Mutations of the tumor suppressor gene discs-large (dlg) lead to postsynaptic structural defects. Here, we report that mutations in dlg also result in larger synaptic currents at fly neuromuscular junctions. By selectively targeting DLG protein to either muscles or motorneurons using Gal-4 enhancer trap lines, we were able to rescue substantially the reduced postsynaptic structure in mutants. Rescue of the physiological defect was accomplished by presynaptic, but not postsynaptic targeting, consistent with our finding that miniature excitatory junctional currents were not changed in dlg mutants. These results suggest that DLG functions in the regulation of neurotransmitter release and postsynaptic structure. We propose that DLG is an integral part of a mechanism by which changes in both neurotransmitter release and synapse structure are accomplished during development and plasticity.

Abstract. The Discs large (Dig) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate k_inase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dig protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dig locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dig protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dig protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Discs large (Dlg) was the first identified member of an increasingly important class of proteins called membrane-associated guanylate kinase homologs (MAGUKs), which are often concentrated at cell junctions and contain distinct peptide domains named PDZ1-3, SH3, HOOK, and GUK. Dlg is localized at and required for the formation of both septate junctions in epithelial cells and synaptic junctions in neurons. In the absence of Dlg, epithelia lose their organization and overgrow. We tested the functions of each domain of Dlg in vivo by constructing transgenic flies expressing altered forms of the protein. In the first set of experiments each domain was examined for its ability to correctly target an epitope-tagged Dlg to pre-existing septate junctions. Based on these results the Hook domain is necessary for localization of the protein to the cell membrane and the PDZ2 is required for restricting the protein to the septate junction. In the second set of experiments each domain was tested for its role in growth regulation and organization of epithelial structure. These results show that PDZ1 and GUK are apparently dispensable for function, PDZ2 and PDZ3 are required for growth regulation but not for epithelial structure, and SH3 and HOOK are essential for both aspects of function. The results demonstrate the functional modularity of Dlg and clarify the functions of individual MAGUK domains in regulating the structure and growth of epithelial tissue.

Postsynaptic targeting of DLG requires a HOOK-dependent association with extrasynaptic compartments, and interactions mediated by the first two PDZ domains. The GUK domain routes DLG between compartments, possibly by interacting with recently identified cytoskeletal-binding partners.