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

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

Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain

<div><p>Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.</p></div

Synaptic currents and accumulation of N-methyl-D-aspartate receptors (NMDARs) at synaptic sites are controlled by phosphorylation of EphB2 Y504.

<p>(A) Experimental approach for the figure is illustrated. (B) Mean traces of whole-cell patch-clamp recording at 50 mV show the evoked and miniature excitatory postsynaptic currents (EPSCs) of primary rat cortical day in vitro (DIV) 21–23 neurons expressing enhanced green fluorescent protein (EGFP) without or with EphB2 wild type (WT) and Y504 mutants induced by light stimulation of neurons that express optogenetic light-sensitive channels (channelrhodopsin-2). (C) Effects of overexpression of EphB2 WT and Y504 mutants on the mean amplitude of evoked, spontaneous, and miniature EPSCs (30 milliseconds after the evoked EPSC peak) in mature cultured neurons. Overexpression of EphB2 WT or Y504E significantly increased amplitude of the NMDAR-dependent component of evoked EPSCs compared to the control or the Y504F mutant (****<i>p <</i> 0001, ANOVA followed by Fisher’s exact test, <i>n</i> = 375, 332, 581, and 200 events for control, EphB2 WT, Y504E, and Y504F, respectively). In addition, the amplitude of Y504E-overexpressing neurons was significantly higher than that of WT-overexpressing neurons (**<i>p <</i> 0.02, ANOVA followed by Fisher’s exact test). (D) Representative sample traces of whole-cell patch-clamp recording at 50 mV show that NMDAR-dependent currents in control neurons and neurons expressing EphB2 WT and Y504E, but not Y504F, are greatly reduced by GluN2B-specific antagonist Ro 25–6981 (Ro25). (E) Cumulative probability histogram of miniature EPSC (mESPC) amplitude for Y504E before and after application of Ro25 (2.5 μM). Inset: mean traces of mEPSCs after treatment with Ro25 (<i>p <</i> 0.001, Kolmogorov—Smirnov [K–S] test, <i>n</i> = 720 for Y504E and <i>n</i> = 427 for Y504E + Ro25). Vertical scale bar = 20 picoamperes (pA); horizontal scale bar = 10 milliseconds. (F) Cumulative probability histogram of mEPSC amplitude Y504F as in (E) (<i>p</i> = 0.0775, K–S test, <i>n</i> = 414 for Y504F and <i>n</i> = 404 for Y504F + Ro25). Vertical scale bar = 10 pA; horizontal scale bar = 10 milliseconds. (G) Model depicting 2 scenarios of VGLUT1 (blue), EphB2 (red), and GluN1 (green) localization at dendritic spines. (H) High-contrast images of dendrites of DIV 21 cortical neurons expressing EGFP, EphB2 short hairpin RNA (shRNA), and RNA interference (RNAi)-insensitive FLAG-tagged EphB2 WT, EphB2-Y504E, or EphB2-Y504F. Top panels show confocal EGFP staining (white), second panels show stimulated emission depletion (STED) EphB2 staining (red), third panels show STED GluN1 staining (green), fourth panels show confocal VGLUT1 (presynaptic marker) staining (blue), and bottom panels show merged images. White arrows indicate examples of triple colocalization of EphB2, GluN1, and VGLUT1. Scale bar = 1 μm, 0.5 μm inset. (I) Quantification of the effects of expression of EphB2 Y504 mutants on localization of VGLUT1 to dendritic spines in DIV 21 rat cortical neurons transfected at DIV 14. Graph shows fraction of spines containing VGLUT1 (not significant, ANOVA followed by Fisher’s exact test). (J) Quantification of the effects of expression of EphB2 Y504 mutants on colocalization with GluN1 in dendritic spines in DIV 21 rat cortical neurons transfected at DIV 14. Graph shows percentage of spines containing colocalized EphB2 and GluN1 puncta as defined by Fig 5G (*<i>p <</i> 0.05, ***<i>p <</i> 0.005, ANOVA followed by Fisher’s exact test). (K) Quantification of the effects of expression of EphB2 Y504 mutants on colocalization with GluN1 at synaptic sites in DIV 21 rat cortical neurons transfected at DIV 14. Graph shows percentage of spines containing triple colocalized puncta as defined by Fig 5G (***<i>p <</i> 0.005, ****<i>p <</i> 0.0005, ANOVA followed by Fisher’s exact test).</p

Expression of EphB2 recruits the N-methyl-D-aspartate receptor (NMDAR) to synaptic regions of the dorsal horn.

