discs large 1


Table of contents

Drosophila Discs large homologs

Other proteins containing the disc large motif are present in Drosophila: Dishevelled, Canoe, INAD and Camguk. INAD, a novel protein mutated in the inactivation no afterpotential D mutation in Drosophila, is a PDZ domain protein, sharing a protein interaction domain with Drosophila proteins Discs large, Dishevelled and Canoe. canoe interacts genetically with Notch and scabrous during eye, bristle and wing development, suggesting that canoe works in conjunction with sca and N in the morphogenesis of these tissues. CNO is a 1893-amino-acid protein with the GLGF/DHR motif. This motif is a conserved sequence in Discs large, Dishevelled, and some other proteins associated with cellular junctions. Since there appears to be a direct physical interaction between Notch receptor and Dishevelled, providing a link between Notch and wingless signaling, perhaps Canoe plays a role in modifying this interaction (Miyamoto, 1995).

MAGUKs have been classified into two subfamilies: Dlg-like with three DHR/PDZ domains and p55-like with a single DHR/PDZ domain. There is now a new subfamily whose members have a novel domain structure: a calcium/calmodulin-dependent protein kinase domain in the N-terminus as well as the DHR/PDZ, SH3 and GUK domains in the C-terminus. These new MAGUKs may regulate transmembrane molecules that bind calcium, calmodulin, or nucleotides. camguk (cmg) is a Drosophila member of this novel MAGUK subfamily. The C-terminal domain of Camguk shows a strong resemblance to p55, with a single DHR domain, an SH3 domain, and a GUK domain with an intact ATP binding site. The DHR domain is 54% identical to that of human p55. At the N-terminus the sequence predicts a domain with striking similarity to calcium/calmodulin-dependent protein kinase (CaM kinase, including the entire putative catalytic domain (37% identity with that of Drosophila CaM kinase type IIbeta). The cmg locus is the same gene as caki previously thought to encode only a brain-specific CaM kinase type II. There are two reported homologs of Camguk: C. elegans lin-2 and rat CASK (Dimitratos, 1997).

The adenomatous polyposis coli gene (APC: see Drosophila APC-like is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, its product binding to the adherens junction protein Beta-catenin. Overexpression of APC blocks cell cycle progression. The APC-beta-catenin complex has been shown to bind to DLG, the mammalian homolog of the Drosophila Discs large tumor suppressor protein. This interaction required both the carboxyl-terminal region of APC and the DLG homology repeat region of mammalian DLG. APC colocalizes with DLG at the lateral cytoplasm in colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the mammalian APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function (Matsumine, 1996).

Invertebrate Discs large homologs and adherins junctions

The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. dlg-1, the C. elegans homolog of the Drosophila tumor-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localization of the Drosophila and vertebrate homologs in septate and tight junctions, respectively. Proper localization of DLG-1 requires the basolateral LET-413 protein (identified as the Drosophila scrib ortholog), but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity is eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localization of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homolog of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organization of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organization of a protein scaffold that controls the assembly of junction components into the zonula adherens (Bossinger, 2001).

How epithelial cell fates become specified is poorly understood. The putative C2H2 zinc-finger transcription factor LIN-26 is required for the differentiation of ectodermal and mesodermal epithelial cells in C. elegans. Ectopic LIN-26 expression during early gastrulation transforms most blastomeres into epithelial-like cells. Specifically, LIN-26 induces the expression of three epithelial markers: the adherens junction protein JAM-1; DLG-1, which is essential for the assembly of JAM-1 at junctions, and CHE-14, which is involved in apical trafficking. Furthermore, ultrastructural studies have revealed that ectopic LIN-26 expression induces the formation of adherens-like junctions. However, ectopic lin-26 expression does not confer any tissue-specific cell fate, such as the epidermal cell fate, as evidenced from the observation that several epidermal-specific genes are not induced. Conversely, epidermal cells display some polarity defects in lin-26 mutants. It is concluded that lin-26 can induce epithelial differentiation and that epitheliogenesis is not a default pathway in C. elegans (Quintin, 2001).

