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.

The activity of proteins can be finely and reversibly tuned by post-translational modifications. The attachment of phosphate groups to tyrosine residues is one of such modifications. While the existence of extracellular phosphoproteins has been known, the functional significance of extracellular phosphorylation is poorly understood. Here we describe a single extracellular tyrosine whose inducible phosphorylation may represent an archetype for a new class of mechanism mediating protein—protein interaction and regulating protein function. We show that the interaction between EphB2—which occurs upon receptor activation by its ligand ephrin-B—and the N-methyl-D-aspartate receptor (NMDAR) depends on extracellular phosphorylation of EphB2. This interaction regulates the localization of the NMDA receptor to synaptic sites in neurons. In vivo, EphB2 is phosphorylated in response to injury, and the subsequent up-regulation of the interaction between EphB2 and NMDA receptors enhances injury-induced pain behavior and mechanical hypersensitivity in mice. Importantly, our study defines a specific extracellular phosphorylation event as a mechanism driving protein interaction and suggests that extracellular phosphorylation of proteins is an underappreciated mechanism contributing to the development and function of the nervous system and synapse.

Funding: National Institute of Mental Health (grant number MH100093). National Institute of General Medicine (grant number GM102575). National Center for Research Resources (grant number RR027990). 100 Women in Hedge Fund Foundation. National Institute on Drug Abuse (grant number DA022727). National Eye Institute Vision Training Grant (grant number EY007035). National Institute of Neurological Disorders and Stroke (grant number NS050276). National Institute of Neurological Disorders and Stroke (grant number NS065926). The Vicki and Jack Farber Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we show that the EphB2 receptor tyrosine kinase undergoes ephrin-B ligand-induced extracellular phosphorylation of tyrosine residues (Y481 and Y504). Sequence analysis indicates that 1 of these amino acids, Y504, is widely conserved in fibronectin type III (FN3) domains of Eph proteins across phylogeny and is present in all Eph proteins known to interact with the NMDAR. In cortical and spinal cord neurons, ephrin-B—dependent induction of the EphB—NMDAR interaction is mediated by extracellular phosphorylation of Y504 on EphB2. The charge of Y504 is both necessary and sufficient for EphB—NMDAR interaction and regulates the amount of NMDARs found at synaptic sites. Virally mediated spinal cord expression of EphB2 or a phosphomimetic EphB mutant that induces the EphB—NMDAR interaction increases NMDAR levels in the dorsal horn of the spinal cord and results in mechanical hypersensitivity. Intrathecal injections of a membrane-impermeable ectokinase inhibitor that blocks the EphB—NMDAR interaction in cortical and spinal cord neurons reduces long-term hypersensitivity induced by EphB2 wild type (WT) but not pathological pain induced by injection of a phosphomimetic EphB mutant. These findings suggest that extracellular phosphorylation of EphB2 regulates NMDAR synaptic localization and function. Together the data suggest extracellular phosphorylation as a novel, dynamic mechanism that regulates protein—protein interactions at synapses to drive assembly of macromolecular complexes.

Underscoring the importance of the EphB—NMDAR interaction, the EphB—NMDAR interaction has been linked to a number of human diseases that are associated with NMDAR dysfunction. The pathological disruption of the ability of the EphB and NMDAR to interact has been linked to NMDAR dysfunction in Alzheimer disease [ 19 , 20 ] and in anti-NMDAR encephalitis [ 18 , 21 ]. A common feature of these findings is the disruption of the ability of EphBs to interact biochemically with the NMDAR and a rescue of the defects associated with the disease state by restoring the EphB—NMDAR interaction. The EphB-dependent enhancement of NMDAR activity associated with the EphB—NMDAR interaction is also linked to disease. EphB-dependent enhancement of NMDAR function plays a key role in sensitization of nociception, leading to chronic neuropathic and malignancy-induced pain through an unknown mechanism [ 22 – 25 ]. Because the interaction between EphBs and the NMDAR occurs in the extracellular space, it is thought to be a promising drug target [ 19 ]. However, despite the apparent importance of the EphB—NMDAR interaction, the molecular mechanisms controlling direct extracellular interaction between these proteins are unknown.

