Cadherin Domains Research Articles

Binding site for p120/delta-catenin is not required for Drosophila E-cadherin function in vivo.

Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. β-catenin links cadherin to the actin cytoskeleton via α-catenin. The role of p120/δ-catenin proteins in regulating cadherin function is less clear. Both β-catenin and p120/δ-catenin are conserved in Drosophila. Here, we address the importance of cadherin–catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or β-catenin–armadillo (DE-cadherin-Δβ) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Δβ did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/δ-catenin does not appear to be required for DE-cadherin function in vivo.

Synthesis of Peptide and Glycopeptide Partial Structures of the Homophilic Recognition Domain of Epithelial Cadherin

Peptide and glycopeptide sequences of the homophilic recognition site of epithelial cadherin (E-cadherin) were synthesized by solid-phase technique based on acid-sensitive Wang anchor according to Fmoc strategy. Fmoc serine building blocks with T N -, T-, (2-6)-sialyl T-antigen and β-N-acetylglucosamine side chains were prepared for the construction of E-cadherin glycopeptides. The T- and (2-6)-sialylT-serine derivatives have been obtained by chemical glycosylations of the T N -antigen serine derivative carrying Fmoc/tert-butyl ester protecting group combination. According to NOESY and ROESY NMR experiments, E-cadherin(glyco)peptides not acylated at the N-terminus prefer turn-type conformations in water.

Diversity of the cadherin-related neuronal receptor family in the nervous system

Gene knockout experiments have revealed that Fyn-tyrosine kinase plays significant roles in neural network formation in the CNS. Using Fyn molecules, we have identified a novel CNR family from the CNS. CNR family proteins are located at the synaptic junction, and function as Reelin receptors during layer formation of the cerebral cortex. CNR family genes are clustered in the mouse and human genomes. The CNR family corresponds to the Pcdhα family, which is followed by the Pcdhβ and Pcdhγ gene clusters. Each of the CNR/ Pcdhα and Pcdhγ gene clusters is separated by variable and constant regions, similar to the arrangement of the BCR and TCR gene clusters. The constant regions are distinct between the CNR/ Pcdhα and Pcdhγ genes in mice and humans. These respective gene clusters have undergone diversification during vertebrate evolution. In tereost fugu fish, the constant regions of the CNR/Pcdhα genes are duplicated, but in the clustered large exons, the Reelin-binding sequences are not conserved in their cadherin domains. Moreover, somatic mutations of CNR family transcripts accumulate during development of the cerebral cortex. Thus, characterization of CNR family genes will provide key insights into our understanding of the diversity and specificity of neural networks in the CNS during phylogeny and ontogeny.

Physical and functional interaction between receptor-like protein tyrosine phosphatase PCP-2 and beta-catenin.

We have previously identified a human receptor protein tyrosine phosphatase of the MAM domain family, termed PCP-2, in human pancreatic adenocarcinoma cells and found that this protein was colocalized with beta-catenin and E-cadherin at cell junctions [Wang, H.-Y., et al. (1996) Oncogene 12, 2555-2562]. Its intracellular part consists of two tandem phosphatase domains and a relatively large juxtamembrane region that is homologous to the conserved intracellular domain of cadherins, suggesting a role in the regulation of cell adhesion. This study reports that PCP-2 was endogenously expressed at the cell surface and upregulated with increased cell density. An in vivo binding assay revealed that PCP-2 could directly interact with beta-catenin through a region in the juxtamembrane domain. Tyrosine phosphorylation of beta-catenin by EGF or active SrcY527F did not disrupt the formation of the PCP-2-beta-catenin complex, while PCP-2 in this complex could cause a significant reduction in the phosphorylation level in beta-catenin. Finally, we showed that PCP-2 was a negative regulator for cell migration. In conclusion, interaction of PCP-2 with its substrate beta-catenin is involved in the process of cell-cell contact.

Calcium-dependent Homoassociation of E-cadherin by NMR Spectroscopy: Changes in Mobility, Conformation and Mapping of Contact Regions

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 μM–5 mM) and calcium (0–5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny.

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.

Mutations in the calcium-binding motifs of CDH23 and the 35delG mutation in GJB2 cause hearing loss in one family.

