Tenascin major
Ten-m is related to the extracellular matrix molecule tenascin (cytotaxin) (Baumgartner, 1994).
Tenascin accessory is a Drosophila gene located at 11A6-9 on the X-chromosome. The deduced protein of 782 amino acids contains eight tenascin-type EGF-like repeats not previously described in Drosophila, but lacks the fibronectin type III repeats and the fibrinogen homology present in the vertebrate tenascin molecules. Tena codes for a large transcript that exhibits extremely long 5' and 3' untranslated regions. Ten-a transcripts show a specific perinuclear localization within cells and are mainly expressed in the central nervous system, in the brain and near muscle attachment sites during embryogenesis. During pupal stages, tena is detected in the eye. These expression patterns are reminiscent of those formed by vertebrate tenascin. Tenascin-type EGF-like sequences are also detected in other loci of Drosophila and in various other organisms, indicating the existence of a family of genes related to tenascin (Baumgartner, 1993).
Chicken teneurin-1 and teneurin-2, two homologs of the Drosophila pair-rule gene product Ten-m and Drosophila Ten-a, have been characterized. The high degree
of conservation between the vertebrate and invertebrate proteins suggests that these belong to a novel family. The vertebrate members of this family have been named teneurins, because of their predominant expression in the nervous system. The expression of teneurin-1 and -2 was investigated by in situ hybridization. Teneurin-1 and -2 are expressed by distinct populations of neurons during the time of axonal growth. The most prominent site of expression of chicken
teneurins is the developing visual system. Recombinant teneurin-2 was expressed to assay its molecular and functional properties. It is a type II transmembrane protein, which can be released from the cell surface by proteolytic cleavage at a furin site. The expression of teneurin-2 in neuronal cells leads to a
significant increase in the number of filopodia and to the formation of enlarged growth cones. The expression pattern of teneurins in the developing nervous system and the ability of teneurin-2 to reorganize the cellular morphology indicate that these proteins may have an important function in the formation of neuronal connections. The fact that within the cytoplasmic domain of chicken teneurin-1 and -2, two potential calcium-binding sites and two putative SH3-domain-binding sites could be identified argues that the teneurins could serve as receptor proteins
transmitting signals to the cell interior upon homo- or hetero-philic binding of a ligand (Rubin, 1999).
The teneurins are a family of four transmembrane proteins expressed in the developing vertebrate nervous system, though the Drosophila teneurin ten-m is also a pair-rule gene. Whole-mount in situ hybridization was used to localize teneurin-4 transcripts in the chicken embryo. The earliest signal is detected at stage 19 in the somites and limb buds. By stage 20 teneurin-4 transcripts are detected in temporal periocular mesenchyme, branchial arches, diencephalon and somites. Teneurin-4 expression in the limbs changes dramatically during development. Between stages 19 and 21 teneurin-4 expression is concentrated proximally in the zone of polarizing activity. Between stages 24 and 26 teneurin-4 is expressed in the mesenchyme of the anterodistal part of the limb. As in Drosophila, vertebrate teneurins are expressed not only in the nervous system, but also in non-neuronal tissues during pattern formation (Tucker, 2000).
Teneurin-2 is a member of a novel family of transmembrane proteins characterized to date in fish, birds, mammals, and Drosophila (e.g., the pair-rule gene product Ten-m). Teneurin-2 is expressed by neurons in the developing avian visual system in a pattern complementary to the expression of teneurin-1 and recombinant teneurin-2 induces morphologic changes in neuronal cells in culture. cRNA probes to two newly identified splice variants and a teneurin-2-specific antibody have been used to determine whether teneurin-2 is also expressed outside the nervous system. Both reverse transcriptase-polymerase chain reaction and in situ hybridization indicate that the three splice variants known so far are coexpressed at sites of pattern formation during development. Teneurin-2 mRNAs and protein are found in the developing limbs, somites, and craniofacial mesenchyme. In addition to expression of teneurin-2 by the apical ectodermal ridge, teneurin-2 transcripts also appear transiently at sites of tendon development. Teneurin-2 expression patterns are strikingly similar to those of fibroblast growth factor 8 (FGF8). In agreement with the overlapping expression pattern, FGF8-coated beads implanted into chicken limb buds induce the ectopic expression of teneurin-2 and soluble FGF8 induces teneurin-2 in limb explant cultures. Thus, teneurin-2 may act downstream of FGF8 during morphogenesis (Tucker, 2001).
