Discs Lost, a Novel Multi-PDZ Domain Protein, Establishes and Maintains Epithelial Polarity

515

359

Nitric oxide (NO) biosynthesis in cerebellum is preferentially activated by calcium influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors, suggesting that there is a specific link between these receptors and neuronal NO synthase (nNOS). Here, we find that PSD-95 assembles a postsynaptic protein complex containing nNOS and NMDA receptors. Formation of this complex is mediated by the PDZ domains of PSD-95, which bind to the COOH termini of specific NMDA receptor subunits. In contrast, nNOS is recruited to this complex by a novel PDZ-PDZ interaction in which PSD-95 recognizes an internal motif adjacent to the consensus nNOS PDZ domain. This internal motif is a structured "pseudo-peptide" extension of the nNOS PDZ that interacts with the peptide-binding pocket of PSD-95 PDZ2. This asymmetric interaction leaves the peptidebinding pocket of the nNOS PDZ domain available to interact with additional COOH-terminal PDZ ligands. Accordingly, we find that the nNOS PDZ domain can bind PSD-95 PDZ2 and a COOH-terminal peptide simultaneously. This bivalent nature of the nNOS PDZ domain further expands the scope for assembly of protein networks by PDZ domains.Efficiency and specificity in cellular signaling cascades is often mediated by assembly of multiprotein transduction networks. Signaling by the calcium/calmodulin regulated neuronal nitric oxide synthase (nNOS) 1 in cerebellar neurons is activated by calcium influx through N-methyl-D-aspartic acid (NMDA) receptors (1, 2), but nNOS is not efficiently stimulated by activation of non-NMDA receptors that also generate calcium influx (3). Therefore, a mechanism must exist to specifically couple NMDA receptor-mediated calcium influx to nNOS.In addition to mediating neuronal functions, nNOS is also found in skeletal muscle, where nNOS localizes to the sarcolemma. Membrane association of nNOS in skeletal muscle is mediated by direct interaction with ␣1-syntrophin, a component of the dystrophin complex. Binding of nNOS to syntrophin occurs through direct interaction of PDZ protein motifs present near the NH 2 termini of both proteins (4). Mice lacking ␣1-syntrophin lose nNOS protein and enzyme activity from muscle membranes (5). Furthermore, patients with Duchenne muscular dystrophy and mdx mice that lack dystrophin evince a selective loss of nNOS from the sarcolemma (6).In neurons, nNOS is also associated with cell membranes. In a detailed ultrastructural analysis of primate visual cortex, nNOS immunoreactivity in spines was concentrated over thick postsynaptic specializations of plasma membranes, often in association with NMDA receptors (7). This synaptic localization of nNOS in brain may be mediated by association with the postsynaptic density protein, PSD-95. Like ␣1-syntrophin, PSD-95 binds directly to nNOS through a PDZ-PDZ interaction that involves the second PDZ domain of PSD-95 (4). Moreover, PSD-95 is a member of a larger family of postsynaptic density proteins (PSD-95/SAP-90, PSD-93/chapsyn-110, and SAP-102) that cluster NMDA receptors and may anchor these...

Section: Discussionmentioning

confidence: 99%

PSD-95 Assembles a Ternary Complex with theN-Methyl-d-aspartic Acid Receptor and a Bivalent Neuronal NO Synthase PDZ Domain

Christopherson

Hillier

Lim

et al. 1999

515

359

“…Although most PDZ domains promote protein-protein interactions through the recognition of peptide motifs at the carboxyl terminus of a protein, which usually ends with a hydrophobic residue (39,40), some PDZ domains are capable of forming homo-or heterodimers (41,42). The dimerization of PDZ domains is based on the recognition of internal peptides.…”

Section: Discussionmentioning

confidence: 99%

A Novel PTB-PDZ Domain Interaction Mediates Isoform-specific Ubiquitylation of Mammalian Numb

Nie

McGlade

2004

LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of mNumb. Numb is a PTB domaincontaining protein that functions as an intrinsic determinant of cell fate in asymmetric cell division. In mammals, four protein isoforms arise from alternative mRNA splicing. Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded. The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the PTB domain (PTBi). We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between the first PDZ domain (PDZ1) of LNX and the PTBi variant. Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and subsequent degradation of the PTBi form of Numb. Interestingly efficient PTBi ubiquitylation not only depends on association with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX. Thus two distinct modes of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms of mNumb.Controlled protein degradation mediated by ubiquitin-dependent proteolysis underlies the regulation of many critical biological events during the life and death of a cell as well as in health and disease (1). Ubiquitylation of a target protein is achieved through a cascade involving at least three enzymes, namely E1, 1 E2, and E3, which activate and transfer ubiquitin to the substrate in a sequential manner. Polyubiquitylated proteins are recognized by the 26 S proteasome and are subsequently degraded into small peptides (2, 3).E3 ubiquitin ligases are responsible for the specific recognition of a multitude of substrates. Two major classes of E3 have been identified to date characterized by the presence of either a HECT (homologous to E6-AP carboxyl terminus) domain or a RING finger domain. HECT-type E3s catalyze ubiquitylation of a substrate by first forming an E3-ubiquitin thioester intermediate followed by the direct transfer of ubiquitin onto the substrate. On the other hand, RING-type E3s function as scaffolds that bring together the E2 and the target substrate to facilitate efficient transfer of the activated ubiquitin moiety from the E2 to the substrate protein (4). In addition to HECT or RING finger domains, E3 ligases also contain protein interaction modules for substrate recognition. Diversity in the repertoire of E3 substrate selection is achieved by linking a variety of protein interaction modules to the HECT or RING domains. For example, many HECT domain-containing E3s share the WW domain, which is involved in protein-protein interactions and has a role in targeting substrates containing a PY motif for ubiquitylation (5, 6). The RING-type E3 family is composed of two distinct groups, single and multisubunit proteins. Single subunit E3s such as c-Cbl contain...

“…RNAi Methods-dronc RNAi was performed using previously described methods (45,46). RNA transcripts were generated using the Ambion Megascript kit with linearized pOT2-dronc templates.…”

Section: Dronc Transgenic Flies and Genetic Interactionmentioning

confidence: 99%

An Essential Role for the Caspase Dronc in Developmentally Programmed Cell Death in Drosophila

Quinn¹,

Dorstyn²,

Mills³

et al. 2000

151

162

Dronc is a caspase recruitment domain-containingDrosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Here we show that ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99 complex (reaper, hid, and grim) and enhanced by mutations in diap1. In contrast to previous reports, we show that the dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitor p35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Dark. Furthermore, extracts made from Dark homozygous mutant flies have reduced ability to process Dronc, showing that Dark is required for Dronc processing. Finally, using the RNA interference technique, we show that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death in Drosophila.