PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors.
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.
Cui
Hong
H
NoNO Inc., Toronto, Ontario, Canada M8X 1R5.
Hayashi
Amy
A
Sun
Hong-Shuo
HS
Belmares
Michael P
MP
Cobey
Carolyn
C
Phan
Thuymy
T
Schweizer
Johannes
J
Salter
Michael W
MW
Wang
Yu Tian
YT
Tasker
R Andrew
RA
Garman
David
D
Rabinowitz
Joshua
J
Lu
Peter S
PS
Tymianski
Michael
M
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*
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