In addition to its maintenance function, lin-26 could act in a redundant manner to induce jam-1, dlg-1, and che-14 expression during epithelial differentiation. Forced expression of the essential GATA factor elt-1 and of the dispensable GATA factor elt-3 can turn on jam-1 expression in most cells, in a manner very similar to that observed with lin-26. A noteworthy difference between elt-1 and elt-3 in contrast to lin-26 is that forced expression of elt-1 or elt-3 also induces the expression of the epidermal collagen gene dpy-7. Thus, a plausible model for the formation of the epidermis in C. elegans is the following. Initially, elt-1 specifies tissue identity and turns on lin-26, elt-3, and possibly the other epidermal-specific GATA factors elt-5 and elt-6. In turn, elt-1, lin-26, and elt-3 act redundantly to induce epithelial-specific genes (jam-1, dlg-1, and che-14), while elt-1 and elt-3 act redundantly to switch on epidermal-specific genes (collagens). By extension, in other tissues where it is expressed, lin-26 could act redundantly to promote epithelial differentiation, while other regulatory genes would confer tissue identity and dictate the type of epithelium being made. A comparable scheme has been described for the genetic hierarchy controlling formation of the intestinal tube-shaped epithelium. First, the GATA factor end-1 together with at least one other gene specifies intestine identity and turns on the GATA factor elt-2, which is essential for intestine differentiation, and for its function during postembryonic development. Both end-1 and elt-2 then act in a redundant manner to turn on several intestinal genes, including jam-1 and the gut esterase gene ges-1. Although ELT-2 can bind the ges-1 promoter in vitro and induce ges-1 expression in vivo if expressed ectopically, ges-1 is normally expressed in elt-2-null mutants (Quintin, 2001 and references therein).

Various vertebrate Discs large homologs and their binding partners

Biology of mammalian ZO1, ZO2 and ZO3, are reviewed at the Polychaetoid site.

Human DLG2 maps distal to BRCA1 at 17q12-q21. The total sequence predicts a protein of 576 amino acids with three conserved regions: a 90-amino-acid repeat domain, a SH3 (src homology region 3) motif, and a guanylate kinase domain. These conserved regions are shared among members of the discs-large family of proteins, including human p55 (a membrane protein expressed in erythrocytes), rat PSD-95/SAP90 (a synapse protein expressed in brain), Drosophila DLG (a septate junction protein expressed in various epithelia), and human and mouse ZO-1 and canine ZO-2, two tight junction proteins. Since allelic loss has been reported in the 17q12-q21 region in breast and ovarian cancer and it appears that BRCA1 is not the target of the losses, the presence of somatic alterations in DLG2 was examined in sporadic breast tumors. No evidence for mutation was found, making it unlikely that DLG2 is involved in sporadic breast cancer (Mazoyer, 1995).

In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). These E6 proteins can bind to the second PDZ domain of the human homolog of the Drosophila Discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers (Kiyono, 1997).

In CNS synapses, the synaptic junctional complex with postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. A prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to DLG tumor suppressor protein. The sequence similarity between DLG and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain (Cho, 1992).

The synapse-associated protein SAP97 is a member of a novel family of cortical cytoskeletal proteins involved in the localization of ion channels at such membrane specializations as synaptic junctions. These multidomain proteins have binding sites for protein 4.1, GKAPs/SAPAPs, voltage- and ligand-gated ion channels and cell-adhesion molecules containing C-terminal T/SXV motifs. An evaluation was carried out of the contribution of individual domains in SAP97 to its selective recruitment and attachment to the cortical cytoskeleton in epithelial cells. The PDZ, SH3 and GK domains, as well as the I3 insert in SAP97, are not essential for subcellular targeting, though both PDZ1-2 domains and the I3 insert affect the efficiency of localization. Instead, the first 65 amino acid residues in SAP97, which are absent from SAP90/PSD-95 and SAP102, direct the selective subcellular localization and can mediate at least one point of attachment for SAP97 to the cytoskeleton that is assembled at sites of cell-cell contact. These data demonstrate that it is the sequences unique to SAP97 that direct its subcellular targeting to the epithelial lateral membrane (Wu, 1998).