The Drosophila gene ten-m/odz is the only pair rule gene identified to date that is not a transcription factor. In an attempt to analyze
the structure and the function of ten-m/odz in mouse, four murine ten-m cDNAs were isolated that code for proteins of 2,700-2,800
amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain
characteristic of transmembrane proteins, located 300-400 amino acids after the NH2 terminus. Approximately 200 amino acids COOH-terminal to this
hydrophobic region are eight consecutive EGF-like domains. The extracellular part of ten-m
molecules consists of a linker domain of ~200 amino acids, a region with eight EGF-like domains of ~250 amino acids, and a COOH-terminal domain of
~2,000 amino acids. The linker domain of all mouse ten-m proteins contains several dibasic amino acid residues that could serve as potential sites for
proteolytic processing of the molecules. One of these sites is conserved in all four mouse ten-m sequences and Drosophila Ten-m/Odz and Ten-a. Drosophila Ten-m/Odz shows higher identity to the mouse ten-m sequences in the linker domain (26%-29%) than in the cytosolic
domain (19%-21%). Between the mouse ten-m sequences, the similarity of the cysteine-free linker domain ranges from 43% to 48%; the EGF domains range from
65% to 72%, and the large cysteine-rich COOH-terminal domain ranges from 58% to 68%. The similarity to Drosophila Ten-m/Odz over the entire COOH-terminal
domain ranges from 30 to 33% (Oohashi, 1999).
Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins
composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene results in membrane-associated staining of
the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 shows that Ten-m1 is a
disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5, which contain an odd number of cysteines. Tandem arrays of multiple EGF-like modules often appear as rod-like structures. Therefore, the short extended rods in the Ten-m1
extracellular domain most likely represent the EGF-like modules, which are located at the NH2 termini of the recombinantly expressed Ten-m1 monomers. This
hypothesis is further supported by the fact that the second and fifth EGF-like domains have an odd number of cysteines, which might enable the formation of
intermolecular disulfide bonds. Experiments show that the EGF-like modules of Ten-m1 with an odd number of cysteines can form intermolecular disulfide bonds, leading to the homodimerization
of Ten-m1. Northern blot and immunohistochemical analyses reveal widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m
genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter binds to specific
regions in many tissues that are partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrate that Ten-m1
can bind to itself (Oohashi, 1999).
The Drosophila pair-rule gene odz (Tenm) has many patterning roles throughout development. Four
mammalian homologs of this gene have been identified, including one previously described as a mouse ER stress response gene, Doc4. The Odz genes encode large polypeptides displaying the hallmarks of Drosophila Odz: a putative signal peptide;
eight EGF-like repeats; and a putative transmembrane domain followed by an 1800-amino-acid stretch without homology to
any proteins outside of this family. The mouse genes Odz3 and Doc4/Odz4 exhibit partially overlapping, but clearly
distinct, embryonic expression patterns. The major embryonic sites of expression are in the nervous system, including the
tectum, optic recess, optic stalk, and developing eye. Additional sites of expression include trachea and mesodermally
derived tissues, such as mesentery, and forming limbs and bone. The expression patterns suggest that each of the genes has its own distinct developmental role. Comparisons of
Drosophila and vertebrate Odz expression patterns suggest evolutionarily conserved functions (Ben-Zur, 2000).
Preliminary results suggest that
the expression of mouse Odz2 is strictly restricted to the
nervous system. Within the brain, it appears in as many
varied sites as do Odz3 and Doc4, and partially overlaps
them, but primarily appears in tissue regions where the
other two are not expressed. However, it should be noted
that Odz3 and Doc4 brain expression persists in adults:
preliminary data shows expression in the hippocampus,
the Purkinje cells of the cerebellum, and other
distinct nuclei and cell types. Similarly in the PNS, the
dorsal root ganglia expression (mainly Doc4) and trigeminal
ganglion expression (mainly Odz3) persist in postmitotic
neurons. Overall, the nervous system expression patterns of
the Drosophila and murine Odz genes suggest functional
conservation between flies and mammals. The CNS is the
site of highest embryonic expression in both cases, portions
of that expression persist into adulthood in both cases, and
sensory PNS structures show expression in both cases (Ben-Zur, 2000).
The Drosophila-murine comparisons extend further, to
include additional expression correlations. Drosophila odz
expression for patterning of the eye is reflected by the
expression of all three mouse Odz genes from around 14.5
dpc in the inner nuclear neuroblastic layer of the developing
eye. Intriguingly, Doc4 and Odz3 are
expressed in the tectum neuroepithelium, and the genes are
very highly expressed in the optic recess and optic stalk,
which is reminiscent of odz expression in processes linking
the compound eye retina to the optic lobes of the ventral
ganglia. The nasal epithelial expression of Doc4 parallels
odz expression in the developing and adult Drosophila
antannae. Interestingly, both Odz3
and Doc4 are highly expressed by the mesenchyme of the
lung primordium (at 11.5 dpc), and this expression is
drastically diminished at later stages (at 12.5 dpc for Odz3
and somewhat later for Doc4). Expression of
Odz in Drosophila and mouse occur in analagous structures,
which have been shown to be patterned by homologous
signaling mechanisms: tracheae in flies and trachea (and
early developing lungs) in the mouse (Ben-Zur, 2000).
