BIOLOGICAL OVERVIEW

Discs large (DLG) is the prototypic member of a growing family of proteins collectively termed membrane-associated guanylate kinase homologs (MAGUKs). DLG is required for septate junction structure, cell polarity, and proliferation control in Drosophila epithelia. Two other Drosophila MAGUK proteins are Dishevelled (involved in segment polarity) and Canoe (involved in Notch signaling).

Other molecules found at septate junctions include the cell adhesion protein Fasciclin III, as well as Expanded, and Coracle. Both Expanded and Coracle are members of the 4.1 family of proteins that includes mammalian ezrin, radixin and moesin. The 4.1 family proteins physically bind cytoskeletal elements. The protein Neurexin is a transmembrane component of septate junctions interacting with Coracle. Neurexin is required for septate junction and blood-nerve barrier formation and function (Baumgartner, 1996).

All MAGUKs contain a series of domains: either one or three copies of an 80-90 amino acid motif called DHR/PDZ (DLG Homologous Region/PSD-95), DLG, ZO-1, and a region with high similarity to guanylate kinases (GUK). Both the DHR/PDZ and the SH3 domains may serve as sites for protein-protein interactions. The subfamily of MAGUKs (DLG-R) includes DLG, as well as mammalian tight-junction proteins ZO-1 and ZO-2 (homologs of Drosophila Polychaetoid). In DLG-R family members there are three DHR/PDZ domains, each with a three-amino acid deficiency in the ATP binding site within the GUK, making unlikely the existence of guanylate kinase catalytic activity (Woods, 1996 and references).

Imaginal disc cells mutant for dlg lack apicobasal polarity and septate junctions. However, the adherens junctions (see Shotgun and Armadillo) are still present: in fact, adherens junctions can be found at various ectopic positions on mutant cell membranes. The dependence of septate junction structure on functional DLG is seen more clearly in the salivary gland, a differentiated epithelial tissue that grows during larval life by cell enlargement rather than by proliferation. Mutant salivary gland cells still have obvious apicobasal polarity, but the septate junctions (usually extending a long distance over the lateral cell membrane) are reduced to a small fraction of their normal length (Woods, 1996).

Both microfilament and microtubule networks are disrupted by loss of the DLG protein. Filamentous actin, enriched at the apical end of the cell in wild-type epithelia, is found throughout the cell in mutants. Organization of tubulin is also severly disrupted. After the loss of the DLG protein, septate junctions of imaginal discs and some larval epithelia show a drastic alterantion in distribution of Coracle and Expanded (two proteins of the band 4.1 family of membrane cytoskeletal proteins). Loss of the DLG protein results in the distribution of COR and EX throughout the cell. In proventriculus cells both COR and EX are normally highly localized at the apicolateral membrane, presumably at the position of the septate junctions. In mutants, nearly all the COR protein is lost rather than mislocalized (Woods, 1996).

Loss of Discs large also affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion. Fasciclin III is highly enriched at the septate junction and present in lower amounts in the lateral cell membrane, but is excluded from the adherens junction. Neuroglian is enriched at the apical end of the cell, reduced in the septate junction, and also reduced on the rest of the lateral cell membrane. Localization of FAS III and Neuroglian in both salivary glands and imaginal discs is dependent of DLG. When septate junctions are completely eliminated in dlg mutants, both proteins are found apparently unrestricted along the cell membrane. In fact Neuroglian appears to have an elevated level of expression compared with wild type, while FAS III levels are reduced (Woods, 1996).

The adherens junction has long been the darling of Drosophila research: one component (Armadillo) is a downstream target of wingless signals while another component (Shotgun, or Drosophila E-cadherin) is involved in homophylic cell adhesion. The septate junction of Drosophila, present in lieu of the tight junction of vertebrates, now rightly deserves more attention. Tight junctions in vertebrates are physically more closely associated with the adherens junction, but in Drosophila, septate junctions assume a more basolateral position. Five components are now associated with septate junctions, FAS III, Neuroglian, DLG, COR and EX. Mutation of DLG disrupts this association, suggesting a central role for DLG in tight junction stucture. DLG mutants show an enhanced level of disc cell proliferation, suggesting a role for the tight junction in regulation of cell growth (Woods, 1996). The recent finding that a vertebrate DLG homolog, ZO-1, undergoes nuclear localization at the site of wounding, suggests a role for MAGUK proteins in nuclear signaling (Gottardi, 1996).