We have ascertained a multi-generation family with apparent autosomal recessive non-syndromic childhood hearing loss (DFNB). Failure to demonstrate linkage in a genome-wide scan with 300 polymorphic markers has suggested genetic heterogeneity for the hearing loss in this family. This heterogeneity could be demonstrated by analysis of candidate loci and genes for DFNB. Patients in one branch of the family (branch C) are homozygous for the 35delG mutation in the GJB2 gene (DFNB1). Patients in two other branches (A and B) carry two new mutations in the cadherin 23 ( CDH23) gene (DFNB12). A homozygous CDH23 c.6442G-->A (D2148N) mutation is present in branch A. Patients in branch B are compound heterozygous for this mutation and the c.4021G-->A (D1341N) mutation. The substituted aspartic acid residues are highly conserved and are part of the calcium-binding sites of the extracellular cadherin (EC) domains. Molecular modeling of the mutated EC domains of CDH23 based on the structure of E-cadherin indicates that calcium-binding is impaired. In addition, other aspartic and glutamic acid residue substitutions in the highly conserved calcium-binding sites reported to cause DFNB12 are also likely to result in a decreased affinity for calcium. Since calcium provides rigidity to the elongated structure of cadherin molecules enabling homophilic lateral interaction, these mutations are likely to impair interactions of CDH23 molecules either with CDH23 or with other proteins. DFNB12 is the first human disorder that can be attributed to inherited missense mutations in the highly conserved residues of the extracellular calcium-binding domain of a cadherin.

Gα12 and Gα13 Negatively Regulate the Adhesive Functions of Cadherin

Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G12 subfamily comprised of Galpha12 and Galpha13 interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator beta-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). Because of the importance of beta-catenin in cadherin-mediated cell-cell adhesion, we examined whether G12 subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G12 proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G12 proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G12 binding ability was not affected by activated G12 proteins, indicating a requirement for direct G12-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G12 proteins reversed the inhibition via a mechanism that was independent of G12-mediated Rho activation. These results validate the G12-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G12 proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

A Provisional Regulatory Gene Network for Specification of Endomesoderm in the Sea Urchin Embryo

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the β-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg2 endomesodermal domain; the blastula-stage separation of the central veg2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg2 endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal–spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.

The cadherin-catenin adhesion system in signaling and cancer.

The adhesion of cells to their neighbors determines cellular and tissue morphogenesis and regulates major cellular processes including motility, growth, differentiation, and survival. Cell-cell adherens junctions (AJs), the most common (indeed, essentially ubiquitous) type of intercellular adhesions, are important for maintaining tissue architecture and cell polarity and can limit cell movement and proliferation. AJs assemble via homophilic interactions between the extracellular domains of calcium-dependent cadherin receptors on the surface of neighboring cells. The cytoplasmic domains of cadherins bind to the submembranal plaque proteins β-catenin or plakoglobin (γ-catenin), which are linked to the actin cytoskeleton via α-catenin (Figure ​(Figure1;1; refs. 1, 2). The transmembrane assembly of cadherin receptors with the cytoskeleton is necessary for the stabilization of cell-cell adhesions and normal cell physiology. Figure 1 The dual role of β-catenin in cell adhesion and transcriptional activation. β-Catenin (β) and plakoglobin (Pg) bind to cadherin adhesion receptors, and via α-catenin (α) they associate with the actin cytoskeleton ... Malignant transformation is often characterized by major changes in the organization of the cytoskeleton, decreased adhesion, and aberrant adhesion-mediated signaling. Disruption of normal cell-cell adhesion in transformed cells may contribute to tumor cells’ enhanced migration and proliferation, leading to invasion and metastasis. This disruption can be achieved by downregulating the expression of cadherin or catenin family members or by activation of signaling pathways that prevent the assembly of AJs. The importance of the major epithelial cell cadherin, E-cadherin (E-cad, the product of the CDH1 gene), in the maintenance of normal cell architecture and behavior is underscored by the observation that hereditary predisposition to gastric cancer results from germline mutations in CDH1. Loss of E-cad expression eliminates AJ formation and is associated with the transition from adenoma to carcinoma and acquisition of metastatic capacity (3). Re-establishment of AJs in cancer cells by restoration of cadherin expression (4) exerts tumor-suppressive effects, including decreased proliferation and motility. In this Perspective, we discuss the molecular mechanisms underlying the role of the cadherin-catenin system in the regulation of cell proliferation, invasion, and intracellular signaling during cancer progression.

