CNRs (v1-v13) show that all the CNRs are transcribed, but there are variations in the expression ratios among the CNRs.     

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.     

The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Here we show that the fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis.     

Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naïve parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32–35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.     

Expression analysis of epithelial cadherin and related proteins in IBH‐6 and IBH‐4 human breast cancer cell lines

Epithelial cadherin (E‐cadherin) is a 120 kDa cell–cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as β‐catenin. Loss of E‐cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down‐regulation of E‐cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH‐6 and IBH‐4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E‐cadherin and related proteins in IBH‐6 and IBH‐4 cell lines was evaluated. In IBH‐6 and IBH‐4 cell extracts, only an 89 kDa E‐cadherin form (Ecad89) was detected, which is truncated at the C‐terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E‐cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH‐6 and IBH‐4 cells showed an intracellular scattered E‐cadherin localization. β‐catenin accompanied E‐cadherin localization, and actin stress fibers were identified in both cell types. E‐cadherin mRNA levels were remarkably low in IBH‐6 and IBH‐4 cells. The E‐cadherin mRNA and genomic sequence encoding exons 14–16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up‐regulation of Twist mRNA was found in both cell lines. In conclusion, IBH‐6 and IBH‐4 breast cancer cells show down‐regulation of E‐cadherin expression with aberrant protein localization, and up‐regulation of Twist; these features can be related to their invasive/metastatic characteristics. J. Cell. Physiol. 222: 596–605, 2010. © 2009 Wiley‐Liss, Inc.