Distribution of OL-protocadherin protein in correlation with specific neural compartments and local circuits in the postnatal mouse brain.

OL-protocadherin (OL-pc) is a cell adhesion molecule that belongs to the cadherin superfamily. A previous study showed that expression of OL-pc mRNA was specific to certain brain nuclei including those of the olfactory and limbic systems, thus suggesting its involvement in neural circuit formation. Here, we examined the distribution of OL-pc protein in the postnatal mouse brain by immunohistochemistry to confirm the possibility of such a role. The results showed that the protein could be mapped to many brain compartments including brain nuclei and higher subdivisions as previously observed for the expression pattern of the mRNA. Sharp boundaries of the distribution were often seen in areas such as the interpedunclar nucleus, cerebellar cortex, and inferior olive. In addition, the protein was detected in some fibers that could not be examined by the previous study using in situ hybridization. For example, prominent staining was noted in the stria medularis, stria terminalis, fasciculus retroflexus, optic tract, and inferior thalamic radiation, structures that seem to connect OL-pc-positive brain regions. These OL-pc-positive brain nuclei and fiber tracts coincide with some local circuits of functional systems such as the olfactory system, nigrostriatal projection, olivo-cerebellar projection, and visual system. These results support the possibility that OL-pc is involved in the formation of specific neural compartments and circuits in the developing brain.

Granule cell raphes in the developing mouse cerebellum.

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.

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.

Lateral clustering of cadherin-4 without homophilic interaction: possible involvement in the concentration process at cell-cell adhesion sites as well as in the cell adhesion activity.

It is thought that the concentration of classic cadherins at cell-cell adhesion sites is essential for generating strong cell-cell adhesion activity, but the mechanism is not well understood. To clarify the structural basis of the concentration process and the cell adhesion activity, we constructed various mutants of cadherin-4 and examined the adhesion properties of the transfectants. A deletion mutant lacking the entire cytoplasmic domain had weak, but significant Ca(2+)-dependent cell adhesion activity. Interestingly, the deletion mutant showed intrinsic cluster formation in the absence of cell-cell adhesion, possible lateral cluster formation. The cytoplasmic domain-deleted cadherin-4 containing the mutation of Trp-2 to Ala, which is known to inhibit the strand dimer formation required for the cell-cell adhesion, retained the possible activity of lateral cluster formation, supporting this notion. These results suggest that the extracellular domain has intrinsic activity of lateral cluster formation. Indeed, deletion of a cadherin repeat in the extracellular domain significantly reduced or abolished the lateral cluster formation as well as the concentration of cadherin-4 at cell-cell contact sites and cell adhesion activity. When transfectants of the cytoplasmic domain-deleted cadherin-4 made cell-cell contact and formed intimate cell-cell adhesion, the lateral clusters of cadherin-4 initially gathered at cell-cell contact sites, and a smooth linear concentration was gradually formed along the cell-cell adhesion interface. The results suggest that the lateral cluster formation is involved in the concentration process of cadherin-4 at cell-cell adhesion sites, hence in the strong cell adhesion activity of cadherin-4 as well.

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

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.

Mutation analysis of cadherin-4 reveals amino acid residues of EC1 important for the structure and function.

To clarify the structural basis of the cell adhesion activity of cadherins, we examined the effects of point mutations of well-conserved amino acid residues in the extracellular domain 1 of cadherin-4 (Cdh4) on the adhesion properties by alanine scanning mutagenesis. Mutations of two well-conserved aromatic amino acid residues in the extracellular domain 1 resulted in abnormal processing of Cdh4 molecules and no cell adhesion activity, whereas mutations of the corresponding aromatic amino acids in the extracellular domain 2 did not show these effects, suggesting a role for the two residues in the extracellular domain 1 in the folding and/or intracellular transport processes of Cdh4. Mutations of the amino acid residues suspected to be involved in strand dimer formation resulted in loss or significant decrease in cell adhesion activity. The mutant Cdh4s showed weak concentration at cell-cell adhesion sites and chemical cross-linking suggested that the strand dimer formation was actually impaired in the mutants. These results are consistent with the zipper model, in which the extracellular domain 1 of Cdh4 has intrinsic strand dimer formation activity in addition to adhesion dimer formation activity, both of which are involved in cell adhesion activity. The zipper model, however, needs further improvement to fully account for the present results.

Protocadherin 2C: a new member of the protocadherin 2 subfamily expressed in a redundant manner with OL-protocadherin in the developing brain.

