MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis.

A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell-cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein-protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent L-leucyl-L-leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell-cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment.

From molecules to mammals: what's NOS got to do with it?

Nitric oxide synthases (NOSs) generate nitric oxide (NO) and the by-product l-citrulline, via the catalytic combination of l-arginine and molecular oxygen. In mammals, there are three NOS genes: nNOS (NOS1), iNOS (NOS2) and eNOS (NOS3). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important processes. The studies of NOS enzymes using knockout and transgenic mouse models have provided an invaluable contribution, highlighting critical roles in neuronal, renal, pulmonary, gastro-intestinal, skeletal muscle, reproductive and cardiovascular biology. This review will outline the data gleaned from complementary knockout and transgenic over-expression models in mice, and focus on the interactions between NOS enzymes and pathophysiology of the vascular system. These studies are a paradigm for the near future, which will involve the translation of an enormous amount of genomic data into physiological insights that penetrate the realms of both health care and biology.

Polarized targeting of peripheral membrane proteins in neurons.

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.

Electron microscopic immunocytochemical detection of PSD-95, PSD-93, SAP-102, and SAP-97 at postsynaptic, presynaptic, and nonsynaptic sites of adult and neonatal rat visual cortex.

Membrane-associated guanylate kinases (MAGUKs) assemble protein complexes at sites of cell-cell contact. At excitatory synapses in brain, MAGUKs localize to the postsynaptic density (PSD) and interact with N-methyl-D-aspartate (NMDA) glutamate receptors and downstream signaling proteins. However, NMDA receptors are not restricted to the PSDs, as electron microscopic immunocytochemical (EM-ICC) results indicate that NMDA receptors also occur at nonsynaptic portions of dendrites, perhaps functioning as reserves for rapid insertion into synaptic membranes in response to appropriate synaptic activity. NMDA receptors also occur in axons, at least in part to support glutamate-dependent enhancement of transmitter release. In this study, a systematic EM-ICC survey was performed to determine whether the distributions of four neuronal MAGUKs-PSD-95, PSD-93, SAP-102, and SAP-97-resemble that of NMDA receptors. Quantitative analysis revealed that the density of PSD-95 over thick PSDs of asymmetric axo-spinous synaptic junctions is 2-3-fold the level in the immediately adjacent cytoplasm of spines and terminals, while symmetric synapses show no association with PSD-95. Similarly, all four MAGUKs occur over PSDs of spines. However, we also detected MAGUK immunoreactivity, albeit more diffusely, along presynaptic membranes and in the cytoplasm of axons and dendritic shafts. In fact, the overall distribution of PSD-95 within the neuropil is equally prevalent along plasma membranes (including synaptic portions) as in the cytoplasm, away from plasma membranes. These results suggest that MAGUKs have dual roles: to maintain receptors at synapses and to regulate shuttling of receptors between nonsynaptic and synaptic sites.

PSD-93 knock-out mice reveal that neuronal MAGUKs are not required for development or function of parallel fiber synapses in cerebellum.

Membrane-associated guanylate kinases (MAGUKs) are abundant postsynaptic density (PSD)-95/discs large/zona occludens-1 (PDZ)-containing proteins that can assemble receptors and associated signaling enzymes at sites of cell-cell contact, including synapses. PSD-93, a postsynaptic neuronal MAGUK, has three PDZ domains that can bind to specific ion channels, including NMDA delta2 type glutamate receptors, as well as Shaker and inward rectifier type K(+) channels, and can mediate clustering of these channels in heterologous cells. Genetic analyses of Drosophila show that MAGUKs play critical roles in synaptic development because mutations of discs large disrupt the subsynaptic reticulum and block postsynaptic clustering of Shaker K(+) channels. It is uncertain whether MAGUKs play an essential role in the development of central synapses. There are four neuronal MAGUKs with overlapping expression patterns in the mammalian brain; however, we find PSD-93 is the only MAGUK expressed in cerebellar Purkinje neurons. Therefore, we targeted disruption of PSD-93 in mouse. Despite the absence of MAGUK immunoreactivity in Purkinje neurons from the knock-outs, these mice have no structural or functional abnormality in cerebellum. Both the dendritic architecture and the postsynaptic localization of PSD-93 interacting proteins remain intact at light and electron microscopic levels in the knock-outs. Postsynaptic Purkinje cell responses, monosynaptic climbing fiber innervation, and cerebellar-dependent behaviors are also normal. Our data demonstrate that MAGUK proteins of the PSD-93/95 family are not essential for development of certain central synapses but may instead participate in specialized aspects of synaptic signaling and plasticity.

The susceptibility of muscle cells to oxidative stress is independent of nitric oxide synthase expression.

