NORMAL GENOMIC

• McLaughlin M, Hale R, Ellston D, Gaudet S, Lue RA, Viel A
• The distribution and function of alternatively spliced insertions in hDlg.
• J Biol Chem. 2002; 277: 6406-12
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• hDlg is the human homolog of the Drosophila Discs-large tumor suppressor. As a member of the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins, hDlg is composed of three PDZ (PSD-95, Dlg, and ZO-1) repeats, an SH3 (Src homology 3) motif, and a GUK (guanylate kinase-like) domain. Additionally, hDlg contains two regions of alternative splicing. Here we identify a novel insertion, I1B, located N-terminal to the PDZ repeats. We further analyze the tissue-specific combinations of insertions and correlate those results with the distribution of protein isoforms. We also identify the functions of the two alternatively spliced regions. The N-terminal alternatively spliced region is capable of binding several SH3 domains and also moderates the level of protein oligomerization. Insertions in the second region are responsible for determining the localization of hDlg, with insertion I3 targeting the protein to the membrane regions of cell-cell contact and insertion I2 targeting the protein to the nucleus.

• Caruana G
• Genetic studies define MAGUK proteins as regulators of epithelial cell polarity.
• Int J Dev Biol. 2002; 46: 511-8
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• Polarized epithelial cells play critical roles during early embryonic development and organogenesis. Multi-domain scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) family are commonly found at the plasma membrane of polarized epithelial cells. Genetic studies in Drosophila melanogaster and Caenorhabditis elegans have revealed that MAGUK proteins regulate various aspects of the polarized epithelial phenotype, including cell junction assembly, targeting of proteins to the plasma membrane and the organisation of polarized signalling complexes. This review will focus on the genetic studies that have contributed to our understanding of the MAGUK family members, Dlg and Lin-2/CASK, in controlling these processes. In addition, our recent genetic analysis of mouse Dlg, in combination with genetic and biochemical studies of Lin-2/CASK by others suggests a model placing Dlg and Lin-2/CASK within the same developmental pathway.

• Mathew D et al.
• Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein.
• Curr Biol. 2002; 12: 531-9
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• 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.

• Laura RP, Ross S, Koeppen H, Lasky LA
• MAGI-1: a widely expressed, alternatively spliced tight junction protein.
• Exp Cell Res. 2002; 275: 155-70
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• Tight junctions are apically localized structures that regulate the passage of small molecules and proteins through intercellular regions of epithelial or endothelial cells. These structures are complex multimolecular assemblages that contain both transmembrane and membrane-associated proteins. MAGUKs (Membrane-Associated Guanylate Kinases) are a family of scaffolding proteins that contain multiple protein interaction domains, including PDZ, SH3, WW, and guanylate kinase motifs, and have been grouped into five discrete subfamilies based on homology. Little is known regarding the most recently described subfamily of MAGUKs, termed MAGIs (MAGUKS with Inverted domain structure). Here we show that two of the three known MAGI isoforms, MAGI-1 and MAGI-3, are present in the tight junctions of cultured epithelial cells. A broader examination of MAGI-1 expression in vivo shows that it is present in the tight junctions of all epithelial cell types examined. Human MAGI-1 transcripts are alternatively spliced at three sites, and two forms are expressed only in nonepithelial tissues, predominantly in brain. The major form that is expressed in cultured colon carcinoma epithelial cells, as well as several epithelial-rich tissues, contains an extended carboxy terminus encoding potential nuclear targeting signals. MAGI-1, ZO-1, and ZO-2 all colocalize in nonpolarized epithelial cells, suggesting that they form a preassembled complex that is incorporated into the tight junction upon polarization. Finally, all of the alternatively spliced forms of MAGI-1 show tight junction localization, and this localization occurs in the absence of the guanylate kinase and WW domains as well as the extended carboxy terminus.