Teneurins are a novel family of transmembrane proteins conserved between invertebrates and vertebrates. There are two members in Drosophila, one in C. elegans and four members in mouse. The analysis of the genomic structure of the human teneurin-1 gene is described. The entire human teneurin-1 (TEN1) gene is contained in eight PAC clones representing part of the chromosomal locus Xq25. Interestingly, many X-linked mental retardation syndromes (XLMR) and non-specific mental retardation (MRX) are mapped to this region. The location of the human TEN1 together with the neuronal expression makes TEN1 a candidate gene for XLMR and MRX. Large parts of the human teneurin-2 sequence were identified on chromosome 5 and sections of human teneurin-4 at chromosomal position 11q14. Database searches resulted in the identification of ESTs encoding parts of all four human members of the teneurin family. Analysis of the genomic organization of the Drosophila ten-a gene revealed the presence of exons encoding a long form of Ten-a, which can be aligned with all other teneurins known. Sequence comparison and phylogenetic trees of teneurins show that insects and vertebrates diverged before the teneurin ancestor was duplicated independently in the two phyla. This is supported by the presence of conserved intron positions between teneurin genes of man, Drosophila and C. elegans. It is therefore not possible to class any of the vertebrate teneurins with either Drosophila Ten-a or Ten-m. The C-terminal part of all teneurins harbours 26 repetitive sequence motifs termed YD-repeats. YD-repeats are most similar to the repeats encoded by the core of the rearrangement hot spot (rhs) elements of Escherichia coli. This makes the teneurin ancestor a candidate gene for the source of the rhs core acquired by horizontal gene transfer.
The Drosophila gene ten-m is the first pair-rule gene not encoding a transcription factor, but an extracellular protein. A highly conserved
chicken homolog called teneurin-1 has been characterized. The C-terminal part harbors 26 repetitive sequence motifs termed YD-repeats. The YD-repeats are most similar to the
core of the rhs elements of Escherichia coli. Related repeats in toxin A of Clostridium difficile are known to bind specific carbohydrates. Recombinantly
expressed proteins containing the YD-repeats of teneurin-1 bind to heparin. Furthermore, heparin lyase treatment of extracts of cells expressing recombinant
YD-repeat protein releases this protein from high molecular mass aggregates. In situ hybridization and immunostaining reveals teneurin-1 expression in neurons of the
developing visual system of chicken and similar expression of ten-m in Drosophila. This phylogenetic conservation of neuronal expression from flies to birds implies fundamental roles for
teneurin-1 in neurogenesis. This is supported by the neurite outgrowth occurring on substrates made of recombinant YD-repeat proteins, which can be inhibited by
heparin. Database searches have resulted in the identification of ESTs encoding at least three further members of the teneurin family of proteins. Furthermore, the human
teneurin-1 gene could be identified on chromosome Xq24/25, a region implied in an X-linked mental retardation syndrome (Minet, 1999).
The localization of alternatively spliced forms of cytotactin was determined
in neural and nonneural tissues using two probe: one (CT) detects all forms of cytotactin mRNA, and the other (VbVc) detects two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increase from E8 through E15 and then gradually decrease until they are barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridize only to the 7.2-kb message. In the cerebellum, the 220-kD polypeptide and 7.2-kb mRNA are the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message containing the VbVc alternatively spliced insert. Cytotactin mRNA is present in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs are more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA is observed in the soma, whereas the protein is present externally along the glial fibers. In the telencephalon, cytotactin mRNAs are found in a narrow band at the edge of a larger region in which the protein is wide-spread. Hybridization with the VbVc probe generally overlaps that of the CT probe in the spinal cord and cerebellum. In contrast, in the outermost tectal layers, differential hybridization is observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, is detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes is observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme express cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells express cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contain cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein (Prieto, 1990).
At least four independent cell binding regions are distributed among the various cytotactin domains. Two of these
are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise
favorable substrates. The adhesive regions map to the fibronectin type III repeats II-VI and
the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differ; the
cells spread on the fibronectin type III repeats as they do on fibronectin, but remain rounded-up on the
fibrinogen domain. The counteradhesive properties of the molecule map to the EGF-like
repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contains a cell
attachment activity that is observed only after proteolysis of the cells. Several cell types are used
in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin.
The different domains exert their effects in a concentration-dependent manner and can be inhibited by
an excess of the soluble molecule, consistent with the hypothesis that the observed properties are
mediated by specific receptors. It also appears that some of these receptors are restricted to
particular cell types. For example, glial cells bind better than neurons to the fibrinogen domain and
fibroblasts bind better than glia and neurons to the EGF fragment. These results provide a basis for
understanding the multiple activities of cytotactin and a framework for isolating different receptors that
mediate the various cellular responses to this molecule (Prieto, 1992).
