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

Abstract

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...

Homers regulate drug-induced neuroplasticity: Implications for addiction

“…While neither deletion of Homer1 nor Homer2 alters total NMDA receptor subunit expression within the NAC or PFC (Table 3) [34,176], Homer2 deletion reduces the NAC plasma membrane expression of NR2a and NR2b NMDA receptor subunits in vivo [38]. Through the Shank-GKAP-PSD95 complex, NR2 subunits colocalize with nitric oxidase synthase-1 (NOS-1) [177][178][179], an enzyme responsible for the synthesis of the retrograde messenger nitric oxide (NO) [180,181]. Thus, one testable hypothesis to account for the effects of Homer deletion upon basal and stimulated corticoaccumbens glutamate transmission relates to alterations in NMDA-mediated NO retrograde signaling [182,183].…”

Nitric oxide in brain: diffusion, targets and concentration dynamics in hippocampal subregions

“…The bivalent PDZ domain of nNOS targets the enzyme to membranes in synapses and interacts with a PDZ domain in neuron-specific PSD-95. Since PSD-95 also binds to glutamate NMDA receptor through PZD domains, a picture emerges in which PSD-95 functionally couples NMDA receptor activation to nNOS activity (Christopherson et al, 1999). In addition to functional evidence coupling NMDA activity with NO Å synthesis, co-localization of nNOS with NMDA receptors and PSD-95 in hippocampal dendritic spines using microscopic immunocytochemical methods optimized to detect synaptic antigens, adds strong support to the supramolecular organization of the signalling pathway across a synapse as a theme for the regulation of NO Å activity in the brain (Burette et al, 2002).…”

Excitatory Amino Acid Receptors of the Electrosensory System: The NR1/NR2BN-Methyl-d-Aspartate Receptor

“…In the PSD, the C-termini of the NMDA receptor NR2 subunits interact with the PDZ (PSD-95/Dlg/ Z0 -1) domains of the PSD-95 family of synaptic scaffold proteins, interactions that are mediated by the sequence ESDV at the C-termini of the NR2 subunits, which forms the binding site for the PSD-95 proteins (Kornau et al 1995;Niethammer et al 1996). These interactions are thought to stabilize the NMDA receptors at the synapse (El-Husseini et al 2000;Roche et al 2001) and to link the receptor to signaling molecules such as NO synthase (Christopherson et al 1999;Sattler et al 1999). The C-terminal 17 residues of AptNR2B are almost identical to those of the mammalian NR2B, with only single conservative Ser-Thr substitution (Fig.…”