• Hong Y, Stronach B, Perrimon N, Jan LY, Jan YN
• Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts.
• Nature. 2001; 414: 634-8
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• Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.

• Lopes C, Gassanova S, Delabar JM, Rachidi M
• The CASK/Lin-2 Drosophila homologue, Camguk, could play a role in epithelial patterning and in neuronal targeting.
• Biochem Biophys Res Commun. 2001; 284: 1004-10
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• Drosophila Camguk (Cmg) is a member of the CAMGUK subfamily of the MAGUK family of proteins which are localized at cell junction and other plasma membrane specialized regions, from worms to mammals. The protein structure of Cmg, as the other CAMGUK proteins, is characterized by only one PDZ domain and an additional CaM kinase domain, similar to CaMKII. While the mammalian ortholog CASKs play an important role in synaptic protein targeting and in synaptic plasticity, the Drosophila Cmg role is unknown. To study its potential role, we reported a detailed analysis of mRNA distribution of the Drosophila cmg gene at cellular and developmental level, during embryonic, larval, pupal and adult stages. The transient cmg transcription in midgut and Malpighian tubules may suggest a potential function in cell junction formation and in epithelial tissue patterning. Interestingly, cmg transcription increases substantially during embryonic neuroblast proliferation, becoming predominant in the developing central nervous system (CNS) during embryonic and postembryonic development stages and in the mature brain. In addition, a high transcriptional level was detected in the eye imaginal discs and in the adult retina, demonstrating a specific and continuous expression of cmg in neuroblasts and photoreceptor neurons, from the onset of cytodifferentiation. Our findings suggest that Cmg could play a potential role in transmembrane protein targeting, particularly in synapses. These observations suggest the existence of a common highly conserved mechanism involved in forming and maintaining proper synaptic protein targeting, which are fundamental features of synaptic plasticity, learning and memory. Through its function, the CaM kinase domain-containing Cmg may be involved in signal transduction cascade. Its potential relation to Calmodulin and CaMKII is discussed.

• Biederer T, Sudhof TC
• CASK and protein 4.1 support F-actin nucleation on neurexins.
• J Biol Chem. 2001; 276: 47869-76
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• Rearrangements of the actin cytoskeleton are involved in a variety of cellular processes from locomotion of cells to morphological alterations of the cell surface. One important question is how local interactions of cells with the extracellular space are translated into alterations of their membrane organization. To address this problem, we studied CASK, a member of the membrane-associated guanylate kinase homologues family of adaptor proteins. CASK has been shown to bind the erythrocyte isoform of protein 4.1, a class of proteins that promote formation of actin/spectrin microfilaments. In neurons, CASK also interacts via its PDZ domain with the cytosolic C termini of neurexins, neuron-specific cell-surface proteins. We now show that CASK binds a brain-enriched isoform of protein 4.1, and nucleates local assembly of actin/spectrin filaments. These interactions can be reconstituted on the cytosolic tail of neurexins. Furthermore, CASK can be recovered with actin filaments prepared from rat brain extracts, and neurexins are recruited together with CASK and protein 4.1 into these actin filaments. Thus, analogous to the PDZ-domain protein p55 and glycophorin C at the erythrocyte membrane, a similar complex comprising CASK and neurexins exists in neurons. Our data suggest that intercellular junctions formed by neurexins, such as junctions initiated by beta-neurexins with neuroligins, are at least partially coupled to the actin cytoskeleton via an interaction with CASK and protein 4.1.

• Mehta S, Wu H, Garner CC, Marshall J
• Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97.
• J Biol Chem. 2001; 276: 16092-9
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• Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization.

• Bachmann A, Schneider M, Theilenberg E, Grawe F, Knust E
• Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.
• Nature. 2001; 414: 638-43
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• The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.