The free radical, nitric oxide (NO.), has been implicated in the pathogenesis of muscular dystrophies because the enzyme, nitric oxide synthase (NOS), which produces NO., binds to the dystrophin-glycoprotein complex (DGC). In various studies of tissue samples from human and animal muscular dystrophies due to DGC defects, correlations between reductions of NOS activity and disease severity have been reported. To test for any direct effect of NOS expression on muscle cell susceptibility, we examined muscle cells in vitro under conditions of experimentally altered NOS activity. There were no differences in susceptibility to oxidative stress between differentiated myotube cultures from wild-type and from neuronal NOS (nNOS)-deficient mice. Likewise, pharmacological inhibition of NOS did not alter cellular susceptibility to oxidative challenges. Overexpression of NOS neither enhanced nor diminished cellular susceptibility to oxidative stress. Finally, we assessed the effect of NOS overexpression on myotube cultures from dystrophin-deficient (mdx) mice. NOS protein was localized to both membrane and cytosolic compartments in the transduced cells. Still, no difference in susceptibility to oxidative stress was found between the NOS-overexpressing cells and control cells. These data suggest that muscle cell susceptibility to oxidative challenges is independent of the level of NOS expression. Therefore, any role NO. may play in the pathogenesis of muscular dystrophies is likely to be independent of its effect on the redox state of the cell.

Actinin-associated LIM protein-deficient mice maintain normal development and structure of skeletal muscle.

The actinin-associated LIM protein, ALP, is the prototype of a large family of proteins containing an N-terminal PDZ domain and a C-terminal LIM domain. These PDZ-LIM proteins are components of the muscle cytoskeleton and occur along the Z lines owing to interaction of the PDZ domain with the spectrin-like repeats of alpha-actinin. Because PDZ and LIM domains are typically found in proteins that mediate cellular signaling, PDZ-LIM proteins are suspected to participate in muscle development. Interestingly the ALP gene occurs at 4q35 near the heterochromatic region mutated in facioscapulohumeral muscular dystrophy, indicating a possible role for ALP in this disease. Here, we describe the generation and analysis of mice lacking the ALP gene. Surprisingly, the ALP knockout mice show no gross histological abnormalities and maintain sarcolemmal integrity as determined by serum pyruvate kinase assays. The absence of a dystrophic phenotype in these mice suggests that down-regulation of ALP does not participate in facioscapulohumeral muscular dystrophy. These data suggest that ALP does not participate in muscle development or that an alternative PDZ-LIM protein can compensate for the lack of ALP.

Contrasting localizations of MALS/LIN-7 PDZ proteins in brain and molecular compensation in knockout mice.

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.

Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins.

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 intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization.

Postsynaptic targeting of MAGUKs mediated by distinct N-terminal domains.

Postsynaptic targeting of PSD-95 has been extensively studied; however, little is known about how other MAGUKs are localized. Proper targeting of PSD-95 requires dual palmitoylation of an N-terminal motif. We now find that the N-termini of closely related PSD-93 and SAP-102 are also involved in postsynaptic targeting. PSD-93 is N-terminally palmitoylated; however, unlike PSD-95, palmitoylation does not explain the necessity of the N-terminus for PSD-93 postsynaptic targeting. Furthermore, when the N-terminus of PSD-95 is replaced with the first 30 or 64, but not the first 10, amino acids of PSD-93, the chimera is targeted to postsynaptic sites independent of palmitoylation. Similarly, when the N-terminus of PSD-95 is replaced with the non-palmitoylated N-terminus of SAP-102, postsynaptic targeting is maintained. These results suggest that MAGUKs contain diverse signals within their N-termini for postsynaptic targeting.

PSD-95 involvement in maturation of excitatory synapses.

PSD-95 is a neuronal PDZ protein that associates with receptors and cytoskeletal elements at synapses, but whose function is uncertain. We found that overexpression of PSD-95 in hippocampal neurons can drive maturation of glutamatergic synapses. PSD-95 expression enhanced postsynaptic clustering and activity of glutamate receptors. Postsynaptic expression of PSD-95 also enhanced maturation of the presynaptic terminal. These effects required synaptic clustering of PSD-95 but did not rely on its guanylate kinase domain. PSD-95 expression also increased the number and size of dendritic spines. These results demonstrate that PSD-95 can orchestrate synaptic development and are suggestive of roles for PSD-95 in synapse stabilization and plasticity.

Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain.

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.

Inward rectifier K(+) channel Kir2.3 (IRK3) in reactive astrocytes from adult rat brain.