<p>(A) Model for effects of EphB2 extracellular phosphorylation on the EphB—NMDAR interaction. (B) Experimental approach using intrathecal injection of lentivirus (LV) to avoid effects outside of the spinal cord. Examination of injection site revised transduced neurons within the dorsal horn. (C) Neuronal viral transduction was confirmed with NeuN labeling (marker for neuronal nucleus) surrounded by EphB2- enhanced yellow fluorescent protein (EYFP) wild type (WT). The left panel was stained for EphB2-EYFP with α-GFP. The middle panel was stained for NeuN. The right panel shows the merged image. Control enhanced green fluorescent protein (EGFP), EphB2-EYFP Y504E, and EphB2-EYFP Y504F also show the distribution surrounding NeuN. Scale bar = 50 μm. (D) Distribution of GluN1 and vGlut2 in the dorsal horn of the adult mouse spinal cord, expressing control EGFP, EphB2-EYFP WT, or Y504E or Y504F mutants. The left panel shows GluN1 (cyan). The middle panels show vGlut2 (red) to mark superficial layers of dorsal horn. The right panel shows a merged image of GluN1 and vGlut2 staining. The dashed yellow line indicates superficial layers of dorsal horn. Scale bar = 50 μm. (E) Quantification of the effects of expression of EphB2 WT and Y504E and Y504F mutants on GluN1 intensity in superficial layers of the dorsal horn of the adult mouse spinal cord (*<i>p <</i> 0.05, ***<i>p <</i> 0.05, ANOVA followed by Tukey’s range test, 17 sections from 3 mice for control, 18 sections from 3 mice for EphB2 WT, 22 sections from 4 mice for Y504E mutant, and 24 sections from 4 mice for Y504F mutant). (F) Quantification of the effects of expression of EphB2 WT and Y504E and Y504F mutants on vGlut2 intensity in superficial layers of the dorsal horn of the adult mouse spinal cord. (***<i>p <</i> 0.0005, ****<i>p <</i> 0.0001, ANOVA followed by Tukey’s range test, 17 sections from 3 mice for control, 18 sections from 3 mice for EphB2 WT, 22 sections from 4 mice for Y504E mutant, and 24 sections from 4 mice for Y504F mutant). AU, arbitrary unit.</p

Extracellular phosphorylation is induced by ephrin-B2 and mediates the EphB—N-methyl-D-aspartate receptor (NMDAR) interaction.

<p>(A) Untransfected cultured rat cortical neurons (day in vitro [DIV] 6–7) were treated with ephrin-B2 (+) or control reagents (-) for 45–60 minutes and either control (C) or K252b. Endogenous EphB2 was immunoprecipitated using α-EphB2 antibodies, and blots (listed top to bottom) were probed for GluN1, EphB2, EphB2 p*Y504, EphB2 p*Y662, and tubulin. Panels on the left show IP samples and right panels show lysates. (B–C) Quantification of the effects of ephrin-B2 treatment after blockade of extracellular kinase activity with K252b on the phosphorylation of Y504 (B) and the EphB—NMDAR interaction (C) in neurons (**<i>p <</i> 0.01, ****<i>p <</i> 0.001, ANOVA followed by Fisher’s exact test; <i>n</i> = 5 experiments for each condition). (D) Untransfected cultured rat spinal cord neurons (DIV 12–14) were treated with ephrin-B2 (+) or control reagents (-) for 45–60 minutes and either control (C) or K252b. Endogenous EphB2 was immunoprecipitated using α-EphB2 antibodies and blots (listed top to bottom) were probed for GluN1, EphB2, EphB2 p*Y504, EphB2 p*Y662, and tubulin. (E–F) Quantification of the effects of ephrin-B2 treatment after blockade of extracellular kinase activity with K252b (****<i>p <</i> 0.001, ANOVA followed by Fisher’s exact test; <i>n</i> = 5 experiments for each condition).</p

Identification of phosphorylated tyrosine in C-terminal fibronectin type III (FN3) (cFN3) of EphB2 extracellular region.

