Spatial distribution of mDLG6 mRNA in embryonic and adult mouse brain

We have isolated mouse DLG6 (mDLG6) cDNA clones by RT-PCR and then by using the RT-PCR products to screen a mouse brain cDNA library. The deduced amino acid sequence of mDLG6 shows 79.2% and 82.7% overall identity to human (hDLG6) and rat DLG6 (rDLG6), respectively. In situ hybridization revealed that mDLG6 mRNA is predominantly expressed in embryonic and adult brain.     

The PDZ protein discs-large (DLG): the 'Jekyll and Hyde' of the epithelial polarity proteins

Discs-large (DLG) is a multi-PDZ domain-containing protein that belongs to the family of molecular scaffolding proteins known as membrane guanylate kinases or MAGUKs. DLG is a component of the Scribble polarity complex and genetic analyses of DLG in Drosophila have identified a role for the protein in several key biological processes including the regulation of apico-basal polarity of epithelial cells, as well as other polarity processes such as asymmetric cell division and cell invasion. Disturbance of DLG function leads to uncontrolled epithelial cell proliferation and neoplastic transformation, thereby defining DLG as a potential tumour suppressor. However, whether mammalian homologues of DLG (DLG1, DLG2, DLG3 and DLG4) also possess tumour suppressor functions is not known. In this minireview, we focus on the biological functions of DLG1 in human epithelial cells and on how the function of this MAGUK relates to its intracellular location. We examine some of the evidence that implies that DLG has both tumour suppressor and, paradoxically, oncogenic functions depending upon the precise cellular context.     

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.     

Synaptic targeting and localization of discs-large is a stepwise process controlled by different domains of the protein

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs) assemble ion channels, cell-adhesion molecules and components of second messenger cascades into synapses, and are therefore potentially important for co-ordinating synaptic strength and structure. Here, we have examined the targeting of the Drosophila MAGUK Discs-large (DLG) to larval neuromuscular junctions. RESULTS: During development, DLG was first found associated with the muscle subcortical compartment and plasma membrane, and later was recruited to the postsynaptic membrane. Using a transgenic approach, we studied how mutations in various domains of the DLGprotein affect DLG targeting. Deletion of the HOOK region-the region between the Src homology 3 (SH3) domain and the guanylate-kinase-like (GUK) domain-prevented association of DLG with the subcortical network and rendered the protein largely diffuse. Loss of the first two PDZ domains led to the formation of large clusters throughout the plasma membrane, with scant targeting to the neuromuscular junction. Proper trafficking of DLG missing the GUK domain depended on the presence of endogenous DLG. CONCLUSIONS: 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.     

Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.

Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding.     

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.     

DLG differentially localizes Shaker K+-channels in the central nervous system and retina of Drosophila

Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs-Large (DLG), a member of the MAGUK (membrane-associated guanylate kinases) family in Drosophila, co-localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C-terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C-terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C-terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Sh channels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C-terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types.     

A functional interaction between CD46 and DLG4: a role for DLG4 in epithelial polarization.

Using a yeast two-hybrid screen, we identified a physical interaction between CD46 and DLG4. CD46 is a ubiquitous human cell-surface receptor for the complement components C3b and C4b and for measles virus and human herpesvirus 6. DLG4 is a scaffold protein important for neuronal signaling and is homologous to the Drosophila tumor suppressor DLG. We show that an interaction between CD46 and DLG4 is important for polarization in epithelial cells. Specifically, we show (i) biochemical evidence for an interaction between CD46 and DLG4, (ii) that this interaction is specific for the Cyt1 (but not Cyt2) domain of CD46, (iii) that both CD46 and an alternatively spliced isoform of DLG4 are polarized in normal human epithelial cells, and (iv) that the polarized expression of CD46 in epithelial cells requires the DLG4-binding domain and alters with expression of a truncated form of DLG4. This is the first identification of a direct and cytoplasmic domain-specific interaction between CD46 and an intracellular signaling molecule and provides a molecular mechanism for the polarization of CD46. These data also indicate that, in addition to the known role for DLG4 in neuronal cells, DLG4 may be important for polarization in epithelial cells.     

Cloning and expression of mars, a novel member of the guanylate kinase associated protein family in Drosophila

The Membrane-Associated Guanylate Kinase (MAGUK) family of anchor proteins are involved in organising a range of molecules such as cell adhesion molecules, receptors, and intracellular signalling molecules at cell junctions. In mammals, the PSD-95/SAP-90/hDlg class of MAGUK proteins bind to a family of Guanylate Kinase Associated Proteins (GKAPs) that have been found at presumptive synaptic sites in neurons. Here we describe the identification of Mars, a novel Drosophila protein belonging to the GKAP family. RT-PCR analysis reveals that Drosophila mars mRNA and protein are predominantly expressed in embryos and in the adult germline. In embryos, mars is expressed in central nervous system and brain, as determined by RNA in situ hybridisation. In testes, mars is strongly expressed in pre-meiotic germ cells, but is not found in somatic or post-meiotic cells, indicating that in addition to their role in neuronal cells, GKAP proteins are also likely to play a role in germline development.     

