title: PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors.
abstract: In neuronal synapses, PDZ domains [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] of PSD-95 proteins interact with C termini of NMDA receptor [NMDAR (NR)] subunits, linking them to downstream neurotoxic signaling molecules. Perturbing NMDAR/PSD-95 interactions with a Tat peptide comprising the nine C-terminal residues of the NR2B subunit (Tat-NR2B9c) reduces neurons' vulnerability to excitotoxicity and ischemia. However, NR subunit C termini may bind many of >240 cellular PDZs, any of which could mediate neurotoxic signaling independently of PSD-95. Here, we performed a proteomic and biochemical analysis of the interactions of all known human PDZs with synaptic signaling proteins including NR1, NR2A-NR2D, and neuronal nitric oxide synthase (nNOS). Tat-NR2B9c, whose interactions define PDZs involved in neurotoxic signaling, was also used. NR2A-NR2D subunits and Tat-NR2B9c had similar, highly specific, PDZ protein interactions, of which the strongest were with the PSD-95 family members (PSD-95, PSD-93, SAP97, and SAP102) and Tax interaction protein 1 (TIP1). The PSD-95 PDZ2 domain bound NR2A-NR2C subunits most strongly (EC50, approximately 1 microM), and fusing the NR2B C terminus to Tat enhanced its affinity for PSD-95 PDZ2 by >100-fold (EC50, approximately 7 nM). IC50 values for Tat-NR2B9c inhibiting NR2A-NR2C/PSD-95 interactions (approximately 1-10 microM) and nNOS/PSD-95 interactions (200 nM) confirmed the feasibility of such inhibition. To determine which of the PDZ interactions of Tat-NR2B9c mediate neuroprotection, one of PSD-95, PSD-93, SAP97, SAP102, TIP1, or nNOS expression was inhibited in cortical neurons exposed to NMDA toxicity. Only neurons lacking PSD-95 or nNOS but not PSD-93, SAP97, SAP102, or TIP1 exhibited reduced excitotoxic vulnerability. Thus, despite the ubiquitousness of PDZ domain-containing proteins, PSD-95 and nNOS above any other PDZ proteins are keys in effecting NMDAR-dependent excitotoxicity. Consequently, PSD-95 inhibition may constitute a highly specific strategy for treating excitotoxic disorders.
authors.affiliation: NoNO Inc., Toronto, Ontario, Canada M8X 1R5.
authors.forename: Hong
authors.initials: H
authors.lastname: Cui
authors.forename: Amy
authors.initials: A
authors.lastname: Hayashi
authors.forename: Hong-Shuo
authors.initials: HS
authors.lastname: Sun
authors.forename: Michael P
authors.initials: MP
authors.lastname: Belmares
authors.forename: Carolyn
authors.initials: C
authors.lastname: Cobey
authors.forename: Thuymy
authors.initials: T
authors.lastname: Phan
authors.forename: Johannes
authors.initials: J
authors.lastname: Schweizer
authors.forename: Michael W
authors.initials: MW
authors.lastname: Salter
authors.forename: Yu Tian
authors.initials: YT
authors.lastname: Wang
authors.forename: R Andrew
authors.initials: RA
authors.lastname: Tasker
authors.forename: David
authors.initials: D
authors.lastname: Garman
authors.forename: Joshua
authors.initials: J
authors.lastname: Rabinowitz
authors.forename: Peter S
authors.initials: PS
authors.lastname: Lu
authors.forename: Michael
authors.initials: M
authors.lastname: Tymianski
mesh_terms: D030562:Databases, Protein; D000076122:Disks Large Homolog 4 Protein; D018683:Excitatory Amino Acid Agents / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018696:Neuroprotective Agents / Q000378:metabolism* / Q000494:pharmacology; D025941:Protein Interaction Mapping; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000378:metabolism*
chemical_list: C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D018683:Excitatory Amino Acid Agents; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D016194:Receptors, N-Methyl-D-Aspartate; C033652:postsynaptic density proteins