<p>(A and B) Tandem mass spectrometry (MS/MS) spectra of peptides (A) ELSEYNATAIK and (B) AGAIYVFQR are shown. Fragments critical for localization of phosphorylation sites are labeled in red. (C) Schematic of the known functional domains of EphB2 receptor. LBD, ligand-binding domain (purple); Cys, cysteine-rich domain (white); FN3, fibronectin type III repeat domain (orange [N-terminal FN3 (nFN3)]and blue [cFN3]); TM, transmembrane domain; JM, juxtamembrane domain (yellow); TK, tyrosine kinase domain (red), SAM (grey), sterile α-motif; PDZ, PSD-95/DLG1/ZO-1 domain (green). (D) Alignment of cFN3 domains of EphB2 with Eph family proteins (Uniprot database) using ClustalW2 software. EphB2 Y504 (red) corresponds to a conserved tyrosine residue, whereas Y481 (blue) is only conserved in mammals (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002457#pbio.2002457.s001" target="_blank">S1E Fig</a>, grey). Yellow indicates identical amino acids with mouse EphB2. (E) Alignment of cFN3 domain of EphB2 with other FN3-containing molecules. Y504 and neighboring residues in identified peptide (red) are well conserved amongst Eph family proteins (55.4% identical amino acids) or FN3-containing molecules (45.5% identical amino acids), whereas Y481 and neighboring residues in identified peptide (blue) are less well conserved amongst species (19.6% and 11.6% identical amino acids for Eph family proteins and FN3-containing molecules, respectively). (F) Top blot shows HEK293T lysates probed with a phospho-specific antibody generated against EphB2 Y504 (α-EphB2 p*Y504). Middle blot shows same lysates probed for EphB2. Bottom blot shows lysates probed for EphB2 p*Y662 (EphB2 kinase activity). Lanes were loaded with lysates of HEK293T cells transfected with full-length (FL) EphB2 wild type (Y) or Y504F (F), kinase-dead (KD) EphB2 wild type (Y) or Y504F (F), and truncated (TR) EphB2 wild type (Y) or Y504F (F). (G) Left blots show synaptosome lysates prepared from wild-type (WT) CD1 mouse brain and right blots show synaptosome lysates prepared from the spinal cord. Gels were loaded with nonsynaptic (S1), crude synaptosomal (P2), and synaptosomal (Syn) fractions. The top 2 blots were probed with α-EphB2 p*Y504 antibody before (upper) and after (lower) incubation with calf intestinal alkaline phosphatase (CIP) overnight to remove phosphate groups. The third and fourth blots were probed with α-EphB2 before and after CIP treatment. The fifth blot was probed with α-GluN1. The bottom blot was probed with α-PSD-95. (H) Blots show synaptosome lysates prepared from WT CD1 mouse brain at P9, P15, or P21. The top blot was probed with α-EphB2 p8Y504 antibody, the second blot was probed with α-EphB2, the third blot was probed with α-GluN1, the fourth blot was probed with α-GluN2B, and the fifth blot was probed with α-PSD-95. The bottom blot was probed with α-GAPDH as a loading control.</p

Phosphorylation of extracellular tyrosine residue 504 of EphB2 is required for interaction with the N-methyl-D-aspartate receptor (NMDAR).