The direct extracellular interaction between the EphB receptor tyrosine kinase and the NMDAR appears to play important roles in the localization, function, and signaling of NMDARs [ 11 ]. The EphB family of RTKs consists of 5 members that bind to transmembrane ephrin-B ligands. EphB1–3 are essential for formation of up to 40% of excitatory synapses in the developing hippocampus and cortex, while in the mature brain EphBs are required for normal levels of synaptic NMDARs [ 12 – 14 ]. Ephrin-B binding to EphBs controls the localization and function of synaptic NMDARs by inducing a direct extracellular domain—dependent interaction with the NMDAR [ 11 , 14 – 17 ]. While in vitro binding assays indicate that EphBs bind the GluN1 subunit of the NMDAR via a direct extracellular interaction [ 11 , 18 ], the domain and molecular mechanism mediating the interaction between these 2 proteins remain undefined.

At excitatory synapses, glutamate receptors must be recruited and stabilized at synaptic sites. Of particular importance are interactions that maintain the proper localization of N-methyl-D-aspartate receptors (NMDARs), glutamate receptors that are essential for synaptic plasticity and development [ 8 ]. The synaptic localization, function, and signaling of NMDARs are regulated by intracellular scaffolding proteins such as PSD-95 [ 9 ], extracellular interacting proteins such as neuroligin-1 [ 10 ], and the EphB receptor tyrosine kinases (RTKs) [ 11 ]. While the mechanisms mediating intracellular interactions are well understood, the mechanisms mediating extracellular protein—protein interactions are not.

Modification of protein function by phosphorylation controls many aspects of cellular function and signaling [ 1 ]. Interestingly, the first evidence for phosphoproteins came from the observation that the secreted milk protein, casein, contained phosphate, suggesting that phosphorylation can occur in the extracellular space [ 2 , 3 ]. Recently, protein kinases that mediate the selective phosphorylation of extracellular serine, threonine, and tyrosine amino acids have been identified. For example, extracellular phosphorylation of serine and threonine residues can be mediated by Fam20C [ 4 , 5 ] and phosphorylation of extracellular tyrosine residues can be accomplished by vertebrate lonesome kinase (VLK or PKDCC), an essential gene expressed throughout the body, including the nervous system [ 6 , 7 ]. Yet despite identification of these kinases, the functional significance of extracellular phosphorylation and whether extracellular phosphorylation of proteins inducibly modulates their function remains largely unexplored.

