Positive surface charge of GluN1 N-terminus mediates the direct interaction with EphB2 and NMDAR mobility

Halley R Washburn,Wei Zhou,Matthew B Dalva,Yu-Ting Mao,Nan L Xia

doi:10.1038/s41467-020-14345-6

Abstract

Localization of the N-methyl-D-aspartate type glutamate receptor (NMDAR) to dendritic spines is essential for excitatory synaptic transmission and plasticity. Rather than remaining trapped at synaptic sites, NMDA receptors undergo constant cycling into and out of the postsynaptic density. Receptor movement is constrained by protein-protein interactions with both the intracellular and extracellular domains of the NMDAR. The role of extracellular interactions on the mobility of the NMDAR is poorly understood. Here we demonstrate that the positive surface charge of the hinge region of the N-terminal domain in the GluN1 subunit of the NMDAR is required to maintain NMDARs at dendritic spine synapses and mediates the direct extracellular interaction with a negatively charged phospho-tyrosine on the receptor tyrosine kinase EphB2. Loss of the EphB-NMDAR interaction by either mutating GluN1 or knocking down endogenous EphB2 increases NMDAR mobility. These findings begin to define a mechanism for extracellular interactions mediated by charged domains.

Highlights

Localization of the N-methyl-D-aspartate type glutamate receptor (NMDAR) to dendritic spines is essential for excitatory synaptic transmission and plasticity
To understand the mechanism underlying the EphB–NMDAR interaction, we tested whether endogenous EphB2 and NMDARs interact at synapses using synaptosomes isolated from mouse brain (Supplementary Fig. 1a)[19,31,32]
30% of vesicular glutamate transporter 1 (vGlut1)-positive synaptosomes colocalized with both EphB2 and GluN1 (29 ± 3.7%; Fig. 1a, b, Supplementary Fig. 1b). To test whether these proteins might be interacting, we conducted immunocytochemistry (ICC) for vGlut1-positive presynaptic sites followed by rolling-circle amplification proximity ligation assays (PLAs) in synaptosomes with antibodies against the extracellular domains of GluN1 and EphB234

Summary

Introduction

Localization of the N-methyl-D-aspartate type glutamate receptor (NMDAR) to dendritic spines is essential for excitatory synaptic transmission and plasticity. We demonstrate that the positive surface charge of the hinge region of the N-terminal domain in the GluN1 subunit of the NMDAR is required to maintain NMDARs at dendritic spine synapses and mediates the direct extracellular interaction with a negatively charged phospho-tyrosine on the receptor tyrosine kinase EphB2. Loss of the EphB-NMDAR interaction by either mutating GluN1 or knocking down endogenous EphB2 increases NMDAR mobility These findings begin to define a mechanism for extracellular interactions mediated by charged domains. The extracellular domains of the GluN2 subunits contain the glutamate-binding site while GluN1 subunits bind co-agonist glycine and are essential for NMDAR function[4]. While the molecules that bind to the GluN2B NTD and the GluN1/GluN2B NTD interface are well characterized[7,8,9,10], binding partners for the

Methods

Results

Conclusion