• Wei X, Ellis HM
• Localization of the Drosophila MAGUK protein Polychaetoid is controlled by alternative splicing.
• Mech Dev. 2001; 100: 217-31
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• The Drosophila membrane-associated guanylate kinase (MAGUK) protein Polychaetoid (Pyd) is required for dorsal closure of the embryo, sensory organ patterning, and cell fate specification in the developing eye. We demonstrate that pyd is alternatively spliced resulting in two isoforms that differ by the presence or absence of exon 6. To determine the role of alternative splicing in Pyd function, we generated antibodies specific for each isoform. We find that the exon 6(+) form of Pyd is localized at adherens junctions of embryonic and imaginal epithelia, while the exon 6(-) form is distributed broadly along the lateral membrane. These results suggest that localization of Pyd is controlled by alternative splicing and raise the possibility that exon 6 represents a distinct protein-protein interaction domain.

• Firestein BL, Rongo C
• DLG-1 is a MAGUK similar to SAP97 and is required for adherens junction formation.
• Mol Biol Cell. 2001; 12: 3465-75
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• Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.

• Saito H et al.
• Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.
• J Biol Chem. 2001; 276: 32051-5
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• Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

• Tseng TC et al.
• VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.
• Biochim Biophys Acta. 2001; 1518: 249-59
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• The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

• DeMarco SJ, Strehler EE
• Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins.
• J Biol Chem. 2001; 276: 21594-600
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• Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.

• Thomas U et al.
• Synaptic targeting and localization of discs-large is a stepwise process controlled by different domains of the protein.
• Curr Biol. 2000; 10: 1108-17
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• 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.

• Tiffany AM, Manganas LN, Kim E, Hsueh YP, Sheng M, Trimmer JS
• PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering.
• J Cell Biol. 2000; 148: 147-58
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• Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.

• Klebes A, Knust E
• A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila.
• Curr Biol. 2000; 10: 76-85
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• BACKGROUND: Specialised cell junctions in epithelia serve as cell-cell adhesion sites and thus contribute to the maintenance of tissue integrity. The Drosophila gene crumbs encodes a transmembrane protein that is required for the biogenesis of the zonula adherens, a belt-like structure encircling the apex of epithelial cells. As previously shown, expression of just the short membrane-bound cytoplasmic domain is sufficient to rescue major defects associated with the loss of crumbs function. RESULTS: The cytoplasmic domain of Crumbs is highly conserved in two putative crumbs homologues in Caenorhabditis elegans. To assess the significance of conserved residues, various point mutations and deletions were introduced into this region. Two functional domains were revealed, an amino-terminal region and the carboxy-terminal amino acids EERLI. Both are necessary for rescue of the crumbs phenotype. The EERLI motif interacts with Discs Lost, a cytoplasmic protein containing PDZ domains. Overexpression of the Crumbs cytoplasmic domain induces a transition from the single-layered epithelium to a multilayered tissue. This transition is associated with redistribution of the Drosophila homologue of the cell adhesion molecule E-cadherin, and depends on the presence of the EERLI motif. CONCLUSIONS: We propose a model in which the interaction of the Crumbs carboxyl terminus with Discs Lost organises a membrane-associated protein complex in the apical cytocortex of epithelial cells. This scaffold mediates the localisation and stabilisation of the zonula adherens component DE-cadherin, a crucial component for the maintenance of epithelial cell polarity and tissue integrity.

• Wu H et al.
• Intramolecular interactions regulate SAP97 binding to GKAP.
• EMBO J. 2000; 19: 5740-51
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• Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.