To identify domains of chicken tenascin-C, required for interaction with cells, a new approach was used: instead of expressing the particular domains of interest, domains were deleted from an otherwise intact tenascin-C molecule and then the mutant molecules thus constructed were scored for any concomitant change in activity. As a starting point for all mutant constructs, the smallest naturally occurring tenascin-C splice variant was expressed in vertebrate cells. The tenascin-C mutant constructions had either deletions of all EGF-like repeats, all fibronectin type III repeats or of the fibrinogen globe. In double mutants the fibronectin type III repeats were deleted together with either the EGF-like repeats or the fibrinogen globe. All tenascin-C variants assemble correctly to hexameric molecules of the expected molecular characteristics. Neither intact tenascin-C nor the mutant missing the fibrinogen globe promote adhesion of chick embryo fibroblasts, whereas both of them (the hexamers containing solely the fibrinogen globe and the EGF-like repeats) are adhesive substrates and even support cell spreading. When tenascin-C is added to the medium of fibroblasts plated into fibronectin-coated wells, cell adhesion is blocked by intact tenascin-C, but not by mutants missing the fibrinogen globe. In neurite outgrowth assays using dorsal root ganglia, processes form on all substrates except on the mutant missing only the fibrinogen globe, where the ganglia fail to adhere. The mutants missing the fibronectin type III repeats allow more rapid neurite outgrowth than all other tenascin-C variants. The mutant consisting essentially of oligomerized EGF-like repeats is as active a substrate for neurite outgrowth as laminin. From the combined data, it is concluded that the activities of intact tenascin-C cannot be mimicked by a domain by domain investigation, but the concerted action of several domains leads to the diverse cellular responses (Fischer, 1997).
Ten-m/Odz/teneurins are a family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes (Feng, 2002; full text of article).
The role of glycosaminoglycans was investigated in fibronectin matrix assembly
and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production fail to assemble a fibronectin matrix, and do not incorporate tenascin-C. Another mutant with reduced heparan sulfate produces a normal fibronectin matrix but fails to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibit the binding of tenascin-C to purified fibronectin in ELISA, and completely block incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocks their ability to incorporate tenascin-C into matrix fibrils. It is concluded that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. The large heparan sulfate proteoglycan, perlecan, co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C maps to the fibronectin type III domains 3-5, but this binding is strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently, when the alternative splice segment is inserted after domain 5, it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils (Chung, 1997).
The integrin alpha 9 subunit is a partner of the beta 1 subunit (See Drosophila Myospheroid), which is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. Alpha 9 beta 1 was expressed on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480. These transfected cells lines were used to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, the binding site for alpha 9 beta 1 localizes to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment is not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA (Yokosaki, 1994).
Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (Egf). Cultured SMCs require TN-C for Egf-dependent growth on type I collagen. In this study, the regulation and function of TN-C was explored in SMCs. A matix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of Egf. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the Egf growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates Egf-dependent survival and growth was then investigated. TN-C interactions with alphavbeta3 integrins modify SMC shape, and Egf-dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of Egf-rs, tyrosine-phosphorylated proteins, and increased activation of Egf-rs after addition of Egf. Cross-linking SMC beta3 integrins replicates the effect of TN-C on Egf-r clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta3 integrin ligands in type I collagen. In turn, alphavbeta3 interactions with TN-C alter SMC shape and increase Egf-r clustering and Egf-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis (Jones, 1997).
Two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan (the extracellular domain of protein-tyrosine phosphatase-zeta/beta), are high-affinity ligands of tenascin-C. Using portions of tenascin-C expressed as recombinant proteins in human fibrosarcoma cells, it can be demonstrated that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen-like globe and that phosphacan binds to this single domain with nearly the same affinity as to native or recombinant tenascin-C. However, maximum binding of neurocan requires both the fibrinogen globe and some of the adjacent fibronectin type III repeats. Binding of phosphacan and neurocan to intact tenascin-C, and of phosphacan to the fibrinogen globe, is significantly increased in the presence of calcium. Chondroitinase treatment of the proteoglycans does not affect their binding to either native tenascin-C or to any of the recombinant proteins, demonstrating that these interactions are mediated by the proteoglycan core proteins rather than through the glycosaminoglycan chains. These results are also consistent with rotary shadowing electron micrographs that show phosphacan as a rod terminated at one end by a globular domain frequently seen apposed to the fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma cells adhere to and spread on deletion variants of tenascin-C containing only the epidermal growth factor-like domains or the fibronectin type III repeats and the fibrinogen globe. In both cases cell adhesion is inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C (Milev, 1997).
Neurocan is a member of the aggrecan family of proteoglycans that is characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein is designed to contain the last downstream glycosaminoglycan attachment site and is expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognizes tenascin-C. The interaction is abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacts with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 are retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yield a KD value of 17 nM for purified tenascin-C. It is concluded that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C (Rauch, 1997).