An important function of MAGUK proteins appears to be conserved between flies and mammals. MAGUK proteins are present in synapses and are important in directing synapse structure. Chapsyn-110 and PSD-95, two mammalian MAGUK proteins, interact at postsynaptic sites to form a multimeric scaffold for the clustering of N-methyl-D-aspartate (NMDA) receptors and Shaker K+ channel subunits (Kim, 1996). Drosophila DLG is expressed at type I glutamatergic synapse of the neuromuscular junction and is associated with both presynaptic and postsynaptic membranes. Type Ib boutons are large and are filled with 40 nm clear vescles thought to contain glutamate. At the postsynaptic site, type Ib boutons are surrounded by an elaborate system of membranes, the subsynaptic reticulum (SSR), a postsynaptic specialization at these synapses. Mutations in dlg alter the expression of dlg and cause striking changes in the structure of the subsynaptic reticulum (Lahey, 1994).

The SSR consists of highly elaborated junctional membranes that surround Type I boutons. Hypomorphic mutations in dlg result in a poorly developed and much simpler SSR. Developmental studies show that in wild type, the surface of this postsynaptic structure increases (about 100-fold) as the target muscle becomes larger (over 100 times in volume) during larval growth (Guan, 1996). In the mutant, the SSR forms normally at initial larval stages, but fails to expand as target muscles grow. Postsynaptic DLG is able to substantially rescue the SSR phenotype. These results support the model that suggests that DLG is involved in adjusting the size of postsynaptic surfaces during development (Burdnik, 1996).

What is the presynaptic function of DLG? DLG is observed in the presynaptic cell prior to its expression in the postsynaptic cell (Guan, 1996). The expression of DLG in the presynaptic cell may target ion channels and other proteins to the presynaptic terminal and activate the release of an agrin-like molecule, which induces the synthesis of rapsyn-like synapse-orienting proteins, such as DLG in the postsynaptic cell. Mutation of dlg result in larger synaptic currents at fly neuromuscular junctions. The enlarged excitatory junctional current (EJC) amplitude in dlg mutants is caused by an increase in the number of vesicles released during each stimulus (quantal content). Rescue of the physiological defect is accomplished by presynaptic, but not postsynaptic targeted expression of dlg. Thus presynaptic dlg expression can rescue the neurotransmitter release phenotype. Additionally, and paradoxically, presynaptic dlg expression can rescue the postsynaptic SSR structure as well (Budnik, 1996).

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. The region between the Sh3 and GUK domains (HOOK, or I3) shows significant sequence similarity between several MAGUKs: in hDlg and p55, it has been shown to bind certain actin-associated proteins of the protein 4.1/ERM (Exrin, Radizin, Moesin) superfamily (Hough, 1997).

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. The functions of each domain of Dlg were tested 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 domain 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 (Hough, 1997).

A three-step model is proposed for Dlg localization to the septate junctions and binding of the proteins important for structure and growth regulation. Step one involves binding of the HOOK domain to a protein 4.1-like protein and membrane localization of Dlg. Step two, mediating Dlg clustering at septate junctions, involves transmembrane protein binding to PDZ1/2 and Step three involves stabilization and binding of proteins involved in additional functions, such as growth regulation. The relative order of these steps is unknown. The effect of dlg mutations may not be an indirect consequence of loss of junctional structure, but may reflect a direct role of the Dlg protein in the signaling events that control proliferation (Hough, 1997).

Structural Domains

DLG contains a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins (Woods, 1989 and 1991).

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the Guanylate kinase pattern.

date revised: 21 APR 97 

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