• Wu Y et al.
• Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase.
• J Biol Chem. 2000; 275: 21477-85
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• PTEN/MMAC is a phosphatase that is mutated in multiple human tumors. PTEN/MMAC dephosphorylates 3-phosphorylated phosphatidylinositol phosphates that activate AKT/protein kinase B (PKB) kinase activity. AKT/PKB is implicated in the inhibition of apoptosis, and cell lines and tumors with mutated PTEN/MMAC show increased AKT/PKB kinase activity and resistance to apoptosis. PTEN/MMAC contains a PDZ domain-binding site, and we show here that the phosphatase binds to a PDZ domain of membrane-associated guanylate kinase with inverted orientation (MAGI) 3, a novel inverted membrane-associated guanylate kinase that localizes to epithelial cell tight junctions. Importantly, MAGI3 and PTEN/MMAC cooperate to modulate the kinase activity of AKT/PKB. These data suggest that MAGI3 allows for the juxtaposition of PTEN/MMAC to phospholipid signaling pathways involved with cell survival.

• Fei K, Yan L, Zhang J, Sarras MP Jr
• Molecular and biological characterization of a zonula occludens-1 homologue in Hydra vulgaris, named HZO-1.
• Dev Genes Evol. 2000; 210: 611-6
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• Zonula occludens-1 (ZO-1) is one of the earliest identified molecular components of tight junctions. Sequence analysis has placed ZO-1 into the broader membrane-associated guanylate kinase (MAGUK) protein family that contains such diverse members as post-synaptic density 95 (PSD-95), Drosophila discs large tumor suppressor gene product (dlg-A), p55, and TamA. Studies in both vertebrates and invertebrates have established that the MAGUK family is involved in a wide variety of cellular functions. These functions involve the regulation of such cellular processes as: (1) tight junction formation, (2) cell proliferation, (3) cell differentiation, and (4) neuronal synapse transmission. Extending these studies, we report the presence of a ZO-1 homologue in Hydra vulgaris, a member of the Cnidaria, the second oldest phylum of the animal kingdom. Hydra ZO-1 (HZO-1) is encoded by a single messenger RNA (mRNA) of approximately 6.0 kb that contains an open reading frame of 5,085 bp. The 191 kDa predicted protein consists of a characteristic MAGUK domain structure, including three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a guanylate kinase (GUK) domain. Western blot analysis using an antibody generated from a synthetic peptide designed from the HZO-1 sequence confirmed the presence of a Hydra protein of the appropriate mass. While whole mount in situ hybridization determined that HZO-1 mRNA was expressed along the entire longitudinal axis of Hydra, cross-sectional analysis established that HZO-1 mRNA expression was restricted to the ectoderm or outer cell layer of the organism's epithelial bilayer. Consistent with this mRNA expression pattern, immunofluorescence studies localized HZO-1 protein to the apical plasma membrane of ectodermal cells. It is unclear what role HZ0-1 has in the cellular physiology of Hydra; however, immunolocalization studies indicate a conserved plasma membrane-associated function(s), as reported for its counterparts in other invertebrate and vertebrate species. These studies establish that the MAGUK family of proteins with a membrane-associated function arose early during metazoan evolution, even before the divergence of protostomes and deuterostomes.

• Nix SL, Chishti AH, Anderson JM, Walther Z
• hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions.
• J Biol Chem. 2000; 275: 41192-200
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• Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.

• Nishimura W, Iizuka T, Hirabayashi S, Tanaka N, Hata Y
• Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting mediated by the carboxyl-terminal PDZ domains.
• J Cell Physiol. 2000; 185: 358-65
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• Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (GK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form tight junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and GK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1.

• Doerks T, Bork P, Kamberov E, Makarova O, Muecke S, Margolis B
• L27, a novel heterodimerization domain in receptor targeting proteins Lin-2 and Lin-7.
• Trends Biochem Sci. 2000; 25: 317-8

• Kamberov E et al.
• Molecular cloning and characterization of Pals, proteins associated with mLin-7.
• J Biol Chem. 2000; 275: 11425-31
• Display abstract

• In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.

• Ide N et al.
• Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells.
• Oncogene. 1999; 18: 7810-5
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• Membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaffolding molecule (S-SCAM) and Postsynaptic density (PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaffolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clarified. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diffusely at cell - cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rac1 small G protein changed the amounts of SAP97/hDLG at cell - cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These findings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is different from that of SAP97/hDLG.