The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming alpha subunit
and two smaller auxiliary subunits: beta1 and beta2. The beta subunits are single membrane-spanning
glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the beta2 subunit has
close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion
molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C
and tenascin-R. The binding of the purified sodium channel and the extracellular domain of
the beta2 subunit to tenascin-C and tenascin-R was investigated in vitro. Incubation of purified sodium channels on
microtiter plates coated with tenascin-C reveals saturable and specific binding with an apparent Kd of
approximately 15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of
tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized
on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged
extracellular domain of beta2 subunits. Both purified sodium channels and the extracellular domain of the
beta2 subunit bind specifically to fibronectin type III repeats 1-2, A, B, and 6-8 of tenascin-C and
fibronectin type III repeats 1-2 and 6-8 of tenascin-R but not to the epidermal growth factor-like domain or
the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular
matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels
in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized
sodium channels in these locations (Srinivasan, 1998).
Latrophilin-1, -2, and -3 (see Drosophila Calcium-independent receptor for α-latrotoxin) are adhesion-type G protein-coupled receptors that are auxiliary alpha-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, this study identified teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. Teneurin are shown to bind to latrophilins with nanomolar affinity, and this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas the current findings suggest a heterophilic interaction mechanism. Thus, whether mammalian teneurins also are homophilic cell-adhesion molecules was tested, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, it was found that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance (Boucard, 2014).
Latrophilin 1 (LPH1), a neuronal receptor of alpha-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca(2+). As an 'adhesion G-protein-coupled receptor,' LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, wLPH1's extracellular domain was used to purify its endogenous ligand. A single protein of approximately 275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. This study shows that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippocampal neurons in cocultures; LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippocampal neurons show that LPH1 and Lasso are enriched in synapses; in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca(2+) signals in presynaptic boutons of hippocampal neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca(2+) signals, which might affect synaptic functions (Silva, 2011).
LPH1 has been implicated in multiple phenomena, including binding of α-LTX, release of neurotransmitters, intracellular signaling, neuronal morphogenesis, and mental conditions. However, further studies of LPH1 require the identification of its endogenous ligand. Using LPH1-affinity chromatography, this study has now isolated such a ligand. Lasso is a splice variant of teneurin-2. It interacts with LPH1 specifically and strongly, but binds very weakly to LPH2 and does not bind to LPH3. Reciprocally, sequencing results and LPH1 binding suggest that LPH1 interacts with Lasso/teneurin-2 only, and not with teneurin-1, -3, or -4 (Silva, 2011).
Both LPH1 and Lasso/teneurin-2 are highly abundant in the brain. Can they mediate neuronal cell interaction? Teneurin is proteolyzed between the TMR and EGF repeats (see Kenzelmann, 2008), and this could preclude its receptor activity. However, only a proportion of teneurin is cleaved, resulting in two bands on reducing SDS gels. The fragment remains anchored on the cell surface, probably as part of the homodimer. This makes Lasso a bona fide cell-surface receptor (Silva, 2011).
Accordingly, Lasso and LPH1 mediate heterophilic cell-cell contacts between expressing cells in cocultures and formation of artificial synapses between fibroblasts and neurons. Moreover, the data indicate that in cultured neurons and mature brain, LPH1 is localized in the presynaptic membranes, whereas Lasso is mostly postsynaptic. Given the size of LPH1 (14 nm) and Lasso (~30 nm), their complex can span the synaptic cleft (20-30 nm), allowing this receptor pair to connect neurons at synapses (Silva, 2011).
The LPH1-Lasso interaction is not purely structural. LPH1 mediates signaling induced by LTXN4C both in model cells expressing exogenous LPH1 and in organotypic hippocampal cultures. This signaling requires the CTF and involves the activation of phospholipase C, production of inositol-trisphosphate, and release of stored Ca2+. This study demonstrates that a soluble C-terminal fragment of Lasso also induces an increase in cytosolic Ca2+ in NB2a cells expressing LPH1 and in hippocampal neurons. Such a rise in cytosolic Ca2+ in neurons could modulate neurotransmitter release (Silva, 2011).
Interestingly, teneurin contains a sequence termed teneurin C-terminal-associated peptide (TCAP) that resembles corticotropin-releasing factor. It is hypothesized that TCAP is cleaved from teneurin and acts as a soluble ligand of unknown receptors (Wang, 2005). Synthetic TCAP regulates cAMP in immortalized neurons and, when injected cerebrally, affects behaviors related to stress and anxiety. However, there is no evidence that TCAP is released in vivo. The current work suggests an alternative possibility: TCAP, being part of Lasso, affects animal behavior by stimulating LPH1. This is supported by LPH being implicated in schizophrenia, anxiety (offspring killing by LPH1−/− mice), and attention deficit/hyperactivity disorder (Silva, 2011).
The current findings raise several interesting questions. Does Lasso send an intracellular signal in response to LPH1 binding? What are the relationships among the four teneurin homologs and the three LPH proteins found in most vertebrates? Can the LPH1–Lasso interaction affect synaptogenesis, neurotransmitter release, or synaptic plasticity? Answering these questions will provide important insights into the physiological functions of these two families of neuronal cell surface receptors (Silva, 2011).
