Cloning and characterization of GUKHolder, a novel synaptically expressed protein that interacts with Discs -Large and SCRIBBLE at the Drosophila neuromuscular junction
Synaptic transmission between a neuron and its target is crucially dependent upon the precise spatial arrangement of proteins in the pre- and postsynaptic apparatus. PDZ domain-containing proteins such as the Drosophila tumor suppressor Discs-Large (DLG) play critical roles in synapse maturation by regulating the assembly of synaptic protein complexes.
DLG is composed of a number of modular domains, including three PDZ-domains, an SH3 domain and an enzymatically inactive Guanylate Kinase-like (GUK) domain. Previous studies have shown that the PDZ domains of DLG mediate clustering of Shaker K+ channels and of the cell adhesion molecule Fasciclin II. However, the function of the GUK domain has been unclear.
To understand the role of the GUK domain, we carried out a yeast-two hybrid screen for interacting partners of the DLG GUK domain. This screen lead to the identification of a novel synapse-associated protein, GUKHolder (GUKH).
GUKH is a 1044 amino acid protein with a molecular weight of 110 kDa. Its sequence includes a GUK-holding domain, a region homologous to the C-terminal of the long isoform of Kelch, a WH1-like domain, and a PDZ-domain binding motif. These latter two features suggest that GUKH may interact not only with DLG, but also with other proteins, including proteins containing PDZ domains.
GUKH is expressed at the larval neuromuscular junction and at epithelial cell borders in partial colocalization with DLG. Further, DLG can be co-immunoprecipitated with GUKH from Drosophila extracts, indicating an in vivo interaction between the two proteins. GUKH has also been shown to interact directly with SCRIBBLE (SCRIB), another synaptically expressed PDZ-domain protein known to have a genetic interaction with dlg in epithelial tissues. Synaptic SCRIB immunoreactivity is mislocalized in both gukh and dlg mutants. gukh, scrib, and dlg mutants all exhibit synaptic bouton defects at the ultrastructural level.
These data indicate that all three proteins are required for proper synapse maturation, and support a model that the three proteins exist in a tripartite complex, with GUKH forming a link between the other two proteins, and further, between the protein scaffolds organized by the two proteins.