Astrocytic inward rectifying K(+) channels that participate in K(+) spatial buffering in the central nervous system have been extensively investigated, but specific gene products have not been fully identified. We studied primary cultured reactive astrocytes of stellate and polygonal morphology from adult rat brains, as well as stellate astrocytes from neonatal rat brains. Single-channel recordings of cell-attached patches revealed that polygonal reactive astrocytes expressed only one hyperpolarization-activated single-channel conductance of 11-15 pS whose open probability was independent of voltage, whereas stellate reactive and stellate neonatal astrocytes exhibited two conductances, 11-15 pS and 24-27 pS. All three subtypes of astrocytes exhibited a hyperpolarization-activated macroscopic inward K(+) current that was strongly rectifying and was abrogated by 1 mM intracellular Mg(2+) introduced during conventional but not perforated patch whole-cell recording. This Mg(2+)-sensitive current comprised the total inward rectifier current in polygonal reactive astrocytes, but only a fraction of the inward rectifier current in stellate reactive and stellate neonatal astrocytes. Because a strongly rectifying, inward rectifier K(+) channel with a single-channel conductance of 11-15 pS that is voltage independent is consistent with features of Kir2.3 (IRK3), we performed immunofluorescence experiments with anti-Kir2.3 and anti-glial fibrillary acidic protein antibodies. Both antibodies co-localized to all three subtypes of astrocytes in primary culture and to reactive astrocytes in situ within brain and gelatin sponge implants. Our data indicate that astrocytes of both polygonal and stellate morphology, from both adult and neonatal rat brain, express Kir2.3 both in vivo and in vitro. Constitutive expression of Kir2.3 regardless of cell morphology or age of origin of the source tissue suggests an important functional role for this channel in astrocytes.

Nitric oxide mediated induction of cytochrome c oxidase mRNA and protein in a mouse macrophage cell line.

Neurons that express neuronal nitric oxide synthase (nNOS) are selectively spared from nitric oxide (NO)-induced cytotoxicity in acute cerebral ischemia and neurodegenerative conditions but the mechanism of this resistance is unknown. To identify specific gene products which may mediate this resistance, we performed polymerase chain reaction (PCR)-based subtractive hybridization on a mouse macrophage cell line treated with either L-NG-nitroarginine methyl ester (L-NAME, 1 mM, 1 h), an inhibitor of NOS, or with diethylamine NONOate (DEA NONO, 200 microM, 1 h), an NO donor. NO-treated cultures showed an acute induction of mRNA (less than 1 h after treatment) and protein (15 min) for the mitochondrial enzyme cytochrome c oxidase (CcO) as shown by Northern or Western blot analysis, respectively. Cytochrome c oxidase activity assay showed constant activity in NO-treated cultures, as compared to L-NAME-treated cultures. NO directly inhibits CcO, the terminal electron acceptor in mitochondrial oxidative respiration. Up-regulation of this enzyme by NO, therefore, appears to maintain vital CcO activity and cellular energy stores, thus contributing to selective sparing of nNOS neurons.

Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs.

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinase (MAGUK) proteins assemble signal transduction complexes at sites of cell-cell contact including synapses. Whereas PSD-95 and PSD-93 occur only at postsynaptic sites in hippocampal neurons, SAP-102 also occurs in axons. In heterologous cells, PSD-95 and PSD-93 mediate cell surface ion channel clustering, but SAP-102 and SAP-97 do not. This selective ion channel clustering activity by MAGUKs is explained by differential palmitoylation, as PSD-93 and PSD-95 are palmitoylated though SAP-97, and SAP-102 are not. Rather than being palmitoylated, we find that N-terminal cysteines from SAP-102 tightly bind to zinc. And, appending the N terminus of SAP-102 to PSD-95 results in localization of the chimera to both axons and dendrites. These data suggest that lipid modifications and heavy metal associations with the N termini of MAGUKs mediate differential functions and subcellular localizations of these synaptic scaffolds.

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by a tyrosine-based trafficking signal.

Synaptic function requires proper localization of proteins at synaptic sites. Targeting of the postsynaptic density protein 95 (PSD-95) relies on multiple signals within the protein, including twelve C-terminal amino acids. We now show that this C-terminal targeting domain of PSD-95 mediates postsynaptic localization through a short tyrosine-based motif followed by a pair of hydrophobic amino acids. Consistent with a role in cellular trafficking, the tyrosine motif resembles the canonical motif for interactions with clathrin adaptor proteins. In fact, we find that the C-terminal targeting domain of PSD-95 is sufficient to mediate clathrin-dependent endocytosis when appended to a transmembrane protein. Furthermore, systematic mutagenesis reveals that endocytosis mediated by this domain depends on both the tyrosine motif and the dihydrophobic amino acid pair. Thus, postsynaptic targeting of PSD-95 requires a tyrosine-based signal that can mediate clathrin-coated vesicle formation.

Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering.

Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH(2) terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH(2) terminus of PSD-95 with alternative palmitoylation motifs at either the NH(2) or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.