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.
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. 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. 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. 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. 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.
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 TiO2, 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. 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. To begin to examine the mechanism of EphB2 phosphorylation, we next asked whether EphBs might be phosphorylated on the cell surface after ephrin-B2 stimulation. Cultured cortical neurons were treated with activated ephrin-B2 for 45–60 minutes and then lysates were probed with the p*Y504 antibody. Consistent with the ephrin-B2-dependent activation of EphB2, ephrin-B2 treatment led to a significant increase in Y504 phosphorylation (Fig 2A and 2B). Blockade of EphB2 kinase activity after cell lysis had no effect on ephrin-B2–dependent phosphorylation of Y504 (S2A and S2B Fig), suggesting that phosphorylation of EphB2 at Y504 occurred prior to cell lysis. EphB2 can be internalized by clathrin-mediated endocytosis after ephrin-B stimulation [30]. To block internalization of EphB2, we blocked clathrin-mediated endocytosis with 450 mM hypertonic sucrose and asked whether ephrin-B2 treatment induced phosphorylation of EphB2 at Y504. Inhibition of endocytosis blocked ephrin-B2–dependent internalization of EphB2 (S2C and S2D Fig) but had no effect on ephrin-B2–induced extracellular tyrosine phosphorylation (p*Y504) of EphB2 (Fig 2A and 2B). These findings suggest that EphB2 undergoes ligand-dependent phosphorylation of Y504 on the cell surface of neurons. To further test whether the extracellular domain of EphB2 was phosphorylated on the surface of neurons, we conducted live-cell cell surface staining experiments. Because our Y504 phospho-specific antibody is not suitable for immunostaining, these experiments were conducted with the broad-spectrum and well-characterized phosphotyrosine antibody PY99. To induce Y504 phosphorylation, day in vitro (DIV) 7–16 cultured neurons were stimulated with activated soluble ephrin-B2 for 45 minutes, and live-cell staining was conducted with α-phosphotyrosine (PY99) or control antibodies recognizing intracellular proteins. As expected, after treatment with ephrin-B2 or control reagents, live-cell staining with intracellular α-actin antibodies only showed weak background staining (S2E Fig). In contrast, ephrin-B treatment of cultured neurons resulted in a marked increase in tyrosine phosphorylation staining on the cell surface at endogenous sites of ephrin-B binding compared to the control (Fig 2C–2F; p < 0. 01, ANOVA followed by Dunn' s multiple comparison test). We next asked whether any phosphotyrosine staining might be due to phosphorylation of Y504. To test this, cultured neurons were transfected at DIV 3 with either EphB2 WT or EphB2 Y504F. After ephrin-B2 treatment, surface staining with PY99 resulted in a significant increase in phosphotyrosine staining in the EphB2 WT-transfected neurons but not Y504F-transfected neurons (Fig 2G and 2H; p < 0. 05, ANOVA followed by Dunn' s multiple comparison test). These findings suggest that the majority of the surface phosphotyrosine signal induced by ephrin-B2 treatment can be attributed to phosphorylation at Y504. To begin to determine whether EphB2 might be phosphorylated by a kinase outside the cell, we blocked kinase activity selectively in the extracellular space by pretreating neurons with a membrane-impermeable ectokinase inhibitor, K252b [31]. In control in vitro experiments, K252b did not block EphB2 kinase activity (S2A Fig). Following 1 hour of K252b treatment of cultured neurons, ephrin-B2–induced surface tyrosine phosphorylation was significantly reduced (Fig 2C and 2D; p < 0. 01, ANOVA followed by Dunn' s multiple comparison test). Moreover, preincubation with the phosphatase inhibitor vanadate potentiated the ephrin-B—induced increase in surface PY99 staining of ephrin-B2 binding sites (Fig 2E and 2F; p < 0. 01, ANOVA followed by Dunn’s multiple comparison test). These findings suggest that ephrin-B2 stimulation of EphBs in neurons results in the tyrosine phosphorylation of the EphB ectodomain in the extracellular space. Both soluble and membrane-attached ectokinases have been identified in several cell types, including neurons [32]. We next sought to determine whether ephrin-B stimulation of neurons might induce a soluble activity sufficient to phosphorylate the extracellular domain of EphB2. To address this, the medium of cultured neurons was replaced with artificial cerebrospinal fluid (ACSF), a solution that contains only salts and glucose. Activated ephrin-B2 or control reagents were added to the ACSF, and the ACSF was collected after 45–60 minutes. The conditioned ASCF was then tested for an activity that could phosphorylate the ectodomain of EphB2 (Fig 2I). Treated media alone failed to phosphorylate the extracellular region of EphB2 (Fig 2J). Remarkably, the addition of 100 μM ATP, 10 mM magnesium acetate, and 10 mM manganese chloride to the ephrin-B2 treated ASCF resulted in robust phosphorylation of the EphB2 ectodomain (Fig 2J and 2K; p < 0. 