E6AP-dependent degradation of DLG4/PSD95 by high-risk human papillomavirus type 18 E6 protein

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.     

Binding of the mammalian homolog of the Drosophila discs large tumor suppressor protein to the ribosome receptor

DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.     

Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis

Epithelial tubes are the functional units of many organs, but little is known about how tube sizes are established. Using the Drosophila tracheal system as a model, we previously showed that mutations in varicose (vari) cause tubes to become elongated without increasing cell number. Here we show vari is required for accumulation of the tracheal size-control proteins Vermiform and Serpentine in the tracheal lumen. We also show that vari is an essential septate junction (SJ) gene encoding a membrane associated guanylate kinase (MAGUK). In vivo analyses of domains important for MAGUK scaffolding functions demonstrate that while the Vari HOOK domain is essential, the L27 domain is dispensable. Phylogenetic analyses reveal that Vari helps define a new MAGUK subgroup that includes mammalian PALS2. Importantly, both Vari and PALS2 are basolateral, and the interaction of Vari with the cell-adhesion protein Neurexin IV parallels the interaction of PALS2 and another cell-adhesion protein, Necl-2. Vari therefore bolsters the similarity between Drosophila and vertebrate epithelial basolateral regions, which had previously been limited to the common basolateral localization of Scrib, Dlg and Lgl, proteins required for epithelial polarization at the beginning of embryogenesis. However, by contrast to Scrib, Dlg and Lgl, Vari is not required for cell polarity but rather is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from sharing only polarity complexes to sharing both polarity and cell-adhesion complexes.     

CASK Deletion in Intestinal Epithelia Causes Mislocalization of LIN7C and the DLG1/Scrib Polarity Complex without Affecting Cell Polarity

CASK is the mammalian ortholog of LIN2, a component of the LIN2/7/10 protein complex that targets epidermal growth factor receptor (EGFR) to basolateral membranes in Caenorhabditis elegans. A member of the MAGUK family of scaffolding proteins, CASK resides at basolateral membranes in polarized epithelia. Its interaction with LIN7 is evolutionarily conserved. In addition, CASK forms a complex with another MAGUK, the DLG1 tumor suppressor. Although complete knockout of CASK is lethal, the gene is X-linked, enabling us to generate heterozygous female adults that are mosaic for its expression. We also generated intestine-specific CASK knockout mice. Immunofluorescence analysis revealed that in intestine, CASK is not required for epithelial polarity or differentiation but is necessary for the basolateral localization of DLG1 and LIN7C. However, the subcellular distributions of DLG1 and LIN7C are independent of CASK in the stomach. Moreover, CASK and LIN7C show normal localization in dlg1^−/− intestine. Despite the disappearance of basolateral LIN7C in CASK-deficient intestinal crypts, this epithelium retains normal localization of LIN7A/B, EGFR and ErbB-2. Finally, crypt-to-villus migration rates are unchanged in CASK-deficient intestinal epithelium. Thus, CASK expression and the appropriate localization of DLG1 are not essential for either epithelial polarity or intestinal homeostasis in vivo.     

Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.     

Formation of complexes between Ca2+.calmodulin and the synapse-associated protein SAP97 requires the SH3 domain-guanylate kinase domain-connecting HOOK region

Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre- and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calcium-dependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K(d) of 122 nm for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.     

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.     

Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.     

Protein kinase C (PKC) isoforms in Drosophila

Six protein kinase C (PKC) genes are present in Drosophila, comprising two classical PKCs (PKC53E and eye-PKC), two novel PKCs (PKC98E and PKCdelta), an atypical PKC (DaPKC), and a PKC-related kinase. Loss of function alleles affecting DaPKC and eye-PKC are available and their mutant phenotypes have been characterized. DaPKC is essential for early embryonic development because it regulates cell polarity and asymmetric cell division. Eye-PKC plays a role in the regulation of visual signaling, a G-protein coupled phospholipase Cbeta-mediated cascade. Both eye-PKC and DaPKC are differentially localized through tethering to multimolecular complexes. DaPKC interacts with partitioning-defective 3 (Par-3) and Par-6 proteins, which contain PDZ (PSD95, DLG, ZO-1) domains. Similarly, eye-PKC is anchored to a PDZ domain containing scaffolding protein INAD. Characterization of these two PKCs in Drosophila revealed a universal mechanism by which PKC is tethered to specific protein complexes for participation in distinct signal transduction processes.