Like the chicken cytotactin gene, the mouse tenascin gene
has a single RNA start site that lies 27 bp downstream of a TATA box. A 4028-bp region of DNA
upstream of the mouse tenascin gene was sequenced and examined for regulatory motifs in common with
the upstream sequence from the chicken cytotactin promoter. Two hundred thirty base pairs of the
proximal promoter regions from both genes have an extended sequence similarity and contain common
regulatory motifs such as two tracts of homopolymeric dA.dT sequence, an octamer motif, an ATTA
(TAAT) motif, which is a common core sequence for binding of homeodomain transcription factors, and
a TATA-box/cap-site region. The conserved proximal promoter region of tenascin is responsible for most of the positive
regulatory activity. In addition, an upstream region (-2478 to -247) represses proximal promoter activity
in mouse fibroblasts and also in chicken embryo fibroblasts. These data indicate that both the structure
and function of the cytotactin/tenascin proximal promoters have remained conserved over 250 million
years (Copertino, 1995).
A promoter within the proximal 250 base pairs upstream of the mouse Tenascin (TN) gene contains several putative regulatory elements that are conserved among vertebrate genes. Four different DNA elements have been identified within this promoter, and they contribute in different ways to TN gene expression in cultured cells. These four elements are: a binding site for Drox proteins, one for nuclear factor1, an octamer motif that binds POU-homeodomain proteins, and a novel TN control element. The nuclear factor 1 and TN control element have positive effects on TN promoter activity and form similar DNA-protein complexes with nuclear extracts from three cell lines. The Krox element has a negative effect on TN promoter activity in one cell line and a positive effect in another. Two DNA binding complexes, one correlated with the negative and the other with the positive activities of the Krox element are found to contain the protein Krox24. The octamer motif is required for induction of TN promoter activity by the POU-homeodomain protein Brn2 (See Drosophila Drifter) in one cell line but is inactive in another (Copertino, 1997).
Approximately 2300 base pairs of the human tenascin-C (TN) gene 5'-flanking
region have been cloned and sequenced. This genomic region contains several potential binding sites for
transcription factors. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts,
showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases
upstreamof the transcription start site are equally active in directing the expression in TN producer and nonproducer cells.
The putative "silencer" elements are present in the -220 to
-2300 region active in both TN producer and nonproducer cell lines. The selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the
TN gene intron 1 in expression vectors. These findings indicate that complex mechanisms
control the transcriptional regulation of TN gene (Gherzi, 1995).
Cytotactin (tenascin) is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and
migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The
promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory
sequences. These include putative binding sites for homeodomain proteins and a phorbol
response element (TRE)/AP-1 element (see Drosophila Jun), a potential target for transcription
factors thought to be involved in growth-factor signal transduction. To determine the effects of
homeobox-containing genes on cytotactin promoter activity, a series of cotransfection
experiments were carried out on NIH 3T3 cells using cytotactin promoter-
reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and
Hox-1.3. Cotransfection with Evx-1 (Drosophila homolog: Even skipped)stimulates cytotactin promoter activity whereas cotransfection in
control experiments with Hox-1.3 have no effect. To localize the sequences required for Evx-1 activation, a series of deletions in the cytotactin promoter were tested. An 89-base-pair region containing a consensus
TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this
TRE/AP-1 site was found to confer Evx-1 inducibility on a minimal promoter. Mutation of
the TRE/AP-1 site abolishes this activity. To explore the potential role of growth factors in cytotactin
promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first
transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine
serum. Although the cells exhibit only low levels of reporter activity under these conditions, cells exposed
for 12 h to 10% fetal bovine serum show a marked increase in reporter activity. Cotransfection
with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum is sufficient,
however, to produce high levels of reporter activity. These findings are consistent with the hypothesis that
Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a
growth-factor signal transduction pathway. More generally, the results support the hypothesis that the
place-dependent expression of morphoregulatory molecules may depend upon local cues provided by
homeobox genes and their encoded proteins (Jones, 1992).
In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun (see Drosophila DJun)
transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to
activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets.
c-Jun-upregulated cDNA clones homologous to the tenascin-C gene have been identified by differential
screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix
protein involved in cell attachment and migration and in the control of cell growth. Transient
overexpression of c-Jun induces tenascin-C expression in primary REF and in FR3T3, an established
fibroblast cell line. Surprisingly, tenascin-C synthesis is repressed after stable transformation by
c-Jun, as compared to that in the nontransformed parental cells. As assessed by using the tenascin-C
(-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation
is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at
position -210. As demonstrated by gel shift experiments and cotransfections of the
reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two
transcription factors bind and synergistically transactivate the tenascin-C promoter. Two other extracellular matrix proteins, SPARC and thrombospondin-1, are c-Jun targets.