Results

Extracellular phosphorylation of the EphB2 RTK EphB receptors interact directly with NMDA-type glutamate receptors through an undefined region of their extracellular domain [11]. The extracellular domain of the EphB receptor consists of a globular domain required for ephrin-B binding, a cysteine-rich domain, and 2 FN3 repeat domains of unknown function (Fig 1C). To study whether the extracellular region of EphB2 undergoes post-translational modification, we took an unbiased mass spectrometry—based approach: liquid chromatography tandem mass spectrometry (LC-MS/MS) in combination with receptor immunoprecipitation (IP) and phosphopeptide mapping (S1A–S1D Fig). FLAG-tagged EphB2 was expressed in the neuroblastoma cell line NG108, treated with either ephrin-B1 or control reagents, and immunoprecipitated. After enrichment of phosphopeptides using TiO 2 , LC-MS/MS identified the known tyrosine phosphopeptides in the juxtamembrane and kinase domains (S1 Table) of EphB2 and 2 tyrosine phosphopeptides (ELSEYNATAIK [Y481] and AGAIYVFQVR [Y504]) from the extracellular portion of EphB2 (Fig 1A–1C). Each extracellular peptide was identified in 4 independent experiments, with Mascot scores of 34 and 63, respectively, and definable separation from the next peptide assigned to that spectrum. Manual inspection of the tandem mass spectrometry (MS/MS) spectrum confirmed that the majority of ion signals present are accounted for by the assigned amino acid sequence and ions critical to localization of the site of phosphorylation are present. The 2 phosphopeptides in the extracellular region of EphB2 were both found in the C-terminal FN3 (cFN3) domain (see Fig 1C for schematic) and correspond to tyrosine residues Y481 and Y504. Interestingly, recent analysis of human non—small cell lung cancer cell line also identified Y481 as undergoing phosphorylation (PhosphoSitePlus; http://www.phosphosite.org), further supporting our analysis. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Identification of phosphorylated tyrosine in C-terminal fibronectin type III (FN3) (cFN3) of EphB2 extracellular region. (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 (S1E Fig, 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. https://doi.org/10.1371/journal.pbio.2002457.g001 Y504 and neighboring amino acid residues in the identified peptide are well conserved among different species (79.0% identical amino acids) (S1E Fig), within the Eph family of proteins (55.4% identical amino acids) (Fig 1D), and in other FN3-containing molecules (Figs 1E and S1F). In contrast, Y481 and neighboring residues in the identified peptide are less well conserved amongst different species (63.6% identical amino acids) (S1E Fig), within the Eph family (19.6% identical amino acids), and in other FN3-containing proteins (Figs 1D and 1E and S1F). Because Y481 is found only in EphB2 and was not conserved among other known NMDAR-interacting Eph family members (Fig 1D), we focused our study on Y504. Our analysis of the F-strand region of cFN3 domains indicates that Y504 is well conserved in proteins that contain homologous domains [11, 26]. Therefore, we next examined mass spectrometry (MS) databases and asked whether FN3-containing molecules that regulate axon guidance and target recognition might also contain phosphorylated tyrosines in FN3 domains that are similar to the cFN3 domain of EphB2. Interestingly, phosphorylated tyrosines have been identified in a number of these proteins at homologous residues. Proteins with previously identified phosphorylation sites include Sidekick1 and Sidekick2 as well as DSCAM1 (S1F Fig). These findings suggest that phosphorylation at EphB2 Y504 may be conserved among various species, Eph family proteins, and other synaptic FN3-containing molecules. To begin to determine whether Y504 might be phosphorylated, we generated a polyclonal phospho-specific antibody to tyrosine 504 of EphB2. Recognition by the phospho-Y504 antibody (p*Y504) was blocked by preabsorption with the immunogenic phospho-Y504 peptide (S1G Fig). In addition, the p*Y504 antibody recognized full-length EphB2 WT and full-length EphB2 Y481F but not nonphosphorylatable EphB2 Y504F, kinase-dead, or intracellular region—truncated EphB2 mutants (Fig 1F and S1H Fig). These results suggest that the antibody selectively recognizes phosphorylated Y504 and that the kinase domain may play a role in the phosphorylation of EphB2 Y504. To test whether Y504 phosphorylation of EphB2 is enriched in the cortex and spinal cord, we purified synaptosomes from these regions of WT mice. To validate our synaptosome purification, blots were probed for EphB2, PSD-95, and GluN1 [27]. The EphB p*Y504 signal was enriched in the synaptosome fraction and migrated at the same molecular weight as EphB2. To confirm that the signal detected was due to the presence of phosphate groups, the same blots were incubated with calf intestinal alkaline phosphatase (CIP) (Fig 1G). Incubation with CIP completely removed the signaling from the p*Y504 antibody but had no effect on the signal from the EphB2 antibody. Thus, EphB2 is likely to be phosphorylated on Y504 at synapses in both the brain and spinal cord (S1I Fig). Finally, to begin to address whether phosphorylation of Y504 on EphB2 might change as synapses mature, we asked whether the phosphorylation of Y504 was developmentally regulated. Synaptosomes were purified from P9, P15, and P21 WT mouse brains and probed for Y504 phosphorylation. We found that the levels of p*Y504 were elevated at P9 and P15 and declined with age. Interestingly, the pattern of p*Y504 parallels decreases in the proportion of NMDARs containing the developmentally regulated NMDAR subunit GluN2B (Fig 1H) [28, 29]. Together these findings indicate that EphB2 is phosphorylated on a highly conserved residue in vitro and in the brain and spinal cord at synaptic sites and this phosphorylation is down-regulated as synapses begin to mature.