Dynamic testicular adhesion junctions are immunologically unique. I. Localization of p120 catenin in rat testis.

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact, but the molecular composition of such junctions is not well known. At prototypical adherens junctions, proteins termed catenins bind to the intracellular domain of classic cadherins and regulate the strength of adhesion. Using a panel of monoclonal antibodies (5A7, 8D11, and 15D2), p120 catenin (p120) was localized in postnatal and adult rat testis cryosections and touch preparations by immunofluorescence. Immunoprecipitation of testis homogenates showed that at least four p120 isoforms were expressed from Postnatal Day 7 through adulthood. Both inter-Sertoli and Sertoli-germ cell junctions were p120-positive, however, individual p120 monoclonals were localized to specific junctions. The 5A7 and 8D11 antibodies colocalized with beta-catenin and plectin at inter-Sertoli and Sertoli-spermatocyte junctions. At inter-Sertoli junctions, p120 was juxtaposed to but did not colocalize with f-actin. Thus, p120 is likely a component of inter-Sertoli desmosome-like junctions. In contrast, the 15D2 monoclonal antibody specifically immunostained Sertoli-round spermatid and inter-Sertoli cell junctions in a dynamic pattern. From the time that round spermatids form to their differentiation into elongate spermatids, Sertoli-round spermatid 15D2 immunostaining cycled from a single mass to a curvilinear pattern, and finally to punctate structures scattered throughout the epithelium. This localization and stage-specific immunostaining pattern indicated that 15D2 recognized Sertoli-round spermatid desmosome-like junctions. Between Sertoli cells, 15D2 immunostained newly formed junctions (at Postnatal Days 21 through 43), but not mature junctions in the adult. From these data, we conclude that p120 is a component of most, if not all, desmosome-like junctions, and that desmosome-like junctions between different cell types contain a unique molecular composition.

Cadherin-like domains in α-dystroglycan, α/ε-sarcoglycan and yeast and bacterial proteins

Dystrophin, a gene product that is mutated in individuals with Duchenne muscular dystrophy, is tethered to the extracellular matrix via membrane-associated multimolecular complexes. In striated muscle cells this complex contains two glycoprotein subcomplexes, the sarcoglycan (SG) and dystroglycan (DG) complexes. Disruption of these large transmembrane complexes has been shown to result in muscle disease. Altered glycosylation of α-DG is associated with two types of congenital muscular dystrophy [1. and 2.] and mutations in the α-DG–binding laminin α2 gene product is linked to a third congenital muscular dystrophy [3.]. Mutations in α-SG (adhalin) and var epsilon-SG result in type 2D limb-girdle muscular dystrophy (LGMD2D) and myoclonus-dystonia syndrome (a CNS disorder), respectively [4. and 5.]. The dystroglycan gene product is cleaved post-translationally to yield two associated glycoproteins [6.]. α-DG represents the highly glycosylated amino-terminal portion, which binds several extracellular molecules, whereas the β-DG carboxy-terminal portion spans the membrane and links to the actin cytoskeleton via dystrophin or its paralogue utrophin. DG and α/var epsilon-SG homologues are known in other vertebrates and invertebrates yet their domain contents and evolutionary heritages have not been reported. Here we reveal that DG and α/var epsilon-SG sequences contain cadherin domain homologues (see legend to Fig. 1). In animals, cadherin domain-containing proteins are adhesion molecules that modulate a wide variety of processes including cell polarization and migration [7.]. Our study also identified cadherin domains in Saccharomyces cerevisiae Axl2p (also known as Sro4p and Bud10p) and several very large proteins from magnetotactic bacteria.

Isolation and Characterization of XKaiso, a Transcriptional Repressor That Associates with the Catenin Xp120ctn in Xenopus laevis

The Armadillo family of catenin proteins function in multiple capacities including cadherin-mediated cell-cell adhesion and nuclear signaling. The newest catenin, p120(ctn), differs from the classical catenins and binds to the membrane-proximal domain of cadherins. Recently, a novel transcription factor Kaiso was found to interact with p120(ctn), suggesting that p120(ctn) also possesses a nuclear function. We isolated the Xenopus homolog of Kaiso, XKaiso, from a Xenopus stage 17 cDNA library. XKaiso contains an amino-terminal BTB/POZ domain and three carboxyl-terminal zinc fingers. The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization and was found localized to the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xenopus p120(ctn) and XKaiso demonstrated their mutual association, whereas related experiments employing differentially epitope-tagged XKaiso constructs suggest that XKaiso additionally self-associates. Finally, reporter assays employing a chimera of XKaiso fused to the GAL4 DNA binding domain indicate that XKaiso is a transcriptional repressor. These data suggest that XKaiso functions throughout development and that its repressor functions may be most apparent in the context of neural tissues. The significance of the XKaiso-p120(ctn) interaction has yet to be determined, but it may include transducing information from cadherin-mediated cell-cell contacts to transcriptional processes within the nucleus.

Mammalian fat3: A Large Protein That Contains Multiple Cadherin and EGF-like Motifs

Using computer-based, motif-trap screening, we have identified a third member of the mammalian fat family, fat3. Human and rat fat3 are also similar to the Drosophila tumor suppressor gene fat. The rat fat3 gene encodes a large protein of 4555 amino acids with 34 cadherin domains, 4 epidermal growth factor (EGF)-like motifs, a laminin A-G motif, and a cytoplasmic domain. Each member of the fat family is differentially expressed in the central nervous system during development. While both fat3 mRNA and fat1 mRNA are abundantly expressed in the fetal brain, the expression of MEGF1/fat2 mRNA is restricted to the postnatal cerebellum. fat3 mRNA and protein expression in the brain peaks at E15 during embryonic development. During this time, robust fat3 immunoreactivity is also observed in the spinal cord. These data suggest that the fat3 protein plays an important role in axon fasciculation and modulation of the extracellular space surrounding axons during embryonic development.

Differential Localization of Chicken FIP2 Homologue, Ag-9C5, in Secretory Epithelial Cells

When hepatocytes polarize, a subset of cellular proteins specifically localizes to the apical cell surface forming the boundary of the bile canaliculus. We have isolated a cDNA encoding a protein recognized by a monoclonal antibody (9C5) that specifically stains the bile canaliculus. The encoded protein (Ag-9C5) is a cytoplasmic protein with three leucine zippers and a zinc finger at the C-terminus. Extensive amino acid sequence similarity indicates that Ag-9C5 is likely the chicken homologue of a human protein, FIP2, which interacts with huntingtin and Rab8. Epitope-tagged Ag-9C5 colocalizes with endogenous Ag-9C5 and other canaliculus marker antigens in transfected organ cultures. In Cos7 cells and MDCK cells Ag-9C5 forms punctate cytoplasmic structures. In intact tissues Ag-9C5 is highly concentrated at the apical surfaces of cells that secrete protein from the apical surfaces, but is found in a fine punctate cytoplasmic pattern in other polarized epithelia. Because this protein has a number of characteristics of proteins that act as scaffolds for assembly of protein complexes (e.g., the cytoplasmic domain of classical cadherins and the FERM superfamily of proteins), it appears that FIP2/Ag-9C5 may act as a scaffold for assembling a complex of proteins that are involved in targeting of some secretory vesicles to defined regions of the cell surface.

Tissue distribution and cell type-specific expression of p120ctn isoforms.

Cadherin-based molecular complexes play a major role in cell-cell adhesion. At the adherens junctions the intracellular domain of cadherins specifically interacts with beta-catenin and p120ctn, members of the Armadillo repeat protein family. Differential splicing and utilization of the alternative translation initiation codons lead to many p120ctn isoforms. Two major p120ctn isoforms are expressed in mouse tissues. In this study we used indirect immunofluorescence to demonstrate significant tissue specificity in expression of the p120ctn isoforms. The short isoform is abundant at cell-cell adhesion junctions in epidermis, palatal, and tongue epithelia, in the ducts of excretory glands, bronchiolar epithelium, and in mucosal epithelia of esophagus, forestomach, and small intestine. In contrast, the long isoform, containing an amino terminus highly conserved within the p120ctn subfamily, is expressed at vascular-endothelial cell junctions in blood vessels, at cell-cell junctions in the serosal epithelium lining the internal organs, in choroid plexus of brain, in the pigment epithelium of retina, and in structures such as the outer limiting membrane of retina and intercalated discs of cardiomyocytes. The tissue- and cell type-specific expression of p120ctn isoforms suggests a role for the long p120ctn isoform in cell structures responsible for stable tissue integrity, compared to the role of the short isoform in cell-cell adhesion in the external epithelia with rapid turnover.

Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs

The terminal sequences of long cDNAs from human brains were subjected to an improved method of motif-trap screening. This process resulted in the identification of three novel genes that encode proteins with 27, 27, and six cadherin domains that we denoted as KIAA1773, KIAA1774 and KIAA1775, respectively. Sequence analysis indicated that the products of these genes were non-classical cadherins. KIAA1773 was found to be a mammalian homologue of the Drosophila dachsous gene but the remaining two genes did not have any likely homologues in public databases. Assessment of their expression in rat tissues indicated that these genes are expressed in highly distinct and tissue-specific patterns. Notably, KIAA1775 is expressed almost exclusively in the olfactory bulb in the rat brain. In situ hybridization further showed that KIAA1775 is strongly expressed by the mitral and tufted cells in the main and accessory olfactory bulbs, suggesting that KIAA1775 may be important in the formation and maintenance of neuronal networks, particularly those in the olfactory bulb. This study clearly shows the importance and usefulness of our cDNA project in search for genes encoding large proteins, as this project has allowed us to identify several novel non-classical cadherin genes that have thus far not been detected by conventional methods.

Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth.

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.

Distinct Regions of the Cadherin Cytoplasmic Domain Are Essential for Functional Interaction with Gα12 and β-Catenin

Heterotrimeric G proteins of the G(12) subfamily mediate cellular signals leading to events such as cytoskeletal rearrangements, cell proliferation, and oncogenic transformation. Several recent studies have revealed direct effector proteins through which G(12) subfamily members may transmit signals leading to various cellular responses. Our laboratory recently demonstrated that Galpha(12) and Galpha(13) specifically interact with the cytoplasmic domains of several members of the cadherin family of cell adhesion molecules (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). This interaction causes beta-catenin to release from cadherin and relocalize to the cytoplasm and nucleus, where it participates in transcriptional activation. Here we report that two distinct regions of the epithelial cadherin (E-cadherin) tail are required for interaction with beta-catenin and Galpha(12), respectively. Deletion of an acidic, 19-amino acid region of E-cadherin abolishes its ability to bind beta-catenin in vitro, to inhibit beta-catenin-mediated transactivation, or to stabilize beta-catenin; causes subcellular mislocalization of beta-catenin; and disrupts cadherin-mediated cell adhesion. On the other hand, deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with Galpha(12); furthermore, Galpha(12) is ineffective in stimulating beta-catenin release from an E-cadherin cytoplasmic domain lacking this putative Galpha(12)-binding region. These findings indicate that Galpha(12) and beta-catenin do not compete for the same binding site on cadherin and provide molecular targets for selectively disrupting the interaction of these proteins with cadherin.

The sequence determinants of cadherin molecules.

The sequence and structural analysis of cadherins allow us to find sequence determinants-a few positions in sequences whose residues are characteristic and specific for the structures of a given family. Comparison of the five extracellular domains of classic cadherins showed that they share the same sequence determinants despite only a nonsignificant sequence similarity between the N-terminal domain and other extracellular domains. This allowed us to predict secondary structures and propose three-dimensional structures for these domains that have not been structurally analyzed previously. A new method of assigning a sequence to its proper protein family is suggested: analysis of sequence determinants. The main advantage of this method is that it is not necessary to know all or almost all residues in a sequence as required for other traditional classification tools such as BLAST, FASTA, and HMM. Using the key positions only, that is, residues that serve as the sequence determinants, we found that all members of the classic cadherin family were unequivocally selected from among 80,000 examined proteins. In addition, we proposed a model for the secondary structure of the cytoplasmic domain of cadherins based on the principal relations between sequences and secondary structure multialignments. The patterns of the secondary structure of this domain can serve as the distinguishing characteristics of cadherins.