• Perego C et al.
• PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells.
• EMBO J. 1999; 18: 2384-93
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• The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial gamma-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.

• McGee AW, Bredt DS
• Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.
• J Biol Chem. 1999; 274: 17431-6
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• 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.

• Bhat MA, Izaddoost S, Lu Y, Cho KO, Choi KW, Bellen HJ
• Discs Lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity.
• Cell. 1999; 96: 833-45
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• Polarization of epithelial cells depends on a hierarchical process whereby specific membrane-associated proteins become targeted to specialized membrane domains. Here, we describe a novel Drosophila protein, Discs Lost (DLT), that plays a crucial role in the polarization of embryonic epithelia during cellular blastoderm formation. At subsequent stages of development, DLT interacts with the apical determinant Crumbs (CRB) and the laterally localized protein Neurexin IV (NRX IV). Mutations in dlt or double-stranded RNA interference lead to aberrant localization of CRB and NRX IV and cause a concomitant loss of epithelial cell polarity. Hence, DLT is required to establish and maintain cell polarity and participates in different molecular complexes that define apical and lateral membrane domains.

• Fanning AS, Anderson JM
• PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane.
• J Clin Invest. 1999; 103: 767-72

• Brenman JE et al.
• Localization of postsynaptic density-93 to dendritic microtubules and interaction with microtubule-associated protein 1A.
• J Neurosci. 1998; 18: 8805-13
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• Postsynaptic density-93 (PSD-93)/Chapsyn-110 is a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ domain-containing proteins. MAGUKs are widely expressed in the brain and are critical elements of the cytoskeleton and of certain synapses. In the ultrastructural studies that are described here, PSD-93 localizes to both postsynaptic densities and dendritic microtubules of cerebellar Purkinje neurons. The microtubule localization is paralleled by a high-affinity in vivo interaction of PSD-93 via its guanylate kinase (GK) domain with microtubule-associated protein 1A (MAP1A). GK domain truncations that mimic genetically identified mutations of a Drosophila MAGUK, discs-large, disrupt the GK/MAP-1A interaction. Additional biochemical experiments demonstrate that intact MAGUKs do not bind to MAP1A as effectively as do isolated GK domains. This appears to be attributable to an intramolecular inhibition of the GK domain by the PDZs, because GK binding activity of full-length MAGUKs is partially restored by a variety of PDZ ligands, including the C termini of NMDA receptor 2B, adenomatous polyposis coli (APC), and CRIPT. Beyond demonstrating a novel cytoskeletal link for PSD-93, these experiments support a model in which intramolecular interactions between the multiple domains of MAGUKs regulate intermolecular associations and thereby may play a role in the proper targeting and function of MAGUK proteins.

• Dimitratos SD, Woods DF, Bryant PJ
• Camguk, Lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains.
• Mech Dev. 1997; 63: 127-30
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• MAGUKs (membrane-associated guanylate kinase homologs) are proteins involved in cell junction organization, tumor suppression, and signalling. Their structure includes one or three copies of a DHR or PDZ domain (discs-large homologous region or PSD-95/SAP90, discs-large ZO-1 homologous domain), an SH3 domain, and a guanylate kinase domain. MAGUKs were 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; we report its sequence and domain structure.

• Lin L, Sahr KE, Chishti AH
• Identification of the mouse homologue of human discs large and rat SAP97 genes.
• Biochim Biophys Acta. 1997; 1362: 1-5
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• The human homologue of the Drosophila discs large (dlg) tumor suppressor gene encodes a 926 amino acid protein, hDlg, which is a member of the MAGUK (Membrane Associated GUanylate Kinase homologues) family of proteins. To facilitate the development of murine model system for functional studies in vivo, the primary structure of the mouse homologue of hDlg has been determined. Dlgh1 encodes a approximately 5.5 kb transcript that is ubiquitously expressed in murine tissues. Mouse mDlg is a 927 amino acid protein that is 95% identical to hDlg and 94% identical to rat synapse associated protein, SAP97. The unusually high conservation of the primary structure of murine and human Dlg proteins across their distinct protein domains suggests a conserved function in vivo.