<p>(A) Model of how extracellular phosphorylation at Y504 modulates the EphB—NMDAR interaction. (B) Live-cell immunostaining of HEK293T for FLAG(-EphB2) and ephrin-B2-Fc—binding sites. HEK293T cells transfected with FLAG-EphB2 wild type (WT), Y504E, or Y504F mutants were incubated with ephrin-B2-Fc for 45 minutes and then α-FLAG antibody was added to cells at 37°C for 10 minutes. After washing, cells were fixed and processed for immunostaining. Left panels were live-cell stained for FLAG-tagged EphB2, middle panels were stained for ephrin-B2-Fc, and right panels show merged images. Quantified in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002457#pbio.2002457.s004" target="_blank">S4A Fig</a>. For surface staining controls, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002457#pbio.2002457.s004" target="_blank">S4B–S4D Fig</a>. Scale bar = 5μm. (C) Coimmunoprecipitation (co-IP) of FLAG-EphB2 and GluN1 with α-GluN1 antibodies from HEK293T. Left control (C) lane has only FLAG-EphB2 WT. Other lanes are transfected with GluN1 and GluN2B and indicated EphB2 constructs. The right WT lane is transfected with FLAG-EphB2 WT, the Y504E lane is transfected with FLAG-EphB2 Y504E, and the Y504F lane is transfected with FLAG-EphB2 Y504F. Antibodies used for detection are shown at the left. IP samples are shown in blots on the left; cell lysates are shown on the right. Tubulin is used as loading control. (D) Quantification of ratio of coimmunoprecipitated EphB2 to total EphB2 in input. EphB2 Y504E coimmunoprecipitated with GluN1 significantly more than EphB2 WT. In addition, GluN1 co-IP of EphB2 Y504E was significantly higher than EphB2 WT (*<i>p <</i> 0.05, ***<i>p <</i> 0.005, ****<i>p <</i> 0.001, ANOVA followed by Fisher’s exact test, <i>n</i> = 4). (E) Co-IP of GluN1 and FLAG-tagged EphB2 with α-EphB2 antibodies from HEK293T. All lanes are transfected with HA-GluN1 and GluN2B. The control lane had only HA-GluN1 and GluN2B alone, the WT lane was transfected with FLAG-EphB2, the E lane was transfected with FLAG-EphB2 Y504E, and the F lane was transfected with FLAG-EphB2 Y504F. Top blots were probed for HA (GluN1), and the bottom blots were probed for FLAG (EphB2). Left panels are IP samples and right panels are lysates. Tubulin was used as a loading control. (F) Quantification of ratio of coimmunoprecipitated GluN1 to total GluN1 in the input. IP of FLAG-tagged EphB2 mutants revealed a significant increase of GluN1 co-IP in lysates from cells expressing EphB2 Y504E, while GluN1 co-IP was significantly reduced in lysates from cells expressing the Y504F EphB2 mutants compared to GluN1 pull-down in EphB2 WT-expressing cells (*<i>p <</i> 0.05, **<i>p <</i> 0.01, ***<i>p <</i> 0.005, ANOVA followed by Fisher’s exact test, <i>n</i> = 5). (G) Cultured rat cortical neurons (day in vitro [DIV] 9) infected with lentivirus harboring either enhanced yellow fluorescent protein (EYFP)-tagged EphB2 WT or Y504 mutants at DIV 2 were stimulated with ephrin-B2-Fc or control. EphB2 was immunoprecipitated with α-GFP antibodies and blots were probed for GluN1 (top left). WT indicates neurons transduced with EphB2-EYFP, Y504E indicates neurons transduced with EphB2 Y504F EYFP, and Y504F indicates neurons transduced with EphB2 Y504F EYFP. Minus sign (-) indicates control treatment, plus sign (+) indicates ephrin-B2 treatment. Top blots were probed for GluN1 (α-GluN1), and bottom blots were probed for transduced EphB2 (α-GFP). Left panels are IP samples and right panels are lysates. Tubulin was used as a loading control. (H) Quantification of ratio of coimmunoprecipitated GluN1 to total GluN1 in input. In control neurons infected with EphB2-EYFP, GluN1 coimmunoprecipitates robustly with EphB2 pull-down after ephrin-B2 treatment (top-left blot in E). In neurons expressing the Y504E mutant, GluN1 coimmunoprecipitates under control conditions without ephrin-B treatment (<i>p</i> = 0.0106, ANOVA followed by Fisher’s exact test, <i>n</i> = 6), and ephrin-B treatment did not potentiate GluN1 co-IP (<i>p</i> = 0.919, <i>n</i> = 6). Y504F mutants demonstrate little pull-down with GluN1 in absence of ephrin-B treatment (<i>n</i> = 6), and ephrin-B stimulation did not potentiate the EphB—NMDAR interaction in Y504 mutants (<i>p</i> = 0.549, ANOVA followed by Fisher’s exact test; <i>n</i> = 6). (I) Alignment of homologous regions of fibronectin type III (FN3) domains of EphB2 and EphA8. (J) IP of FLAG-EphB2 and FLAG-EphA8 from HEK293T cells cotransfected with HA-GluN1 and GluN2B. The first 3 lanes are transfected without NMDAR. The last 3 lanes are transfected with HA-GluN1 and GluN2B. The Control lane has only HA-GluN1 and GluN2B alone, B2 is transfected with FLAG-EphB2, and the A8 lane is transfected with FLAG-EphA8. Top blots were probed for α-GluN1, and bottom blots were probed for FLAG (EphB2). Left panels are IP samples and right panels are lysates. Tubulin was used as a loading control. (K) Quantification of ratio of coimmunoprecipitated GluN1 to FLAG IP. Immunoprecipitation from HEK293T cells transfected with FLAG-EphB2 or FLAG-EphA8 and GluN1 and GluN2B revealed that both EphA8 and EphB2 can effectively co-IP the GluN1 subunit of the NMDAR (*<i>p <</i> 0.05 versus control, ANOVA followed by Fisher’s exact test, <i>n</i> = 4).</p

Pain sensitivity is regulated by EphB2 Y504 and extracellular phosphorylation.

