BIOLOGICAL OVERVIEW

Ten-m is the first example of a known pair-rule gene product acting from outside the cell. Every odd-numbered body segment is deleted in Ten-m mutants. The protein is also a putative transmembrane tyrosine kinase substrate. The protein is under control of fushi tarazu and even-skipped (Baumgartner, 1994 and Levine, 1994). Ten-m alters wingless and engrailed expression through odd-paired's effects on paired and sloppy paired (Baumgartner, 1994).

Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin alpha2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand Tiggrin (and the Tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the alphaPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alphaPS2 m8 splice variant. The betaPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the betaPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure (Graner, 1998).

Curiously, the ten-m gene is expressed in an embryonic pair-rule pattern, and ten-m mutants display pair-rule patterning defects. Since the protein influences expression of downstream genes, it must communicate its presence to the cell nucleus. However, it does not appear that integrin signal transduction is important in early embryonic segmentation. PS integrins are not strongly expressed at this time, and, more importantly, mutations in integrin subunit genes do not cause segmentation phenotypes (Graner, 1998 and references).

Ten-m is later localized (among other places) at muscle attachment sites, where integrins are known to accumulate. This localization of Ten-m in vivo, as well as the demonstration of TENM-RGD interactions with PS2 integrins in vitro, suggests that Ten-m may function with PS2 integrins in muscle attachment. Interestingly, the heparan sulfate-containing protein D-syndecan also localizes to muscle attachments, and Ten-m contains a consensus heparin-binding sequence near the RGD, suggesting the potential of a Ten-m-syndecan-integrin complex. Syndecan proteoglycans recently have been shown to be important for signal transduction in vertebrate cell focal adhesions (Graner, 1998 and references).

The available data, although very suggestive, do not demonstrate unequivocally that Ten-m serves as an integrin ligand at muscle attachment sites. However, other potential PS2 ligands, such as Tiggrin, also accumulate at muscle attachment sites, and genetic studies of tiggrin suggest considerable functional redundancy among the extracellular matrix components there. Because of this redundancy, a direct genetic demonstration of a role for Ten-m in muscle attachment may require simultaneous disruption of multiple genes encoding matrix proteins, and the early embryonic phenotype of ten-m mutants will further complicate such an analysis. One potential approach might be to demonstrate a dominant genetic effect of ten-m mutations in a background that has been sensitized for loss of function phenotypes by viable mutations in other genes that encode proteins important for muscle attachment or other integrin-dependent processes (Graner, 1998).

GENE STRUCTURE

Exons - six

PROTEIN STRUCTURE

Amino Acids - 2515

Structural Domains

The extracellular domain contains eight tenascin like EGF repeats. These are followed by a putative transmembrane region (Levine, 1994). The putative intracellular domain contains putative fibronectin domains (Baumgartner, 1994).

Ten-m possesses some, but not all, of the features common to most vertebrate tenascins. For example, Ten-m is a secreted glycoprotein with eight tenascin-type EGF-like repeats and putative fibronectin-type III repeats (Baumgartner, 1994). Ten-m lacks a tenascin C-terminal fibrinogen-like domain, and the Ten-m RGD sequence is found 72 amino acids from the C terminus. Recombinant protein fragments containing this RGD sequence promote RGD-dependent, PS2 integrin-mediated cell spreading better with cells expressing the PS2C splice variant than with cells expressing the PS2m8 variant (Graner, 1998).

Levine (1994) reported a partial cDNA sequence from the ten-m gene; this partial sequence stops short of the final 325 amino acids and thus does not include the RGD tripeptide near the C terminus; it also includes 216 N-terminal residues not reported by Baumgartner. Levine ascribed properties to the presumed polypeptide that are significantly different from those deduced by Baumgarter; for example, Levine suggests that Odd Oz is a transmembrane phosphoprotein with tenascin homology in its putative extracellular domain, and the earlier study also proposes that the polypeptide is cleaved into smaller mature proteins. These apparent discrepancies have yet to be resolved, and it is possible that the protein functions in different forms. In any case, Baumgartner found that a Ten-m polypeptide could be found in conditioned media from Drosophila cells, and so a secreted form is present in at least some instances (Graner, 1998).

date revised:  13 September 97

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