The PSD-95/SAP90 family of PDZ-containing proteins is directly involved in the clustering of specific ion channels at synapses. Channel clustering depends on a conserved N-terminal domain of PSD-95 that mediates multimerization and disulfide linkage of PSD-95 protomers. This N-terminal multimerization domain confers channel clustering activity on a single PDZ domain. Channel clustering depends on the aggregation of PDZ domains achieved by head-to-head multimerization of PSD-95, rather than by concatenation of PDZ domains in PSD-95 monomers. This mechanism predicts that PSD-95 can organize heterogeneous membrane protein clusters via differential binding specificities of its three PDZ domains. PSD-95 and its relative chapsyn-110 exist as disulfide-linked complexes in rat brain, consistent with head-to-head multimerization of these proteins in vivo (Hsueh, 1997).

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 binds to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins (Irie, 1997).

Ion channels and associated signal transduction cascades are clustered at excitatory synapses by PSD-95 and related PDZ-containing proteins. Mechanisms that target PSD-95 to synaptic membranes, however, are unknown. Here, PSD-95 is shown to partition in brain homogenates as an integral membrane protein. Metabolic labeling of brain slices or cultured cells demonstrates that PSD-95 is modified by thioester-linked palmitate, a long chain fatty acid that targets proteins to cell membranes. In fact, PSD-95 is a major palmitoylated protein in intact cells, and palmitoylated PSD-95 partitions exclusively with cell membranes. Mutagenesis indicates that palmitoylation of PSD-95 occurs on conserved N-terminal cysteines 3 and 5. Palmitoylation-deficient mutants of PSD-95 do not partition as integral membrane proteins and do not participate in PDZ-ion channel interactions in vivo. This work identifies palmitoylation as a critical regulatory mechanism for receptor interactions with PSD-95 (Topinka, 1998).

The PSD-95 family of PSD-95/Discs large/ZO-1 (PDZ) domain-containing proteins plays a role in the clustering and localization of specific ion channels and receptors at synapses. Previous studies have shown that PSD-95 forms multimers through an N-terminal region (termed the N-segment) and that the multimerization of PSD-95 is critical for its ability to cluster Shaker-type potassium channel Kv1.4 in heterologous cells. The PSD-95 N-segment has been shown to function as a multimerization domain only when located at the N-terminal end of a heterologous protein. A pair of N-terminal cysteines, Cys3 and Cys5, is essential for the ability of PSD-95 to self-associate and to form cell surface clusters with Kv1.4. However, PSD-95 mutants lacking these cysteine residues retain their ability to associate with membranes and to bind to Kv1.4. Unlike wild type PSD-95, the cysteine mutant of PSD-95 cannot form a ternary complex with Kv1.4 and the cell adhesion molecule Fasciclin II. These results suggest that the N-terminal cysteines are essential for PSD-95 multimerization and that multimerization is required for simultaneous binding of multiple membrane protein ligands by PSD-95 (Hsueh, 1999).

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS, see Drosophila NOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ-PDZ domain interaction between PSD95 and nNOS. A novel nNOS-associated protein, CAPON, is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes (Jaffrey. 1998).

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 is most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, known as the rod spherules and cone pedicles respectively, are strongly labeled. Electron microscopy, using preembedding immunocytochemistry, shows PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling is also present in the inner plexiform layer (IPL). It has a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy shows that PSD-95 is concentrated in processes that are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad is labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for either SAP 102 and PSD-93, two other members of the family of synapse-associated proteins. All three are found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 are found presynaptically, whereas SAP 102 is located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They are found to be colocalized in synaptic hot spots in the IPL (Koulen, 1998).