<p>(A) Experimental approach for panels B–C is illustrated. (B) Quantification of the effects of intrathecal (I.T.) injection of ephrin-B2-Fc (0.2 μg) on mechanical sensitivity in adult mice (***<i>p <</i> 0.001, ANOVA followed by Dunnet’s multiple comparison test, <i>n</i> = 6 mice). (C) Co-immunoprecipitation (co-IP) of GluN1 and EphB1 with α-FLAG (EphB1) antibody from HEK293T. All lanes are transfected with HA-GluN1 and GluN2B. The control lane is transfected with enhanced green fluorescent protein (EGFP), the wild type (WT) lane is transfected with EphB1 WT, the E lane is transfected with EphB1 Y502E, and the F lane is transfected with EphB1 Y502F. Top blots were probed for GluN1, and bottom blots were probed for EphB1. The left panels are IP samples pulled down with α-FLAG antibody, and the right panels are lysates. Tubulin was used as a loading control. (D) Experimental approach for panels (E–G) is illustrated. (E) Adult mice underwent unilateral plantar incision. 24 hours following incision, their spinal cords were separated into ipsilateral (+) and contra-lateral (-) sides to injury. Endogenous EphB1 was immunoprecipitated with α-EphB1 antibody from these tissues. Blots were probed for GluN1 and EphB1. EphB1 is expressed in the spinal cord and interacts with the N-methyl-D-aspartate receptor (NMDAR) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002457#pbio.2002457.ref025" target="_blank">25</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002457#pbio.2002457.ref061" target="_blank">61</a>]; therefore, for these experiments we used antibodies against EphB1. (F) Quantification of the effects of unilateral plantar incision on the EphB—NMDAR interaction (<i>p <</i> 0.05, Mann—Whitney U test, 8 spinal cords pooled per sample, <i>n</i> = 3). (G) Adult mice underwent plantar incision. Mice were given intrathecal injections of either vehicle control (black) or K252b (1 μg, purple) at the time of plantar incision and again 24 hours following incision (<i>n</i> = 6 mice for control and <i>n</i> = 5 mice for K252b) (for all graphs, *<i>p <</i> 0.05, **<i>p <</i> 0.01, ***<i>p <</i> 0.001, ****<i>p <</i> 0.0001, 1-way or2-way ANOVA followed by Dunnett’s multiple comparison test). (H) Experimental approach for panels (I–K) is illustrated. (I) Intrathecal injections of lentivirus transducing EGFP (black), enhanced yellow fluorescent protein (EYFP)-tagged EphB2 WT (blue), EphB2 Y504E (green), or EphB2 Y504F (red) were made into adult mice. Graph shows quantification of the effects of these injections on mechanical sensitivity in mice 1–4 weeks after injection of the virus (<i>n</i> = 8 mice for control before viral injection and <i>n</i> = 7 for 1–4 weeks after viral injection; <i>n</i> = 8 mice for EphB2 WT; <i>n</i> = 8 for EphB2 Y504E before viral injection; <i>n</i> = 7 for 2–4 weeks after viral injection; <i>n</i> = 8 for EphB2 Y504F). (J) Intrathecal injections of lentivirus transducing EGFP (black), EYFP-tagged EphB2 WT (blue), or EphB2 Y504E (green) were made into adult mice. Graph shows quantification of the effects of these injections on mechanical sensitivity in mice 1–4 weeks after injection of the virus (<i>n</i> = 6 mice for control, <i>n</i> = 8 mice for EphB2 WT, <i>n</i> = 6 for EphB2 Y504E before viral injection and <i>n</i> = 4 for 1–4 weeks after viral injection). Bars indicate the period of significant effects on sensitivity in EphB2 WT or Y504E-injected animals. (K) Four weeks after intrathecal injection of lentivirus transducing EGFP (black), EYFP-tagged EphB2 WT (blue), or EphB2 Y504E (green), mice were given intrathecal injections with K252b (1 μg), and effects on mechanical sensitivity were monitored at 1, 3, and 24 hours after injection (<i>n</i> = 6 mice for control before K252b injection and <i>n</i> = 3 mice for control after K252b injection; <i>n</i> = 6 mice for EphB2-WT before K252b injection and 1 hour after K252b injection; <i>n</i> = 5 mice for EphB2-WT 3 hours and 24 hours after K252b injection; <i>n</i> = 6 mice for EphB2-Y504E). Bars indicate period of significant effects on sensitivity in EphB2 WT or Y504E-injected animals following K252b injection. K252b reduced mechanical sensitivity in EphB2 WT—transduced but not in EphB2 Y504E—transduced mice.</p