Dynamic and specific interaction between synaptic NR2-NMDA receptor and PDZ proteins

Abstract

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using highresolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A-or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions. lateral diffusion | glutamate receptor | trafficking | biomimetic multivalent ligand | development T he identification of the cellular mechanisms involved in the regulation of glutamate receptor trafficking is crucial to our understanding of synaptic maturation and plasticity. One common paradigm of these processes is the activation of the calciumpermeable postsynaptic NMDA receptors (NMDARs). In the neocortex, the most abundant types of NMDARs are composed of NR1 subunits associated with NR2A (enriched in synapses) and/or NR2B subunits (1). Rapid changes in the synaptic 2A/2B NMDAR ratio have been reported during connection refinements and synaptic plasticity (2), and several key molecular interactions have been shown to control the trafficking of intracellular and membrane NMDARs (3-6).The intracellular proteins that interact with the C terminus of the subunits, through direct binding or modification of the phosphorylation state, are likely candidates for regulating the synaptic retention of NMDARs. Indeed, intracellular domains of NR2 subunits provide a binding motif for proteins of the postsynaptic density such as PSD-95 and SAP102 (7-10). The binding of the NR2B subunit C terminus to PDZ domain-containing scaffold proteins regulates, in part, the synaptic retention of this receptor (8,9,(11)(12)(13)(14). For the 2A-NMDARs, which make up the majority of synaptic NMDARs, the role of such interactions in synaptic retention remains controversial. Indeed, long-term expression of NR2A subunits with a truncated or mutated C terminus does not affect synaptic NMDAR currents in cerebellar or hippocampal neurons (9, 15), whereas deletion of the NR2A subunit C terminus sequence significantly reduces NMDAR synaptic signaling (11,14,16,17). Currently, there is no simple explanation for this discrepancy, and the use of long-term expression of exogenous NR subunits and lack of good pharmacological tools to discriminate betwee...

Identification of the SNARE complex mediating the exocytosis of NMDA receptors

“…Following the exocytosis of pH-NR1, the pHluorin tag will be exposed to the extracellular solution with pH 7.4 and its fluorescence will dramatically increase. Previous studies showed that neither pHluorin nor EGFP fluorescent tag at the N terminus of NR1 affected its trafficking in neurons (26)(27)(28).…”

“…Following the exocytosis of pH-NR1, the pHluorin tag will be exposed to the extracellular solution with pH 7.4 and its fluorescence will dramatically increase. Previous studies showed that neither pHluorin nor EGFP fluorescent tag at the N terminus of NR1 affected its trafficking in neurons (26)(27)(28).To visualize the exocytosis of NR1, dissociated hippocampal neurons grown on glass coverslips were cotransfected with pH-NR1 and untagged NR2A subunit to form NMDA receptor complexes, and imaged 24-48 h after transfection. Following the photobleaching of preexisting fluorescence on the plasma membrane Significance NMDA receptors are ionotropic glutamate receptors and crucial for excitatory synaptic transmissions in the brain.…”

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

“…Based on these findings, one may predict that favoring the presence of perisynaptic NMDAR in the spine area would increase their potential binding to D1R and thereby modulate the size of D1R clusters. To address this question, we artificially increased the amount of perisynaptic NMDAR by blocking their synaptic retention with a biomimetic ligand (TAT-[N2A 15 ], 10 μM, 20 min) that prevents their Cterminal interaction with cytosolic PDZ domain-containing proteins (22). Strikingly, this treatment resulted in a rapid and significant increase in D1R cluster intensity (Fig.…”

Identification of N-Methyl-d-aspartic Acid (NMDA) Receptor Subtype-specific Binding Sites That Mediate Direct Interactions with Scaffold Protein PSD-95

“…They were NR2A(1381-1390), RCPSD-PYKHS, termed SH3, and corresponded to the amino acid sequence around the SH3 binding domain; SH3 scrambled, DSYSRPHCKP; NR2A(1450 -1464), NRRVYKKMPSIESDV, termed PDZ, and corresponded to the amino acid sequence at the C-terminal ESDV domain and the same as used previously (18), and PDZ scrambled, KRSMNIVEKDPVRYS.…”