BIOLOGICAL OVERVIEW

Toll is a transmembrane receptor and a member of the 12 gene dorsal group responsible for dorsoventral polarity in the fly. The ligand for Toll is Spätzle, and immediate targets include Pelle, Tube, Dorsal and Cactus. These proteins are held in a state of readiness while in the unfertilized egg. They are primed to carry out the transition from egg to zygote after fertilization occurs. Spätzle is available only in the ventral portion of the egg in the extracellular perivitelline space; when subjected to proteolysis, Spätzle becomes an active ligand for Toll.

Toll signals are first picked up by Tube, a protein in contact with the cell membrane. The signal is transduced to Pelle and subsequently to Cactus, which until its destruction holds Dorsal in the cytoplasm. With Cactus's bond destroyed, Dorsal enters the nucleus where it can serve as activator and repressor of genes involved in dorso-ventral polarity.

Toll is required zygotically in the development of a number of tissues, but Spätzle has not been documented as the ligand in these circumstances. Toll's mammalian homolog is Interleukin-1 (IL-1) receptor, involved in the activation of the immune response. Similarly, the Toll-Cactus-Dorsal system in the fly also activates an immune response.

The extracellular region of the Toll protein does not bear any similarity to the extracellular ligand-binding portion of the IL-1 receptor. Instead, it carries leucine rich repeats (LRR) that are found in molecules as diverse as proteoglycans, adhesion molecules, enzymes, tyrosine kinase receptors and G-protein coupled receptors. LRRs in the fly are found in Toll, Chaoptin and Connectin adhesion molecules and the Slit secreted protein. All these have roles in cell differentiation, morphogenetic events and the migration of cells and axons (Ollendorff, 1994).

The localized activation of Toll was first suggested based on the results of injection of wild-type cytoplasm into mutant flies. Wherever the Toll rescuing activity occurs defines the region from which ventral structures arise. Rescuing activity is not localized ventrally, but distributed uniformly in the wild-type embryo. This implies that the Toll gene product is normally present throughout the embryo but its activity is somehow restricted to ventral regions (Anderson, 1985 and Hashimoto, 1991). It is now understood that the ventral stimulation of Toll is caused by Spätzle, activated only on the ventral side of the egg (Morisato, 1994 and Roth, 1994).

Toll is dynamically expressed later in development by the embryonic musculature. Growth cones of RP3 and other motoneurons normally grow past muscle cells expressing Toll on their surface and innervate more distal muscle cells (muscles 6 and 7), which have down-regulated their Toll expression. The RP3 growth cone likely responds positively to Fasciclin III, an Ig-like cell adhesion molecule expressed on the target muscle cells, but still manages to avoid targeting errors in embryos lacking Fas III. Toll protein preferentially accumulates at muscle-muscle contact sites or "clefts," (the apposition between muscle cells). Later, the Toll-positive ventral muscle cells gradually lose Toll. Late-arriving growth cones innervate the clefts just as Toll expression becomes undetectable (Rose, 1997).

Reciprocal genetic manipulations of Toll proteins can produce reciprocal RP3 phenotypes. In Toll null mutants, the RP3 growth cone sometimes innervates the wrong muscle cells, including those that are normally Toll-positive. In contrast, heterochronic misexpression of Toll in the musculature leads to the same growth cone reaching its correct target region but delaying synaptic initiation. It is proposed that Toll acts locally to inhibit synaptogenesis of specific motoneuron growth cones and that both temporal and spatial control of Toll expression is crucial for its role in development (Rose, 1997).

The LRR (leucine-rich repeats) motif is shared by a number of other Drosophila cell surface molecules: Connectin, Chaoptin, 18-wheeler, Kekkon and Toll-like, as well as mammalian neural receptors, such as NLRR-1 and GARP. Structural analyses indicate that the LRR motifs could mediate protein-lipid as well as homophilic and heterophilic protein-protein interactions. The structurally related Connectin protein, when ectopically expressed in some of the ventral muscle cells, can function as a repulsive signal to motoneuron growth cone. All this evidence suggests a pivotal role for LRR proteins in axon guidance (Rose, 1997 and references).

GENE STRUCTURE

Bases in 5' UTR - 574

Bases in 3' UTR - 1257

PROTEIN STRUCTURE

Amino Acids - 1097

Structural Domains

A 5.3 kb poly(A)+ ovarian transcript of Toll was purified by hybrid selection with cloned DNA. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains two blocks (for a total of 15 repeats) of a 24 amino acid, leucine-rich sequence found in both human and yeast membrane proteins. The transmembrane domain is between residues 804 and 828. There are 17 potential glycosylation sites and 17 cysteine residues in 3 clusters (Hashimoto, 1988).

The Toll protein has sequences held in common with the human membrane receptor platelet glycoprotein 1b (Gp1b). These sequences in Toll form disulphide linked extracellular domains that are important for the binding of ligands in the perivitelline space of the embryo. Expression of Toll protein induced in a non-adhesive cell line promotes cellular adhesion, a property held in common with the related Drosophila glycoprotein Chaoptin. Toll protein in such aggregates accumulates at sites of cell-cell interaction, a characteristic displayed by other cellular adhesion molecules (Keith, 1990).

Unusual properties are found for a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide forms an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed. The finding that Toll-LRR forms intramolecular ß-sheet structures supports the view that LRRs can participate in protein-protein interactions and homotypic cellular adhesion. It could be that LRRs expressed on the cell surface are initially of disordered structure and that interactions with similarly disordered LRRs on an adjacent cell causes the formation of an extended and stable intermolecular ß structure. Such a mechansim could provide a molecular basis for cellular adhesion mediated by LRRs (Gay, 1991).

date revised: 3 July 97  

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