• Dobrosotskaya I, Guy RK, James GL
• MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains.
• J Biol Chem. 1997; 272: 31589-97
• Display abstract

• Membrane-associated guanylate kinase (MAGUK) proteins participate in the assembly of multiprotein complexes on the inner surface of the plasma membrane at regions of cell-cell contact. MAGUKs are characterized by three types of protein-protein interaction modules: the PDZ domain, the Src homology 3 (SH3) domain, and the guanylate kinase (GuK) domain. The arrangement of these domains is conserved in all previously known MAGUKs: either one or three PDZ domains in the NH2-terminal half, followed by the SH3 domain, followed by a COOH-terminal GuK domain. In this report, we describe the cDNA cloning and subcellular distribution of MAGI-1, a MAGUK with three unique structural features: 1) the GuK domain is at the NH2 terminus, 2) the SH3 domain is replaced by two WW domains, and 3) it contains five PDZ domains. MAGI-1 mRNA was detected in several adult mouse tissues. Sequence analysis of overlapping cDNAs revealed the existence of three splice variants that are predicted to encode MAGI-1 proteins with different COOH termini. The longest variant, MAGI-1c, contains three bipartite nuclear localization signals in its unique COOH-terminal sequence and was found predominantly in the nucleus of Madin-Darby canine kidney cells. A shorter form lacking these signals was found primarily in membrane and cytoplasmic fractions. This distribution, which is reminiscent of that seen for the tight junction protein ZO-1, suggests that MAGI-1 may participate in the transmission of regulatory signals from the cell surface to the nucleus.

• Beatch M, Jesaitis LA, Gallin WJ, Goodenough DA, Stevenson BR
• The tight junction protein ZO-2 contains three PDZ (PSD-95/Discs-Large/ZO-1) domains and an alternatively spliced region.
• J Biol Chem. 1996; 271: 25723-6
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• The complete cDNA sequence for canine ZO-2, a tight junction-specific protein, is presented. A single open reading frame encodes a polypeptide of 1,174 amino acids with a predicted molecular mass of 132,085 daltons. As noted previously (), ZO-2 is a member of the membrane-associated guanylate kinase-containing (MAGUK) protein family, a family which includes an additional tight junction-associated protein, ZO-1. These proteins contain a region homologous to guanylate kinase, an SH3 domain, and variable numbers of PSD-95/discs-large/ZO-1 (PDZ) domains, shown to be involved in protein-protein interactions. ZO-2 and ZO-1 contain three PDZ domains in the N-terminal half of the molecule. Between the first and second PDZ domains, ZO-2 displays a basic region (pI = 10.27) containing 22% arginine residues. Both ZO-1 and ZO-2 have proline-rich C-terminal regions that are not homologous to other MAGUK family members. Sequence analysis of multiple ZO-2 cDNAs reveals a 36-amino acid domain in this C-terminal region present in only some of the cDNAs. Overall, ZO-2 is highly homologous to ZO-1, showing 51% amino acid identity; however, the C-terminal ends of the molecules show only 25% amino acid identity. This suggests that the C-terminal ends of ZO-1 and ZO-2 have different functions.

• Niethammer M, Kim E, Sheng M
• Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases.
• J Neurosci. 1996; 16: 2157-63
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• Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.

• Hata Y, Butz S, Sudhof TC
• CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins.
• J Neurosci. 1996; 16: 2488-94
• Display abstract

• Neurexins are neuronal cell surface proteins with hundreds of isoforms. In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, we identified a single interacting protein called CASK. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 and that contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in brain in synaptic plasma membranes but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca2+ - activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins.