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PSD-95 bridges channels with components of the Ras pathway

The PSD-95/SAP90 family of proteins has recently been implicated in the organization of synaptic structure. A novel Ras-GTPase activating protein, SynGAP, has been isolated that interacts with the PDZ domains of PSD-95 and SAP102 in vitro and in vivo. SynGAP is selectively expressed in brain and is highly enriched at excitatory synapses, where it is present in a large macromolecular complex with PSD-95 and the NMDA receptor. SynGAP stimulates the GTPase activity of Ras, suggesting that it negatively regulates Ras activity at excitatory synapses. Ras signaling at the postsynaptic membrane may be involved in the modulation of excitatory synaptic transmission by NMDA receptors and neurotrophins. These results indicate that SynGAP may play an important role in the modulation of synaptic plasticity (Kim, 1998).

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, both p135 SynGAP (an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. SynGAP, a Ras GTPase activating protein, is nearly as abundant in the PSD fraction as PSD-95 itself. SynGAP can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the PSD fraction and its GAP activity is reduced after phosphorylation. Thus, SynGAP and CaM kinase II constitute a signal transduction complex associated with the NMDA receptor. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus (Zhang, 1999).

The results presented here support the notion that differential expression of PSD-95-binding proteins in different neurons helps to determine the composition of signal transduction complexes formed by association with PSD-95 at glutamatergic PSDs. The resulting distinct compositions of these complexes will likely define the nature of local biochemical signaling associated with activation of NMDA receptors. The selective localization of citron suggests that, in hippocampus, PSDs of glutamatergic synapses made onto inhibitory interneurons contain cytoskeletal regulatory machinery that is not present at glutamatergic synapses made onto excitatory principal neurons. Furthermore, CaM kinase II is not detectable in these same PSDs but is present in the postsynaptic complex of excitatory synapses made onto glutamatergic neurons in the hippocampus. CaM kinase II can phosphorylate and regulate the GluRA/1 subunit of AMPA-type glutamate receptors and the synaptic Ras GTPase-activating protein SynGAP and can phosphorylate the NR2A and NR2B subunits of the NMDA receptor. This regulation by CaM kinase II is absent from the postsynaptic side of glutamatergic synapses on hippocampal inhibitory neurons. Thus, the modes of regulation of synaptic structure (by citron) and of synaptic strength (by CaM kinase II or citron) at glutamatergic synapses will differ dramatically between excitatory and inhibitory neurons. High citron expression found only in GABAergic neurons appears to be a unique feature of the hippocampus. In other brain regions, such as the thalamus and cerebral cortex, citron and CaM kinase II are often found together in excitatory neurons. Thus, the composition of signal transduction machinery at the postsynaptic membrane of glutamatergic synapses varies among neurons throughout the brain in ways that cannot be classified simply. Furthermore, findings regarding the mechanisms of signal transduction and plasticity at hippocampal synapses may not always generalize to synapses in other areas of the brain (Zhang, 1999).

Proteins of the membrane-associated guanylate kinase family play an important role in the anchoring and clustering of neurotransmitter receptors in the postsynaptic density (PSD) at many central synapses. However, relatively little is known about how these multifunctional scaffold proteins might provide a privileged site for activity- and cell type-dependent specification of the postsynaptic signaling machinery. Classically, the Rho signaling pathway has been implicated in mechanisms of axonal outgrowth, dendrogenesis, and cell migration during neural development, but its contribution remains unclear at the synapses in the mature CNS. Evidence is presented that Citron, a Rho-effector in the brain, is enriched in the PSD fraction and interacts with PSD-95/synapse-associated protein (SAP)-90 both in vivo and in vitro. Citron colocalization with PSD-95 occurs, not exclusively but certainly, at glutamatergic synapses in a limited set of neurons, such as the thalamic excitatory neurons; Citron expression, however, cannot be detected in the principal neurons of the hippocampus and the cerebellum in the adult mouse brain. In a heterologous system, Citron was shown to form a heteromeric complex not only with PSD-95 but also with NMDA receptors. Thus, Citron-PSD-95/SAP-90 interaction may provide a region- and cell type-specific link between the Rho signaling cascade and the synaptic NMDA receptor complex (Furuyashiki, 1999).

PSD-95 and Nitric oxide synthase

Interaction of PDZ-containing domains mediates synaptic association of Neuronal nitric oxide synthase (nNOS) (See Drosophila Nitric oxide synthase). nNOS is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. The N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in PSD-95 protein and a related protein PSD93. nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interaction also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin associated protein. nNOS isoforms lacking a PDZ domain, do not asssociate with PSD-95 in brain or with skeletal muscle sarcolemma (Brenman, 1996).

The efficiency with which N-methyl-D-aspartate receptors (NMDARs) trigger intracellular signaling pathways governs neuronal plasticity, development, senescence, and disease. Excessive Ca influx triggers excitotoxicity, damaging neurons in diverse neurological disorders. Rapid Ca2+-dependent neurotoxicity is triggered most efficiently when Ca2+ influx occurs through NMDARs, and cannot be reproduced by loading neurons with equivalent quantities of Ca2+ through non-NMDARs or voltage-sensitive Ca2+ channels (VSCCs). This suggests that Ca2+ influx through NMDAR channels is functionally coupled to neurotoxic signaling pathways. In cultured cortical neurons, suppressing the expression of the NMDAR scaffolding protein PSD-95 (postsynaptic density-95) selectively attenuates excitotoxicity triggered via NMDARs, but not by other glutamate or calcium ion (Ca2+) channels. NMDAR function is unaffected, because receptor expression, NMDA currents, and 45Ca2+ loading are unchanged. Suppressing PSD-95 blocks Ca2+-activated nitric oxide production by NMDARs selectively, without affecting neuronal nitric oxide synthase expression or function. Thus, PSD-95 is required for efficient coupling of NMDAR activity to nitric oxide toxicity, and imparts specificity to excitotoxic Ca2+ signaling. This raises the possibility that the preferential activation of neurotoxic Ca2+ signals by NMDARs is determined by the distinctiveness of NMDAR-bound MAGUKs, or of the intracellular proteins that they bind (Sattler, 1999).

ZO-1 and ZO-2 - Proteins associated with the cytoplasmic surfaces of the zonula occludens or tight junction

Tight junctions form an intercellular barrier between epithelial cells, serve to separate tissue compartments, and maintain cellular polarity. Paracellular sealing properties vary among cell types and are regulated by undefined mechanisms. Sequence of the full-length cDNA for human ZO-1 (Drosophila homolog: polychaetoid), the first identified tight junction component, predicts a protein of 1736 aa. The N-terminal 793 aa domain is homologous to the product of DLG and to PSD-95, a 95-kDa protein located in the postsynaptic densities of rat brain.  All three proteins contain both an src homology 3 region (SH3 domain), previously identified in membrane proteins involved in signal transduction, and a region homologous to guanylate kinase. ZO-1 contains an additional 943-aa C-terminal domain that is proline-rich (14.1%) and contains an alternatively spliced domain, whose expression was previously shown to correlate with variable properties of tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions (Willott, 1993).

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. Individual components of the tight junction assemble in a temporal sequence, with membrane assembly of the cytoplasmic plaque protein ZO-1 occurring 12 hours before that of cingulin, a 140 kDa cytoplasmic constituent of junctions. Subsequently, two alternatively spliced isoforms of ZO-1 (alpha+ and alpha-), differing in the presence or absence of an 80 residue alpha domain associate with the junction. The temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1alpha- mRNA is present in oocytes and all preimplantation stages, whilst ZO-1alpha+ transcripts are first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms are detected in trophectoderm and ICM cells. Immunoprecipitation of 35S-labelled embryos also shows synthesis of ZO-1alpha- throughout cleavage, whereas synthesis of ZO-1alpha+ is only apparent from the blastocyst stage. In addition, both isoforms are phosphorylated at the early blastocyst stage (Sheth, 1997).

The pattern and timing of membrane assembly of the two isoforms is also distinct. ZO-1alpha- is initially seen in punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesce laterally along the membrane until by the late morula stage they completely surround each cell with a zonular belt. ZO-1alpha+ however, is first seen as perinuclear foci in late morulae, before assembling at the tight junction. Membrane assembly of ZO-1alpha+ first occurs during the 32-cell stage and is zonular just prior to the early blastocyst stage. Both isoforms are restricted to the trophectoderm lineage. Membrane assembly of ZO-1alpha+ and blastocoele formation are sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. The tight junction transmembrane protein occludin co-localizes with ZO-1alpha+ at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. The late expression of ZO-1alpha+ and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo (Sheth, 1997).

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) were introduced into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 are concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 is specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/alpha, beta catenin complex. In contrast, C-ZO-1 is localized along actin stress fibers. Recombinant N-ZO-1 can bind directly to alpha catenin, but not to beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin is approximately 0.5 nM. On the other hand, recombinant C-ZO-1 cosediments with actin filaments in vitro, with a dissociation constant of approximately 10 nM. The cadherin-based cell adhesion activity of NZ-EL cells was compared with that of parent EL cells. Cell aggregation assay reveals no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, is significantly suppressed in NZ-EL cells. It is concluded that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with alpha catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system (Itoh, 1997).

Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated by growth factors and oncogenes, although the underlying mechanisms are poorly understood because cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signaling motifs. A yeast two-hybrid protein interaction screen has been used to identify proteins that bind to the carboxy-terminal tail of Cx43; in this way, the zona occludens-1 (ZO-1) protein was isolated. ZO-1 is a 220 kDa peripheral membrane protein containing multiple protein interaction domains including three PDZ domains and a Src homology 3 (SH3) domain. The interaction of Cx43 with ZO-1 occurs through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associates with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy reveals that endogenous Cx43 and ZO-1 colocalize at gap junctions. It is suggested that ZO-1 serves to recruit signaling proteins into Cx43-based gap junctions (Giepmans, 1998).

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2; adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras (see Drosophila Ras) results in the perturbation of cell-cell contacts. The ALL-1 fusion partner from chromosome 6 (AF-6) has been identified as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. The roles of Ras and AF-6 were investigated in the regulation of cell-cell contacts. AF-6 accumulates at the cell-cell contact sites of polarized MDCKII epithelial cells and has a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy reveals a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 interacts with the Ras-binding domain of AF-6; this interaction was inhibited by activated Ras. AF-6 accumulates with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells results in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras (Yamamoto, 1997).

Mammalian Protein kinase C (See Drosophila PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibit development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, is paralleled by a delay in the sorting of the tight-junction protein, Zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction is not detectably affected. ZO-1 is phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevents approximately 85% of the phosphorylation increase. In vitro measurements indicate that ZO-1 may be a direct target of PKC. Membrane-associated PKC activity more than doubles during junction assembly, and immunocytochemical analysis reveals a pool of PKC zeta that appears to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins is detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may well turn out to be occludin. Neither the mass of this complex nor the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were PKC dependent (Stuart, 1995).

The glucocorticoid and transforming growth factor-alpha (Drosophila homolog: Spitz) regulation of growth and cell-cell contact was investigated in a mammary epithelial tumor cell line. In cell monolayers, dexamethasone coordinately suppresses DNA synthesis, stimulates monolayer transepithelial electrical resistance (TER), and decreases the paracellular leakage of inulin or mannitol across the monolayer. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevents the glucocorticoid growth suppression response and disrupts tight junction formation without affecting glucocorticoid responsiveness. DNA synthesis is not a requirement for the growth factor disruption of tight junctions. The ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells; TGF-alpha causes ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, these results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha can override both effects of glucocorticoids. These results have uncovered a novel functional "cross-talk" between glucocorticoids and TGF-alpha, which potentially regulates the proliferation and differentiation of mammary epithelial cells (Buse, 1995).

Under certain conditions ZO-1 can be detected in the nucleus. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells. ZO-1 can be found in nuclei of intestinal epithelial cells only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. The nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homolog signal transduction (Gottardi, 1996).

ZO-1 is a 210-225-kDa peripheral membrane protein associated with the cytoplasmic surfaces of the zonula occludens or tight junction. A 160-kD polypeptide, designated ZO-2, has been found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations. ZO-2 was isolated by bulk coimmunoprecipitation with ZO-1. ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase-like domains that are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, DLG, and a synapse-associated protein from rat brain, PSD-95/SAP90. A polyclonal antiserum, raised against a unique region of ZO-2, exclusively labels the cytoplasmic surfaces of tight junctions, indicating that ZO-2 is a tight junction-associated protein. ZO-2 localizes to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Heart, a non-epithelial tissue, shows ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes, previously shown to contain ZO-1. Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction (Jesaitis, 1994)

The epithelial character of neuroepithelial cells was investigated in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, is detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappears from all neuroepithelial cells. Correspondingly, functional tight junctions are present in the neural plate (Embryonic Day 8), but not in the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increases from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincides with that of N-cadherin (see Drosophila Cadherin-N), whose expression increases concomitantly with the disappearance of occludin. It is proposed that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons (Aaku-Saraste, 1996).

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. Identified was a single open reading frame of 2,694 bp, coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. This protein contains three PDZ domains, and in addition a src homology (SH3) domain and a region similar to guanylate kinase, making it homologous to ZO-1 and ZO-2, as well as the Discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct express the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. This protein colocalizes with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrate that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. It is proposed that this protein be named ZO-3 (Haskins, 1998).

At tight junctions (TJs), claudins with four transmembrane domains are incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs to the immunoglobulin superfamily, is also localized at TJs, but it remains unclear how JAM is integrated into TJs. Immunoreplica electron microscopy has revealed that JAM shows an intimate spatial relationship with TJ strands in epithelial cells. In L fibroblasts expressing exogenous JAM, JAM is concentrated at cell-cell adhesion sites, where there are no strand-like structures, but rather characteristic membrane domains free of intramembranous particles are detected. These domains are specifically labeled with anti-JAM polyclonal antibody, suggesting that JAM forms planar aggregates through their lateral self-association. Immunofluorescence microscopy and in vitro binding assays have revealed that ZO-1 directly binds to the COOH termini of claudins and JAM at its ZO-1's PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing polarity-related protein, PAR-3, is directly bound to the COOH terminus of JAM, but not to that of claudins. These findings led to a molecular architectural model for TJs: small aggregates of JAM are tethered to claudin-based strands through ZO-1, and these JAM aggregates recruit PAR-3 to TJs (Itoh, 2001).

Table of contents

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

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