Using cDNA of human protocadherin 2A (pc2A; originally known as protocadherin 2) as a probe, we cloned a new member of the protocadherin 2 subfamily from mouse brain cDNA libraries and named it protocadherin 2C (pc2C). It was similar to pc2A throughout its entire coding region, and its C-terminal region was highly conserved. The locus of the pc2C gene was on the mouse chromosome 18C where the pc2A gene is located, suggesting that genes of the pc2 subfamily form a gene cluster. The expression of pc2C was restricted to the nervous system, and the expression started in the embryonic stage and increased up to the adult stage. The expression pattern was quite similar to that of OL-protocadherin, a distinct class of protocadherin, although the timing and relative strength of expression were different. These results suggest that pc2C may be involved in neural development along with other classes of protocadherins.

Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems of the developing mouse brain.

We cloned a novel protocadherin cDNA, which we named OL-protocadherin (OL-pc), from mouse brain cDNA libraries. Its cytoplasmic region showed no similarities to other protocadherins, indicating that it belongs to a novel subfamily of protocadherins. Experiments using transfectants showed that OL-pc is a homophilic cell-cell adhesion molecule. The molecular mass of OL-pc was 140 kDa in the brain. Expression of OL-pc mRNA was specific to the nervous system, changing over time from the embryonic stage to the adult stage. The OL-pc expression seemed to be restricted to a subset of functionally related brain nuclei and regions such as the nuclei in the main olfactory system, the limbic system, and the olivocortical projection. There were at least two distinct patterns of distribution for the OL-pc protein. First, it was localized in particular brain nuclei or compartments, such as the stripes of the developing cerebellum. Second, it was found at the synapse in regions such as the glomeruli of the olfactory bulb. In addition, the OL-pc protein seemed not to be detected or was detected only weakly in some regions, such as hippocampus in which the mRNA was expressed at high levels. These results indicate that the expression of OL-pc is developmentally regulated in a subset of the functional systems and that it may be involved in the formation of the neural network by segregation of the brain nuclei and mediation of the axonal connections.

N-cadherin expressed on malignant T cell lymphoma cells is functional, and promotes heterotypic adhesion between the lymphoma cells and mesenchymal cells expressing N-cadherin.

Cadherins are Ca2+-dependent cell-cell adhesion molecules, and are involved in the formation and maintenance of the organocellular architecture. Using a combination of molecular biologic and biochemical methods, we analyzed cadherins expressed on cultured human malignant lymphoma cell lines (adult T cell lymphomas, human T cell leukemia virus type 1-negative T cell lines, and thymus-derived lymphoma cell lines), and obtained evidence that N-cadherin is the major cadherin expressed on these cells. These cells were found to form cell aggregates in a Ca2+-dependent manner, and more importantly to coaggregate and adhere with cells expressing N-cadherin, suggesting that N-cadherin on lymphoma cells is functionally active. Therefore, N-cadherin expressed on lymphoma cells could underlie the frequent invasion of these cells into the mesenchymal tissue in the skin and the central nervous system.

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.

Molecular properties and chromosomal location of cadherin-8.

Cloning of rat cadherin-8 cDNA demonstrated two types of cDNAs. The overall structure of the protein defined by one type of the cDNA is essentially the same as that of classic cadherins, whereas the protein defined by the other type of cDNA ends near the N-terminus of the fifth repeat of the extracellular domain (EC5) and contains a short unique sequence at the C-terminus. The same truncated type of cDNA was also obtained from a human cDNA library. In Northern blot analysis of rat brain mRNA, a probe for EC5 detected multiple bands of about 3.5-4.3 knt, whereas a probe for the alternative form hybridized with a band of about 3.5 knt. Western blot experiments showed that an antibody against the extracellular domain of rat cadherin-8 stained a band of about 95 kDa and a faint band of about 130 kDa in rat brain extract. These results suggest that cadherin-8 is expressed in two forms, a complete form and a truncated form without a transmembrane domain or cytoplasmic domain, in brain. The complete form of cadherin-8 expressed in L cells was about 130 kDa in molecular mass and was located at the cell periphery, mainly at the cell-cell contact sites. However, we failed to express the truncated form in L cells. The transfectants of the complete form showed weak cell adhesion activity. The complete form of cadherin-8 was sensitive to trypsin digestion, and Ca2+ did not protect cadherin-8 from digestion, in contrast to the classic cadherins. The complete form of cadherin-8 coprecipitated with beta-catenin, but did not immunoprecipitate well with alpha-catenin or gamma-catenin. Cadherin-8, as well as cadherin-11, was mapped to a specific region of chromosome 8 that also includes cadherins-1, -3, and -5.

Protocadherins and diversity of the cadherin superfamily.

Recent cadherin studies have revealed that many cadherins and cadherin-related proteins are expressed in various tissues of different multicellular organisms. These proteins are characterized by the multiple repeats of the cadherin motif in their extracellular domains. The members of the cadherin superfamily are divided into two groups: classical cadherin type and protocadherin type. The current cadherins appear to have evolved from a protocadherin type. Recent studies have proved the cell adhesion role of classical cadherins in embryogenesis. In contrast, the biological role of protocadherins is elusive. Circumstantial evidence, however, suggests that protocadherins are involved in a variety of cell-cell interactions. Since protocadherins, and many other new cadherins as well, have unique properties, studies of these cadherins may provide insight into the structure and biological role of the cadherin superfamily.

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 Ca(2+)-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.

Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins.

Cell adhesion and several other properties of a recently identified cadherin-related protein, protocadherin Pcdh2, were characterized. A chimeric Pcdh2 in which the original cytoplasmic domain was replaced with the cytoplasmic domain of E-cadherin was expressed in mouse L cells. The expressed protein had a molecular mass of about 150 kDa and was localized predominantly at the cell periphery, as was the wild-type Pcdh2. In a conventional cell aggregation assay, the transfectants showed cell aggregation activity comparable to that of classical cadherins. This activity was Ca(2+)-dependent and was inhibited by the addition of anti-Pcdh2 antibody, indicating that the chimeric Pcdh2, and probably the wild-type Pcdh2, has Ca(2+)-dependent cell aggregation activity. Mixed cell aggregation assay using L cells and different types of transfectants showed that the activity of Pcdh2 was homophilic and molecular type specific and that Pcdh2 was transfectants did not aggregate with other types of transfectants or with L cells. In immunoprecipitation, the chimeric Pcdh2 co-precipitated with a 105 kDa and a 95 kDa protein, whereas wild-type Pcdh2 co-precipitated with no major protein. Pcdh2 was easily solubilized with non-ionic detergent, in contrast to the case of classical cadherins. On immunofluorescence microscopy, the somas of Purkinje cells were diffusely stained with anti-human Pcdh2 antibody. Mouse Pcdh1 and Pcdh2 were mapped to a small segment of chromosome 18, suggesting that various protocadherins form a gene cluster at this region. The present results suggest that Pcdh2, and possibly other protocadherins as well as protocadherin-related proteins such as Drosophila fat, mediate Ca(2+)-dependent and specific homophilic cell-cell interaction in vivo and play an important role in cell adhesion, cell recognition, and/or some other basic cell processes.

Cloning, expression, and chromosomal localization of a novel cadherin-related protein, protocadherin-3.

To study the diversity of the protocadherin family, the cDNA clones for a novel protocadherin were isolated by screening rat brain cDNA libraries with a cDNA fragment obtained by PCR, and some of the properties were then characterized. The overall structure of the protein defined by the clone is similar to that of previously identified protocadherins; however, the cytoplasmic domain is distinct from those of previously cloned protocadherins or any other protein sequences in the data bank. We named this protocad herin-3 (Pcdh3) since this is the third protocadherin of which the entire coding sequence has been determined. Most of the deduced amino acid sequences of other cDNA clones obtained by the screening show high homology with but are distinct from that of Pcdh3, indicating that most of these sequences correspond to homologous but different protocadherins. These results demonstrate that Pcdh3 and the protocadherins defined by these clones constitute a protocadherin subfamily. Chromosome mapping indicates that mouse Pcdh3 is located in a specific region of mouse chromosome 18, close to the location of previously cloned protocadherins, suggesting that various protocadherins form a cluster in this region. In situ hybridization results showed that Pcdh3 and its related proteins were expressed at various areas in brain. The expressed Pcdh3 protein from the cDNA in mouse L cells was about 100 kDa in molecular weight and was localized at cell-cell contact sites. In contrast to the classical cadherins, however, the expressed Pcdh3 was sensitive to trypsin even in the presence of Ca2+, and the transfectants did not show strong Ca(2+)-dependent cell aggregation activity. These results indicate the structural and possibly functional diversity of the protocadherin family and suggest a distinctive biological role for Pcdh3.