The glutamate receptor subunit delta2 has a unique distribution at the parallel fiber-Purkinje cell synapse of the cerebellum, which is developmentally regulated such that delta2 occurs at both parallel fiber synapses and climbing fiber synapses early in development but is restricted to parallel fiber synapses in adult animals. To identify proteins that might be involved in the trafficking or docking of delta2 receptors, a yeast two-hybrid library was screened with the cytosolic C terminus of delta2 and a member of the postsynaptic density (PSD)-95 family of proteins and isolated. Members of this family are known to interact with the extreme C termini of NMDA receptors. It was found that delta2 binds specifically to PSD-93, which is enriched in Purkinje cells. In addition, PSD-93 causes delta2 to cluster when they are coexpressed in heterologous cells, and clustering is disrupted by point mutations of delta2 that disrupt the delta2-PSD-93 interaction. Ultrastructural localization of PSD-93 and delta2 shows they are colocalize at parallel fiber synapses; however, PSD-93 is also present at climbing fiber synapses of the adult rat, in regions where delta2 is not found, indicating that the presence of PSD-93 alone is not sufficient for determining the synaptic expression of delta2 (Roche, 1999).

Chapsyn-110, a member of the membrane-associated putative guanylate kinase (MAGUK) family and related to Drosophila DLG, binds directly to the N-methyl-D-aspartate (NMDA) receptor and Shaker K+ channel subunits. In rat brain, chapsyn-100 protein shows a somatodendritic expression pattern that overlaps partly with PSD-95 but that contrasts with the axonal distribution of SAP97, other MAGUK proteins. Chapsyn-110 associates tightly with the postsynaptic density in brain, and mediates the clustering of both NMDA receptors and K+ channels in heterologous cells. Chapsyn-110 andd PSD-95 can heteromultimerize with each other and are recruited into the same NMDA receptor and K+ channel clusters. Thus, chapsyn-110 and PSD-95 may interact at postsynaptic sites to form a multimeric scaffold for the clustering of receptors, ion channels, and associated signalling proteins (Kim, 1996)

MAGUKs are diverse. Various neuronally expressed MAGUKs can bind via their first two PDZ domains to neuronal NO-synthase or to the cytoplasmic tails of NMDA-type glutamate receptors or to Shaker-type potassium channels: this binding may result in clustering of the respective membrane proteins. At tight junctions, ZO-1 associates with the transmembrane protein occludin and with ZO-2. Regulated interactions of ZO-1 with catenins have been shown to be involved in the assembly of tight junctions. In epithelia Drosophila Discs large 1 exerts its tumor suppressive function at septate junctions, which are thought to be functional analogs of vertebrate tight junctions. The similarity between ZO-1 and Discs large 1 supports this view. PSD-95/SAP90, SAP97/hdlg and SAP102 mammalian synapse-associated proteins are structurally more closely related to Discs large 1 than they are to ZO-1 or ZO-2, and DLG and SAP97/hdlg exhibit a widespread tissue distribution. SAP97 also binds to protein 4.1, an observation that parallels the co-localization of DLG and Coracle at septate junctions. In Drosophila synaptic bouton structure is severely affected in mutant larvae carrying mutant dlg. SAP97 and SAP102 were tested for functional homology to DLG by heterologous expression in the fly. SAP97 and SAP102 localize to subapical regions within the columnar epithelium of Drosophila imaginal discs. Both SAP97 and SAP102 can suppress tumor formation in dlg mutants; the two proteins mimic DLG at larval neuromuscular junctions. Neuronal expression of SAP97 or SAP102 is required for morphological restoration of synaptic boutons, indicating that presynaptic DLG is essential for establishing structurally intact motor nerve terminals at larval neuromuscular junctions. It is concluded that the synaptic function of DLG as well as its tumor suppressing function can be carried out by SAP97 or SAP102 (Thomas, 1997a).

The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. A novel cellular PDZ domain-containing protein, 9BP-1, binds to wild-type, but not to a transformation-defective, C-terminal mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contains such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. The 9ORF1 protein interacts directly with the PDZ domain-containing protein hDlg/SAP97 (hDlg), which is a mammalian homolog of the Drosophila Discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. In forming complexes, the 9ORF1 protein preferentially associates with the second PDZ domain of hDlg, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possess PDZ domain-binding motifs at their C termini. Significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bind hDlg in vitro. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins (Lee, 1997).

An oriented peptide library technique was used to investigate the peptide-binding specificities of nine PDZ domains from different sources. Each PDZ domain selects peptides with hydrophobic residues at the carboxyl terminus. Individual PDZ domains select unique optimal motifs defined primarily by the C-terminal three to seven residues of the peptides. One family of PDZ domains, including those of the Drosophila Discs large protein, select peptides with one consensus motif; another family of PDZ domains, including those of LIN-2, p55 and Tiam-1, select another. The three PDZ domains of DLG recognize similar motifs, whereas PDZ domains of PTPbas recognize different motifs. On the basis of crystal structures of the mammalian PSD-95-3 PDZ domain, the specificities observed with the peptide library can be rationalized (Songyang, 1997).

A 90 kDa synapse-associated protein, SAP90, is localized at the presynaptic termini of inhibitory GABAergic synapses. SAP90 is a mosaic protein composed of three 90 amino acid residue repeats, an SH3 domain and a region homologous to guanylate kinases. SAP90 shares domain specific homology with DLG and related proteins. The further characterization of cDNA clones encoding components of synaptic junctions has lead to the identification of a 97 kDa protein, called SAP97, that exhibits a strong overall sequence similarity to SAP90. SAP97 is localized in the presynaptic nerve termini of excitatory synapses of the rat hippocampal formation. In other brain regions, SAP97 is found in and along bundles of unmyelinated axons. SAP97 is not restricted to the CNS; it is also present at the basal lateral membrane between a variety of epithelial cells. In cultured T84 cells it is restricted to the cytoplasmic surface of the plasma membranes between adjacent cells, but not at the edges of cells lacking cell-cell contact, suggesting a role for SAP97 in cell adhesion (Muller, 1995).

Postsynaptic density 95 (PSD-95/SAP-90) is a membrane associated guanylate kinase (GK) PDZ protein that scaffolds glutamate receptors and associated signaling networks at excitatory synapses. Affinity chromatography identifies cypin as a major PSD-95-binding protein in brain extracts. Cypin is homologous to a family of hydrolytic bacterial enzymes and shares some similarity with collapsin response mediator protein (CRMP), a cytoplasmic mediator of semaphorin III signalling. Cypin is discretely expressed in neurons and is polarized to basal membranes in intestinal epithelial cells. Overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD-95 and SAP-102, an effect not produced by overexpression of other PDZ ligands. In fact, PSD-95 can induce postsynaptic clustering of an otherwise diffusely localized K+ channel, Kv1.4. By regulating postsynaptic protein sorting, cypin may influence synaptic development and plasticity (Firestein, 1999).

Two-hybrid searches with the tumor suppressor MMAC1/PTEN (see Drosophila Pten) have isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibits both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and 200,000 proteins, but promotes coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions (Adey, 2000).

hDlg, the human homolog of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, a 322-aa serine/threonine kinase has been identified that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T/SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T/SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2/cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation (Gaudet, 2000).

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. A novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. It is proposed that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo (Hanada, 2000).

Membrane-associated guanylate kinase homologues (MAGUKs) are generally found under the plasma membrane of cell-cell contact sites and function as scaffolding proteins by linking cytoskeletal and signaling molecules to transmembrane receptors. The correct targeting of MAGUKs is essential for their receptor-clustering function; however, the molecular mechanism of their intracellular transport is unknown. The guanylate kinase-like domain of human discs large protein has been shown to bind directly within the amino acids 607-831 of the stalk domain of GAKIN, a kinesin-like protein of broad distribution. The primary structure of the binding segment, termed MAGUK binding stalk domain, is conserved in Drosophila kinesin-73 and some other motor and non-motor proteins. This stalk segment is not found in GKAP, a synaptic protein that interacts with the guanylate kinase-like domain, and unlike GKAP, the binding of GAKIN is not regulated by the intramolecular interactions within the discs large protein. The recombinant motor domain of GAKIN is an active microtubule-stimulated ATPase. Overexpression of green fluorescent protein-fused GAKIN in Madin-Darby canine kidney epithelial cells induced long projections with both GAKIN and endogenous discs large accumulating at the tip of these projections. Importantly, the accumulation of endogenous discs large was eliminated when a mutant GAKIN lacking its motor domain was overexpressed under similar conditions. Taken together, these results indicate that discs large is a cargo molecule of GAKIN and suggest a mechanism for intracellular trafficking of MAGUK-laden vesicles to specialized membrane sites in mammalian cells (Asaba, 2003).

Discs large homologs: Structural studies

Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or inter-molecular fashion to complete the SH3 fold. A model for MAGUK oligomerization is proposed in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization (McGee, 2001).

Assembly of ordered structures such as the PSD requires interaction among many proteins. A fundamental question is how these interactions are coordinated and regulated to achieve spatial and temporal specificity. Why do these scaffolds only assemble at the correct membrane sites? Previous studies have suggested that the intermolecular SH3-GK interactions may contribute to MAGUK scaffolding, and that this process may be regulated by extrinsic factors. Using the structure of the PSD-95 SH3-GK module, this model has been refined by proposing that the intermolecular interactions observed for MAGUKs are mediated by 3D domain swapping of structural components of the split SH3 fold. It is hypothesized that ligand binding may constrain the flexibility of the hinge region, thereby promoting the switch from intra- to inter-molecular assembly (McGee, 2001).

This model offers potential advantages as a scaffolding mechanism: (1) because 3D domain swapping regenerates complete folds, this mechanism facilitates oligomerization without occluding sites on the SH3 and GK folds that may bind associated signaling proteins; (2) regulatory proteins with the appropriate subcellular localization could direct the correct temporal and spatial assembly of interlocked MAGUK networks; (3) heteromeric 3D domain swapping of MAGUKs, perhaps directed by sets of regulatory proteins, could provide combinatorial scaffold diversity, which could specify differential protein recruitment. This model of regulated assembly is consistent with the function of MAGUK proteins and is one of several mechanisms that may participate in the proper assembly of supramolecular signaling complexes at cell junctions. The structure of the SH3-GK module reveals how a simple, modular protein fold, such as the SH3 fold, can be used in diverse modes to mediate protein complex assembly. This module provides one of the first structural examples in which normally discrete protein domains have evolved into an integrated functional unit (McGee, 2001).

PSD-95/SAP90 is a member of the MAGUK superfamily. In excitatory synapses, PSD-95 clusters receptors and ion channels at specific sites in the postsynaptic membrane and organizes downstream signaling and cytoskeletal molecules. The crystal structures have been determined of the apo and GMP-bound forms to 2.3 and 2.0 Å resolutions, respectively, of a fragment containing the SH3, HOOK, and guanylate kinase (GK) domains of PSD-95. An intramolecular interaction is observed between the SH3 and GK domains involving the formation of a sheet including residues N- and C-terminal to the GK domain. Based on amino acid conservation and mutational data available in the literature, it is proposed that this intramolecular interaction is a common feature among MAGUK proteins (Travares, 2001).

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. This study identifies palmitate cycling on PSD-95 at the synapse; palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. Rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity (El-Husseini, 2002).

Mutation of Discs large homologs

The characterization of the zebrafish homolog of the human gene DLG3 is described. The zebrafish dlg3 gene encodes a membrane-associated guanylate kinase containing a single PDZ domain. This gene was cloned using a gene-trap construct inserted in the gene's first intron. The insertion co-segregates with a viable mutation called humpback (hmp), which leads to formation of ankylotic vertebrae in adult fish. Insertion and mutation have both been mapped to chromosome 12, in a segment that is syntenic with region p12 to q12 of human chromosome 17. The hmp mutant phenotype, however, appears to be due to two point mutations in the guanylate kinase domain rather than to the transgene insertion itself. The results of this study are discussed in the light of the possible function of the guanylate kinase domain (König, 1999).

Dlgh1 (discs large homolog 1) is a mammalian homolog of the Drosophila tumor suppressor Discs large 1, and is a member of the membrane-associated guanylate kinase (MAGUK) scaffolding proteins that contain three PSD-95/Dlg/ZO-1 (PDZ) domains. Discs large 1 is involved in epithelial polarization and cell-cell adhesion complex formation during Drosophila development. However, the functions of Dlgh1 during mammalian development remain to be elucidated. Dlgh1-knockout mice were generated and it was found that homozygous Dlgh1-knockout mice developed various abnormalities in their renal and urogenital organs. The kidneys and ureters were hypoplastic and the lower ends of the ureters were ectopic. In addition, the vagina and seminal vesicle, which are derived from the lower part of the Müllerian and Wolffian duct, respectively, were absent. Unexpectedly, loss of Dlgh1 function in the developing ureters did not disrupt cell-cell junctional complexes, but did impair cellular proliferation in the epithelium. These results suggest a novel role for Dlgh1 in regulating epithelial duct formation and morphogenesis during mammalian development. Although congenital absence of the vagina associated with other variable Müllerian duct abnormalities has been reported in humans, its mechanism has not yet been clarified. These findings might contribute to a better understanding of such abnormalities (Iizuka-Kongo, 2007).

Epithelial tubes represent fundamental building blocks of metazoan organisms; however, the mechanisms responsible for their formation and maintenance are not well understood. This study shows that the evolutionarily conserved coiled-coil MAGUK protein Dlg5 is required for epithelial tube maintenance in mammalian brain and kidneys. Dlg5-/- mice develop fully penetrant hydrocephalus and kidney cysts caused by a deficiency in membrane delivery of cadherin-catenin adhesion complexes and loss of cell polarity. Dlg5 travels with cadherin-containing vesicles and binds to syntaxin 4, a t-SNARE protein that regulates fusion of transport vesicles with the lateral membrane domain. It is proposed that Dlg5 functions in plasma membrane delivery of cadherins by linking cadherin-containing transport vesicles with the t-SNARE targeting complex. These findings show that Dlg5 is causally involved in hydrocephalus and renal cysts and reveal that targeted membrane delivery of cadherin-catenin adhesion complexes is critical for cell polarity and epithelial tube maintenance (Nechiporuk, 2007).

Transcriptional regulation of PSD-95

Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. In the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD-Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of gamma-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD-Eos complex provides a molecular basis for activity-dependent synaptic plasticity (Bao, 2004).

DHHC proteins are PSD-95 palmitoylating enzymes

Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast (Roth, 2002). Twenty three mammalian DHHC proteins have been isolated and a subset were found to specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galphas, or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduces palmitoylation and synaptic clustering of PSD-95 and diminishes AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation (Fukata, 2004).

Regulation of subcellular distribution of Discs large homologs

Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, has been identified as a physiological substrate for the p38gamma MAP kinase (SAPK3/p38gamma) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38gamma-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress (Sabio, 2005).

Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. mPins (mammalian homologue of Drosophila Partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR - MAGUK (N-methyl-D-aspartate receptor - membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs (Sans, 2005).

mPins is a ubiquitously expressed protein that is critical for the regulation of mitotic spindle organization in dividing cells. mPins interacts with several functionally distinct proteins, including NuMA, Ras, LKB1 and Galphai. The finding that mPins interacts with the PSD-95 family adds another group of important proteins to those whose trafficking depends on mPins. Drosophila Pins is required for asymmetric division of sensory organ precursor cells (pI) and dividing neuroblasts. Whereas the roles of Pins in cell division are relatively well-characterized, the function of mPins in the mature mammalian central nervous system remains enigmatic. The related protein, AGS3, may affect cocaine-induced plasticity by regulating G-protein signalling in the prefrontal cortex. The data show that mPins and AGS3 are both expressed in the developing hippocampus but have different subcellular localizations, perhaps because mPins, but not AGS3, interacts with SAP102. Moreover, AGS3 is down-regulated in adult hippocampus and seems to be absent from the PSD, whereas mPins is expressed throughout development and is enriched in synaptic membranes. mPins and AGS3 are found in different domains throughout the cell body and dendrites in primary cultures of hippocampal neurons. mPins, but not AGS3, redistributes into punctate structures after ionomycin or NMDA treatment, suggesting that calcium signalling functions in trafficking of mPins complexes. These findings strongly suggest that these two orthologues of Drosophila Pins have different functions in neurons (Sans, 2005).