Thus, these results strongly suggest that the regulation of the extracellular matrix composition plays a
central role in c-Jun-induced transformation (Mettouchi, 1997).
EMX2 and PAX6 are expressed by cortical progenitors and specify area patterning. Representational difference analysis (RDA) was used to compare expressed RNAs from wild type and Emx2−/− cortex and 41 unique clones were identified. Using secondary screening by in situ hybridization, five genes were selected for further analysis, Cdk4, Cofilin1, Crmp1, ME2, and Odz4, involved in neuronal proliferation, differentiation, migration, and axon guidance. Each exhibits differential expression in wild type cortex. Odz4 is one of four members of a vertebrate gene family homologous to the Drosophila pair-rule patterning gene, Odd Oz (Odz), a transmembrane receptor. Odz genes are expressed in complementary patterns in cortex, as well as in nuclei-specific patterns in thalamus that relates to their area-unique cortical expression. In addition, each of the genes analyzed shows different expression patterns in wild type cortex, Emx2, and Pax6 mutant cortex, consistent with potential roles in area patterning. These findings identify potential targets of EMX2 that might account for its function and the defects in Emx2−/− cortex, and suggest that the Odz family of transmembrane proteins influences cortical area patterning downstream to EMX2 and PAX6 (Li, 2006).
CDK4 stimulates growth rate and is expressed exclusively by proliferating cells within neocortex. Down-regulated Cdk4 expression in Emx2−/− and Sey/Sey is consistent with decreased cortical sizes of both mutant mice. At E18.5, Cdk4 expression within ventricular zone (vz)/subventricular zone (svz) becomes broader and more diffused in Emx2−/− cortex while thinner in Sey/Sey cortex. This might be caused secondarily by different structural abnormalities of the mutant cortices (Li, 2006).
Absence of functional EMX2 leads to defective cell migration in telencephalon. Transplantation studies support that EMX2 mutation results in abnormal cortical milieu that impairs tangential cell migration from ganglionic eminence (GE) into neocortex. Defective radial migration is attributed to defective Cajal–Retzius cells that produce Reelin. Radial migration is robustly affected in Emx2−/− neocortex, while the penetration of impaired Reelin expression varies from zero to severe. Disorganized cortical lamination in Emx2−/− seems to be the result of more than defective Cajal–Retzius cells (Li, 2006).
Sema3A-mediated signaling plays important roles in directing axon guidance and neuronal migration. CRMP1, a component of Sema3A-receptor complex, is expressed in rostral-high to caudal-low gradient in neocortex. Cortical Crmp1 expression is down-regulated with flattened-out expression gradient in Emx2−/−, which might compromise neuronal sensitivity to semaphorins. Cell migration and axon guidance of cortical neurons would subsequently be affected, which has impact on cortical arealization (Li, 2006).
Cofilin1 regulates actin polymerization/depolymerization, which is the major machinery of cell motility. Cofilin1 is expressed in rostral-high to caudal-low gradient in cortex, and the expression is up-regulated with a flattened gradient in Emx2−/− while down-regulated in Sey/Sey. EMX2 and PAX6 might be involved in regulating neuronal motility by determining the dynamics of cytoskeletal components such as actin (Li, 2006).
bHLH transcription factors play important roles in a variety of developmental processes, including corticogenesis. ME2 belongs to class A bHLH family (Daughterless homolog) and is expressed by both proliferating and differentiating neurons. Within neocortex, ME2 is initially expressed in rostral-low to caudal-high gradient that becomes reversed as cortex matures. Cortical expression of ME2 is decreased and shifts caudally in Emx2−/− while becoming enhanced and shifts anteriorly in Sey/Sey. ME2 stimulates expression of Id2 that is capable of promoting expression of neurotrophin receptor, p75. EMX2 and PAX6 might regulate neocortical arealization indirectly through the neurotrophin-signaling pathway that is involved in neuronal differentiation, regeneration, survival, apoptosis, and cell migration (Li, 2006).
Odd Oz is a pair rule gene crucial for patterning Drosophila body plan which mouse orthologue, Odz4, is identified by RDA as down-regulated in Emx2−/−. In Drosophila, ODZ expression coincides with the developmental transition from syncytial to cellular blastoderm, which represents the first step in shifting segmentation mechanism from internuclear to intercellular signaling (Li, 2006).
In mouse, expression gradients of Odz2, Odz3, and Odz4 are increasing from lateral-anterior to medial-posterior neocortex. Genomics screens have also identified Odz2 and Odz4 as caudal genes. Odz1 expression gradient is decreasing from lateral-anterior to medial-posterior similar to Pax6 expression. While Emx2 and Pax6 expressions are restricted to proliferating neural progenitor cells, Odz4 is expressed by both proliferating and differentiating neurons. Odz1, Odz2, and Odz3 expressions are detected only in differentiating cells within cortical plate. The expression gradients suggest possible involvement of Odz in neocortical arealization. Indeed, Odz expressions are decreased and shrunk in Emx2−/− while increased and expanded in Sey/Sey cortex. EMX2 and PAX6 are not required to initiate or maintain Odz expression, but rather responsible for controlling the cortical position and area range that are permissive for Odz expression or settling of Odz-expressing cells (Li, 2006).
