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The single transmembrane protein Gogo, which is expressed in photoreceptor cells, is required in the retina for axon-axon repulsive interactions and appropriate column and target layer selection in the optic lobe of the Drosophila brain. The evolutionary conservation of this molecule across different species implies a high functional relevance.

The protein structure of Gogo, comprising protein interaction domains present in other axon guidance receptors, strongly indicates a function as a receptor or cell adhesion molecule. Within the extracellular domain, two conserved regions were shown to be essential and sufficient for Gogo function in photoreceptor axons, the Tsp1 and the newly identified GOGO domain (Chapter 3.3). Both domains show the ability for protein interaction. Eight conserved cysteines within in the GOGO domain possibly form four disulfide-bonds to assemble immunoglobulin-like protein interaction domains (Takayanagi et al., 2006). Also, the Tsp1 domain shows the ability to interact with multiple cell-surface or extracellular proteins, including matrix glycoproteins and proteoglycans (Adams and Tucker, 2000).

So far, there is no evidence that Gogo’s extracellular domain is able to promote homophilic binding among Gogo proteins, as shown in S2 cell aggregation assays (chapter 3.6). In addition, homophilic interactions are neither required for the repulsion among R8 axons nor in axon-target interaction (chapter 3.6, 3.10). All results obtained suggest a heterotypic interaction, in which Gogo could act in response to an as yet unidentified ligand.

The strict requirement of the cytoplasmic domain, which was demonstrated in rescue experiments in two different stages during development, axon-axon interaction in larvae (chapter 3.10) and axon-target interaction in adults (chapter 3.3), argues against a merely adhesive role of Gogo. In contrast, it was shown for some adhesion molecules (such as N-Cad) that the cytoplasmic domain is not needed for homophilic adhesion and functionality. Therefore, their function can be achieved without intracellular signaling (Yonekura et al., 2007). The opposite is known for repulsive guidance receptors, such as Eph receptors or Dscam. Although the physical binding

Discussion

of the extracellular domains to their ligands can be achieved without the cytoplasmic domain, still the repulsive response triggered by these receptors strictly requires the cytoplasmic domain for intracellular signal transduction (Feldheim et al., 2004; Labrador et al., 1997; Matthews et al., 2007; Wojtowicz et al., 2004; Zhu et al., 2006). The requirement of Gogo’s cytoplasmic domain argues against a simple adhesive role and implies that cytoplasmic signaling is required for Gogo function. The lack of an intracellular catalytic domain (as for example a kinase domain) does not contradict intracellular signaling events, as shown for the well described family of Robo receptors. Similar to Gogo, Robo proteins contain a poorly conserved cytoplasmic domain without any obvious catalytic activity (Kidd et al., 1998). But they contain short conserved cytoplasmic sequence motifs, which are thought to be binding sites for various signaling proteins, as for example Abl/Ena/Vasp, Dock/Nck or the Rho GAPs (GTPase activating proteins) Vilse/crGAP (Bashaw et al., 2000; Fan et al., 2003; Hu et al., 2005; Lundstrom et al., 2004; Wong et al., 2001).

Similarly, there is no overall conservation within the Gogo cytoplasmic domain, except for a specific short motif, which is shared by all Gogo orthologues (chapter 3.2). The short sequence contains a highly conserved tripeptide motif, Tyr-Tyr-Asp (YYD) that may serve as a putative regulatory site through phosphorylation of the Tyrosine and/or protein interaction domain. It was shown that a similar tripeptide motif Asp-Arg-Tyr (DRY) is conserved in mammalian odorant receptors, which belong to the family of G protein coupled receptors (GPCRs). Olfactory sensory neurons expressing odorant receptors mutant for the DRY motif are deficient in both axon targeting and G protein coupling (Imai et al., 2006). In principle, it is feasible that the highly conserved Gogo tripeptide also holds the potential to trigger an intracellular signaling pathway which could even be conserved across different species. For this reason, it would be interesting to investigate a possible functional role of this short cytoplasmic motif. Rescue experiments using either deletions of the motif or site-directed mutagenesis manipulating the potential phosphorylation-sites could be applied in order to investigate this question.

Although Gogo expression was detected in brain neurons other than R cells by in situ and antibody staining (chapter 3.5), the gogo transheterozygous mutants were rescued by the exclusive Gogo expression in R neurons (chapter 3.6). R axon targeting is therefore not dependent on Gogo expression within the brain. This result not only excludes the possibility of homophilic axon-target interaction, but also shows the autonomous requirement in R cells. In addition, a cell-autonomous function of Gogo was shown for single axons in mosaic animals. Also in pupae, single gogo mutant R8 axons show defects and fail to extend into the medulla column (Figure 3-16). Vice versa, single isolated WT R8 axons, which are surrounded by misprojecting and stopping gogo mutant axons, constituting a reverse MARCM situation, correctly innervate the medulla (Figure 3-15B). All these examples nicely demonstrate that Gogo acts in a cell-autonomous manner in R8 axons.

Both lines of evidence, the autonomous function and the requirement of the cytoplasmic domain, provide a strong argument that Gogo could act as a novel receptor in axon guidance.

Discussion