The MAGUKs do not compete with the other known interacting proteins of mPins suggesting that the association of these other interacting proteins may indirectly influence the trafficking of the MAGUK and its associated proteins, such as NMDARs. Both Ras and Galphai are particularly interesting in this context. Ras has been implicated in the trafficking of GluRs. Characterized as molecular switches that alternate between GTP-bound ('on') and GDP-bound ('off') forms, these proteins are involved in the reorganization of synaptic structure. G-proteins, such as Galphai, influence NMDAR trafficking through metabotropic GluRs. In this study, it is shown that Galphai proteins function in NMDAR trafficking through a direct interaction with the mPins-SAP102 complex. mPins mediates G-protein signalling through binding to Galphai1-3GDP, thereby inhibiting binding of Galphai to Gßγ (and consequently enhancing Gßγ signalling in the absence of a G-protein-coupled receptor). mPins shifts between a closed state, when the N- and C-terminal halves of the protein bind to one another, and an open state when NuMA binds to mPins to switch it open, allowing the binding of Galphai. SAP102, similarly to SAP97, may exist in the cytoplasm as a folded molecule in which the GK domain is folded onto the SH3 domain. The data suggest that SAP102 binds to mPins in its closed state, as the two proteins localized in ring-like structures in COS cells. Therefore, mPins could be required upstream of, or in parallel to, the NR2B-SAP102 interaction. It is also shown that SAP102-mPins complexes have a different fate from that of NR2B-SAP102-mPins complexes, since the three proteins form clusters in COS cells and synaptic clusters in spines. These data suggest that NMDARs can open the SAP102-mPins complexes. Interestingly, cotransfection of the linker region of mPins with NR2B and SAP102 results in the formation of ternary complexes that are rapidly degraded, suggesting that interaction of Galphai with GoLoco domains (or an unidentified protein with TPR domains) is important for stabilization of the complex. mPins can bind four Galphai molecules, and it is unclear at present whether all of the sites need to be occupied for proper folding and targeting of mPins. As a modulator of G-protein signalling, the possibility cannot be excluded that Galphai binds to the NMDAR-MAGUK-mPins complex at synapses after activation of a G-protein-coupled receptor. Studies have suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) can exhibit some of their effects through interactions with heterotrimeric G-proteins in addition to their ionic channel function. For instance, it has been shown that AMPA can induce dissociation of Galphai1 from the Galphai1/ß heterotrimeric complex and its association with GluR1 through an adaptor protein. In light of the current data, the possibility exists that Galphai signalling proteins may also be recruited to certain MAGUK-mPins complexes through simultaneous dissociation from AMPARs (Sans, 2005).

The results suggest that the NMDAR associates indirectly through SAP102 with two molecular complexes -- the exocyst and mPins-Galphai complexes -- and that these associations are necessary for proper trafficking of receptors in neurons. The results also suggest that this complex is formed in the ER in heterologous cells and early in the secretory pathway in neurons. Although this has not been demonstrated directly for native proteins, an association of MAGUK with AMPARs in the ER (or cis-Golgi) has been shown for native AMPARs in brain by using the endo-H sensitivity of immature AMPARs, so such an association is not unprecedented. These results suggest that NMDARs are trafficked as part of a large complex from their site of synthesis in the cell body to the postsynaptic membrane, presumably in a transport vesicle. The identification of other components of the SAP102 cargo complex (containing NMDARs, the exocyst and mPins-Galphai complexes) will undoubtedly help to clarify the steps involved in trafficking of NMDARs from assembly and ER exit to transport in dendrites and spines in normal and disease states (Sans, 2005).

Table of contents

discs large 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.