The arealization of neocortex depends on neuronal differentiation, positioning, and wiring. Migratory cells get instructions from diffusible chemical factors, electrical currents, or extracellular matrix (EM). Similar guiding mechanisms have been used by the neuronal growth cone to lead axons from cell soma to target tissue. Axons must grow upon solid substrate and growth cone orients with particular receptors for factors deposited in EM. There are three major EM components: collagen, proteoglycans, and large glycoproteins. ODZ has eight EGF repeats highly homologous to tenascin that is a large glycoprotein resembling fibronectin. ODZ4 is isolated as insoluble complex with cell membrane/EM during immunoprecipitation analyses. Electron microscopy combined with analytical ultracentrifugation demonstrates that ODZ can form homodimers through EGF repeat domains (Li, 2006).
ODZ may play important roles in axon guidance and cell migration. In vitro transfection studies suggested that homophilic binding of ODZ2 may be involved in the formation of synapses and fasciculation. During rat olfactory bulb regeneration, ODZ2 may facilitate precise wiring between receptor neurons and glomeruli. Interestingly, these data show that Odz2, Odz3, and Odz4 are strongly expressed in both visual cortex and dLG that are connected. ODZ may be involved in correct path-finding of TCAs to subplate, which underlies the neocortical area shift in Emx2−/− (Li, 2006).
At E18.5, there is a dramatic decrease and caudal shift of Odz4 expression in Emx2−/− dcp while the opposite is observed in Sey/Sey. Expression of Odz4 in layer 5 is, however, enhanced and evenly distributed throughout Emx2−/− cortex while there is no obvious change in Sey/Sey. Neurons of layer 5 project axons to specific subcortical targets, such as superior colliculus and spinal cord. Conceivably, corticotectal and corticospinal projections have to adjust to the shifted areas where ODZ4 might be involved (Li, 2006).
Odz1 separated from the other Odz members early during evolution and Odz1 expression pattern shifts from initially increasing to later decreasing gradient along the anterior–posterior axis during corticogenesis, suggesting that Odz1 may be important for the development of anterior cortical areas. Odz1 is also expressed in gradient by subplate cells that establish the initial connections between thalamic nuclei and neocortex. The graded expression of Odz1 polarizes the subplate, which may serve as guidance cues for invading TCAs. Interestingly, Odz1 is mapped to human X-chromosome in area Xq25, a region to which X-linked lymphoproliferative disease (XLP), X-linked mental retardation syndromes (XLMR), and “non-specific” mental retardation (MRX) have been mapped (Li, 2006).
Investigation on Odz is made complicated by the unsettled debate about whether this receptor family represents type I or type II transmembrane proteins, an important issue to resolve before designing any reasonable function studies. Based on expression data, it would be expected that Odz2, Odz3, and Odz4 may play overlapping roles in defining posterior cortical areas, while all their functions may not be redundant. Odz4 is the only Odz gene expressed in vz/svz which indicates possible roles in determining the fate of cortical progenitor cells. Odz4 is mapped to a region on mouse chromosome 7 where lethal ENU mutations exed (extraembryonic ectoderm development) and pid (preimplantation development) are localized. The mutant phenotype correlates with Odz4 expression patterns, suggesting that Odz4 might be indispensable for survival. It would be important to study in detail the functions of each Odz gene for a better understanding of cortical arealization (Li, 2006).
A mouse homolog of the Drosophila pair-rule gene Odd Oz (Odz4) maps to the critical region of the l7Rn3 locus on mouse chromosome 7. Odz4 is an excellent candidate for this allelic series because (1) it spans the entire critical region, (2) the phenotypes correlate with embryonic expression, (3) the complex genetic inheritance of the alleles is consistent with complex transcriptional regulation, and (4) one allele has a mutation in a conserved amino acid. Odz4 uses five alternate promoters that encode both secreted and membrane-bound proteins. Intragenic complementation of the l7Rn3 alleles is consistent with these multiple-protein isoforms. Further, the allelic series shows that Odz4 is required to establish the anterior-posterior axis of the gastrulating mouse embryo and is necessary later for mesoderm-derived tissues such as somites, heart, and skeleton. Sequencing of RT-PCR products from five of the six alleles reveals a nonconservative amino acid change in the l7Rn3m4 allele. This amino acid is important evolutionarily, as it is conserved to Drosophila. Together, these data indicate that Odz4 is mutated in the l7Rn3 allele series and performs roles in the mouse brain, heart, and embryonic patterning similar to those of its Drosophila counterpart (Lossie, 2005; full test of article).
continued: see Tenascin major Evolutionary Homologs part 2/2
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