Extracellular phosphorylation of EphB2 is required for interaction with the NMDAR EphBs are required for normal levels of synaptic NMDARs [14]. EphB2 binds to the NMDAR in both the cortex and the spinal cord [23] via an extracellular domain—dependent interaction that requires ephrin-B [11]. Therefore, we asked whether phosphorylation of EphB2 Y504 might be required for the EphB—NMDAR interaction in cortical and spinal cord neurons. In these experiments, dissociated cortical or spinal cord neurons were treated with soluble activated ephrin-B2 to activate the endogenous EphB—NMDAR interaction. We then conducted experiments to determine whether endogenous EphB2 Y504 was phosphorylated by ephrin-B2 treatment and whether pharmacological blockade of this phosphorylation might block the EphB—NMDAR interaction. To determine whether the EphB2–NMDAR interaction requires Y504 phosphorylation, endogenous extracellular kinase activity was blocked with K252b. Neurons were then treated with activated ephrin-B2 for 45–60 minutes, endogenous EphB2 was immunoprecipitated in a RIPA buffer, and blots were probed for endogenous GluN1 and p*Y504. Ephrin-B2 treatment of DIV 6–7 cultured cortical and spinal cord neurons effectively induced the EphB—NMDAR interaction and phosphorylation of Y504 (Fig 3A–3F). Pretreatment of neurons for 1 hour with K252b caused a significant decrease in phosphorylation of Y504 (p < 0.001, ANOVA followed by Fisher’s exact test; Fig 3B and 3E). K252b treatment did not alter the ability of ephrin-B2 treatment to stimulate EphB2 intracellular phosphorylation (Fig 3D and S3A Fig), suggesting that K252b does not alter the ability of ephrin-B to activate the EphB kinase. However, K252b treatment did cause a significant decrease in the ephrin-B—induced EphB—NMDAR interaction (**p < 0.01, ****p < 0.001, ANOVA followed by Fisher’s exact test; Fig 3C and 3F). These effects were particularly robust in cortical neurons but resulted in significant decreases in both cortical and spinal cord neurons. These data indicate that blockade of endogenous p*Y504 blocks the ability of EphB2 to interact with the NMDAR and suggest that the extracellular phosphorylation of EphB2 at Y504 is required for the EphB—NMDAR interaction. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Extracellular phosphorylation is induced by ephrin-B2 and mediates the EphB—N-methyl-D-aspartate receptor (NMDAR) interaction. (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 (**p < 0.01, ****p < 0.001, ANOVA followed by Fisher’s exact test; n = 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 (****p < 0.001, ANOVA followed by Fisher’s exact test; n = 5 experiments for each condition). https://doi.org/10.1371/journal.pbio.2002457.g003 Induction of phosphorylation requires ATP hydrolysis. If phosphorylation of Y504 is necessary for the EphB—NMDAR interaction, we expect that blocking extracellular ATP hydrolysis should block the interaction. Neurons were treated with the nonhydrolyzable ATP analogue ATPγS (1 μM) for 1 hour before ephrin-B treatment, and the effect on the EphB—NMDAR interaction was determined by co-IP. Bath application of ATPγS significantly reduced the ephrin-B—induced EphB—NMDAR interaction without affecting intracellular tyrosine phosphorylation of EphB2 (***p < 0.005, ****p < 0.001, ANOVA followed by Fisher’s exact test; S3B–S3D Fig). These findings suggest that phosphorylation of EphB2 at Y504 is required for the EphB—NMDAR interaction and that ephrin-B—dependent induction of this interaction can be blocked by blocking ATP hydrolysis in the extracellular space.

Phosphorylation of EphB2 is necessary and sufficient for the EphB—NMDAR interaction Phosphorylation of Y504 appears to be required for the EphB—NMDAR interaction in neurons. Therefore, we tested whether the phosphorylation of Y504 within the cFN3 of EphB2 might be necessary and sufficient for the EphB—NMDAR interaction (Fig 4A). To test this possibility, we generated an EphB2 phosphomimetic mutant (Y504E) and a nonphosphorylatable mutant (Y504F) and then examined the interaction between EphB2 mutants and GluN2B-containing NMDARs in HEK293T cells by co-IP. The ability of surface EphB2 to bind to ephrin-B2 was not altered by mutation of Y504 (Fig 4B and S4A–S4D Fig, p > 0.05, ANOVA), suggesting that the structure of the extracellular domain remains intact after mutation of this residue. In HEK293T cells, mutations to Y504 modulated the rate of removal of EphB2 from the cell surface; however, these effects were not seen in neurons (S5A–S5G Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Phosphorylation of extracellular tyrosine residue 504 of EphB2 is required for interaction with the N-methyl-D-aspartate receptor (NMDAR). (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 S4A Fig. For surface staining controls, see S4B–S4D Fig. 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 (*p < 0.05, ***p < 0.005, ****p < 0.001, ANOVA followed by Fisher’s exact test, n = 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 (*p < 0.05, **p < 0.01, ***p < 0.005, ANOVA followed by Fisher’s exact test, n = 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 (p = 0.0106, ANOVA followed by Fisher’s exact test, n = 6), and ephrin-B treatment did not potentiate GluN1 co-IP (p = 0.919, n = 6). Y504F mutants demonstrate little pull-down with GluN1 in absence of ephrin-B treatment (n = 6), and ephrin-B stimulation did not potentiate the EphB—NMDAR interaction in Y504 mutants (p = 0.549, ANOVA followed by Fisher’s exact test; n = 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 (*p < 0.05 versus control, ANOVA followed by Fisher’s exact test, n = 4). https://doi.org/10.1371/journal.pbio.2002457.g004 Mutation of the second phosphotyrosine identified by MS (Y481) had no effect on the ability of EphB2 to interact with the NMDAR, suggesting that this amino acid is not required for the EphB—NMDAR interaction (S4E and S4F Fig). In contrast, mutations of EphB2 at Y504 resulted in profound changes. Expression of the phosphomimetic EphB2 Y504E mutant resulted in a significant increase in EphB2 co-IP when pulled down with GluN1 antibody, while expression of the nonphosphorylatable EphB2 Y504F mutant caused a significant decrease in co-IP compared to WT (Fig 4C and 4D; p > 0.001, ANOVA followed by Fisher’s exact test). Similar effects were observed when antibodies against FLAG (FLAG-EphB2 or FLAG-EphB2 mutants Y504E or Y504F) were used to immunoprecipitate GluN1 (Fig 4E and 4F). These results indicate that the charge of this residue within the extracellular domain of EphB2 is necessary and sufficient for the EphB—NMDAR interaction in HEK293T cells. To test the effect of EphB2 Y504 mutants in neurons, DIV 2 cultured cortical neurons were transduced with lentiviruses expressing enhanced yellow fluorescent protein (EYFP)-tagged EphB2 WT, EphB2-Y504E, or EphB2-Y504F. Neurons were then challenged with ephrin-B2 treatment at DIV 9 for 45–60 minutes, and the ability of the GluN1 subunit of the NMDAR to co-IP with EphB2 was tested. In the control group, cultured neurons were transduced with EphB2 WT. In these neurons, little EphB—NMDAR interaction was detectable without ephrin-B stimulation, but ephrin-B stimulation induced a significant increase in the co-IP of GluN1 with EYFP-tagged EphB2 WT (Fig 4G and 4H; p = 0.0056, ANOVA followed by Fisher’s exact test). In contrast, neurons transduced with Y504E mutant EphB2 showed robust GluN1 pull-down even in the absence of ephrin-B treatment (Fig 4G and 4H; p = 0.0106, ANOVA followed by Fisher’s exact test), and ephrin-B treatment of neurons transduced with EYFP-tagged EphB2 Y504E did not further increase the EphB—NMDAR interaction (Fig 4G and 4H; p = 0.919, ANOVA followed by Fisher’s exact test). These findings suggest that Y504 phosphorylation is sufficient to induce the EphB2–NMDAR interaction in neurons. Neurons transduced with the Y504F mutant form of EphB2 failed to show GluN1 co-IP with EphB2, either with or without ephrin-B treatment (Fig 4G and 4H; p = 0.549, ANOVA followed by Fisher’s exact test). These data indicate that phosphorylated EphB2 Y504 is both necessary and sufficient for the EphB—NMDAR interaction in neurons and suggest that extracellular phosphorylation of a tyrosine in the cFN3 domain of EphBs may drive protein—protein interactions. To test whether phosphorylation of specific tyrosine residues and surrounding amino acids in the FN3 domain might provide a mechanism to mediate protein—protein interactions, we examined whether other Eph proteins with sequences similar to that found in EphB2 might interact with the NMDAR. EphB1–3 all interact with the NMDAR and have a similar set of amino acids near Y504 (XpYVXQVR), while the sequences of EphA3 and EphA4, which do not interact with the NMDAR, differ [11]. Interestingly, the sequence of EphA8, which was not previously known to interact with the NMDAR, is identical to EphB2 in this region (Fig 4I). To test whether EphA8 can associate with the NMDAR, we generated a FLAG-tagged EphA8 expression construct and coexpressed it along with GluN1 and GluN2B in HEK293T cells. We found that FLAG-EphA8 efficiently coimmunoprecipitates with the NMDAR from HEK293T cell lysates (Fig 4J and 4K). EphA8 is only 49.0% homologous to EphB2 outside of this region, while EphA4, which does not interact with the NMDAR, is 58.5% homologous; thus, these findings suggest that tyrosines with surrounding amino acids similar to EphB2 might be used to predict the ability of proteins to interact with the NMDAR.

Y504 regulates NMDAR localization in spinal cord Despite normal levels of NMDARs in EphB triple-knockout mice, in these animals NMDARs are redistributed from synaptic sites to extrasynaptic locations, suggesting that EphBs play an important role in regulating NMDAR localization [14]. To test whether Y504 might regulate the recruitment of NMDARs in vivo, neurons in the dorsal horn of the spinal cord were transduced by intrathecal injection of lentivirus coding for either EYFP-tagged EphB2 WT or the constitutively interacting EYFP-tagged EphB2 Y504E mutant (Fig 6A and 6B). This method results in transduction of approximately 60% of NeuN-positive cells in the region near the injection site [35]. Consistent with our previous findings, injection of the virus resulted in a focal infection of neurons (NeuN-positive cells, Fig 6C) within the dorsal aspect of the spinal cord near the injection site (Fig 6B–6D). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Expression of EphB2 recruits the N-methyl-D-aspartate receptor (NMDAR) to synaptic regions of the dorsal horn. (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 (*p < 0.05, ***p < 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. (***p < 0.0005, ****p < 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. https://doi.org/10.1371/journal.pbio.2002457.g006 The superficial nociceptive layers of the dorsal horn are vGlut1-negative and vGlut2-positive [36, 37]. To determine whether transduction of EYFP-tagged EphB2 results in changes in NMDAR expression within the nociceptive region of the spinal cord, sections were stained for GluN1 and vGlut2. In mice transduced with EYFP-tagged EphB2 WT or Y504E, the expression of the GluN1 subunit of the NMDAR was significantly up-regulated in the superficial layers of the dorsal horn compared to control and Y504F-injected mice (p < 0.05, ANOVA followed by Tukey’s range test; Fig 6D and 6E). In addition to the increase in NMDAR levels, there was also a significant increase in vGlut2 intensity (p < 0.0005, ANOVA followed by Tukey’s range test; Fig 6D and 6F). These changes in presynaptic vesicle markers are consistent with the known function of EphB2 in the induction of presynaptic terminal formation. Evidence suggests that different domains might mediate the role of EphB2 in synapse formation and the EphB—NMDAR interaction, with the ephrin-B binding domain of EphB2 being essential for induction of presynaptic differentiation [12, 38]. Consistent with this model, transduction of EphB2 Y504F resulted in a significant increase in the intensity of vGlut2 but not in GluN1 levels (Fig 6E and 6F). Regardless, these findings suggest that Y504 of EphB2 regulates the localization of the NMDAR in the spinal cord.