005, ANOVA followed by Fisher’s exact test). This activity was absent in control ACSF with ATP added or ephrin-B2–treated media without ATP and blocked by heating the ephrin-B2–treated ACSF to 73°C (Fig 2J and S2F and S2G Fig). Taken together with the results from the live-cell staining experiments, these findings indicate that EphB2 Y504 is phosphorylated on the cell surface, likely by an ephrin-B2–induced soluble activity that requires ATP. 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. 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 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). 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. Early in development, EphBs function to control synapse formation [13], but later in neuronal development, EphBs are key regulators of NMDAR localization and function at synapses [14]. To test whether the extracellular phosphorylation of EphB2 Y504 is required for EphB-dependent NMDAR function and synaptic accumulation, we asked if mutation of EphB2 at Y504 might alter the NMDAR-dependent synaptic currents in cortical neurons (Fig 5A). To avoid effects on synapse development, neurons were transfected at DIV 14 with enhanced green fluorescent protein (EGFP) with or without EYFP-tagged EphB2 WT, Y504E, or Y504F constructs [14]. Cultures were infected at DIV 10 with adeno-associated virus transducing channelrhodopsin-2 to enable optical activation [33] (Penn Vector Core). To evaluate the strength of evoked NMDAR currents, we measured both spontaneous and optogenetically evoked synaptic currents in neurons at DIV 21–23 using whole-cell patch-clamp recording at 50 mV. Expression of EYFP-tagged EphB2 WT and Y504E both caused a significant increase in amplitude of the NMDAR-dependent component of excitatory postsynaptic currents (EPSCs) compared to the control or the Y504F mutant (Fig 5B and 5C; NMDAR component measured 30 milliseconds after the evoked EPSC peak; ****p < 0. 001, ANOVA followed by Fisher’s exact test). In addition, the amplitude of Y504E-expressing neurons was significantly higher than that of WT-expressing neurons (**p < 0. 02, ANOVA followed by Fisher’s exact test). To determine whether these effects were due to changes to the NMDAR at synaptic sites, spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of tetrodotoxin and blockers of GABAergic channels. Consistent with previous work [14], no significant changes in mEPSC frequency were observed between conditions (S6A and S6B Fig). As expected, expression of EYFP-tagged EphB2 WT and Y504E both caused a significant increase in amplitude of the NMDAR-dependent component of mEPSC compared to control or Y504F mutants (S6C and S6D Fig). These changes in mEPSC amplitude are attributable specifically to the increase of synaptic NMDARs because treatment with the NMDAR antagonist D-2-amino-5-phosphonovalerate (D-APV) (50 μM) blocked the effects of EphB overexpression and reduced mEPSC amplitude to a similar size in all transfection conditions (S6E and S6F Fig; NMDAR component measured 15–25 milliseconds after the mEPSC peak; p < 0. 01; ANOVA followed by Fisher’s exact test). Cumulative probability histograms of events from neurons transfected with EphB2 Y504E or EphB2 Y504F indicate that the NMDAR-dependent component of mEPSC amplitude is greater in EphB2 Y504E than in EphB2 Y504F—expressing neurons (S6F Fig, p < 0. 0001, Kolmogorov—Smirnov [K–S] test). APV blocks all NMDARs, but previous work suggests that EphBs may preferentially act on GluN2B-containing NMDARs in younger neurons [14]. Therefore, we asked whether mutations to EphB2 at Y504 that enhance (Y504E) or prevent (Y504F) the EphB—NMDAR interaction might drive or exclude GluN2B-containing NMDAR from synapses. We selectively blocked GluN2B-containing NMDAR with the GluN2B-specific antagonist Ro 25–6981 (Ro-25) and determined the effect on the NMDAR component of mEPSCs in cultured cortical neurons. Whole-cell patch-clamp recordings of mEPSCs revealed that NMDAR-dependent currents in neurons transfected with EphB2 Y504E decreased significantly with selective inhibition of GluN2B-containing NMDARs (Fig 5D and 5E, p < 0. 001, K–S test). However, Ro-25 treatment had no significant effect on mEPSC amplitude in Y504F-overexspressing neurons (Fig 5D and 5F). These data suggest that EphB2 preferentially recruits GluN2B-containing NMDARs to synaptic sites. We next asked whether the accumulation of NMDARs at synaptic sites is altered by expression of EphB2 Y504 mutants in mature neurons. To test this, we conducted 2-color super-resolution imaging using stimulated emission depletion (STED) microscopy of FLAG-EphB2 or FLAG-EphB2 mutants (Y504E and Y504F) and endogenous GluN1 combined with 2-color confocal imaging of vGlut1 and cell-filling EGFP expressed in DIV 21–23 cortical neurons [34]. EphBs are required for normal numbers of dendritic spine synapses and the proper number of NMDARs at synaptic sites [13]. Therefore, we focused on analyzing spine synapses. Synapses were defined by the presence of 1 or more vGlut1 puncta that contacted a spine (Fig 5G). Consistent with our previous findings and physiological data, the percentage of vGlut1-positive spines did not differ between the transfection conditions, with approximately 80% of imaged spines containing vGlut1 puncta (Fig 5I) [14]. Using 2-color STED super-resolution imaging, we determined the proportion of vGlut1-positive spines containing FLAG-EphB2 and GluN1 puncta. Approximately 30% of spines contained FLAG-EphB2 WT puncta that colocalized with 1 or more puncta of GluN1 (Fig 5G, 5H and 5J). We next examined the impact of the expression of FLAG-EphB2 Y504E. FLAG-EphB2 Y504E interacts with the NMDAR independent of ephrin-B ligand activation; therefore, we expect that expression of FLAG-EphB2 Y504E would increase the number of synapses where EphB2 and the GluN1 subunit of NMDAR colocalize. Indeed, STED analysis revealed that both the fraction of spines containing colocalized FLAG and GluN1 puncta and the fraction of spines with GluN1 colocalization with EphB2 at synaptic sites were significantly increased in Y504E-expressing neurons compared with EphB2 WT- or Y504F-expressing neurons (*p < 0. 05, **p < 0. 01, ANOVA followed by Fisher’s exact test; Fig 5G, 5H, 5J and 5K). Interestingly, in these experiments, expression of EphB2 Y504F did not appear to act in a dominant negative manner. Although we knocked down endogenous EphB2, the lack of an effect of EphB2 Y504F might be due to the presence of other EphB proteins or might reflect the presence NMDAR-interacting proteins such as PSD-95. Regardless, these findings indicate that phosphorylation of Y504 enables EphB2 to recruit or retain NMDARs to spine synapses. 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). 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. Ephrin-B—EphB signaling to the NMDAR is implicated in pain plasticity, leading to pathological pain states [39,40]. Given the impact of overexpression of EphBs on NMDAR levels in the dorsal synaptic region of the spinal cord that is associated with pain, we next examined whether the EphB—NMDAR interaction might be enhanced in models of pain. Consistent with previous reports on the role of ephrin-Bs in induction of pain and pain plasticity [40,41], intrathecal injection of activated ephrin-B2, which activates EphBs and the EphB—NMDAR interaction [11] (Fig 7A, 0. 2 μg), induced robust, sustained mechanical hypersensitivity in mice (p < 0. 0001, ANOVA followed by Dunnett’s multiple comparison test; Fig 7B). Thus. injection of activated ephrin results in hypersensitivity. Because EphB1 is expressed more highly than EphB2 in the spinal cord and is known to interact with the NMDAR (S7C Fig) [23,42,43], we tested whether the region homologous to Y504 in EphB2 was also important for the previously described EphB1–NMDAR interaction. EphB1 contains a homologous tyrosine residue at Y502 (Fig 1D). Therefore, we hypothesized that the charge of this residue might regulate the EphB1–NMDAR interaction. Consistent with this model, compared to FLAG-EphB1, WT co-expression of FLAG-EphB1 Y502E with GluN1 and GluN2B in HEK293T cells resulted in increased coimmunoprecipitation, while the interaction was significantly reduced when FLAG-EphB1 Y502F was coexpressed (Fig 7C and S7A Fig). We next asked whether EphB1 is also phosphorylated at Y502. HEK293T cells were transfected with either EphB1 WT or EphB1 Y502F, and lysates were probed with the p*Y504 antibody. Consistent with selective phosphorylation of Y502, only WT EphB1 was recognized by the p*Y504 antibody (S7B Fig). These findings suggest that the ability of both EphB1 and EphB2 to interact with the NMDAR is mediated by phosphorylation of homologous tyrosine residues (Y502 and Y504 respectively). Next, we asked whether injury-induced mechanical hypersensitivity might induce the EphB—NMDAR interaction (Fig 7D). Consistent with a role for the EphB—NMDAR interaction, unilateral plantar incision (a mouse model sensitive to NMDAR receptor blockade [44]) resulted in a significant increase in the coimmunoprecipitation of the GluN1 subunit of the NMDAR with EphB1 (51. 3 ± 26. 4-fold increase, p < 0. 05, Mann—Whitney U test, 8 hemispinal cords pooled per sample, n = 3, Fig 7E and 7F). In the spinal cord, detection of phosphorylation of EphB1 and EphB2 was difficult because of the small amounts of tissue available from each animal and levels of EphB expression. However, by pooling samples we were able to observe modest effects on EphB2 Y504 and Y502 phosphorylation in spinal cord samples after injury (S7C Fig). Importantly, the increase in the EphB—NMDAR interaction was only observed in tissue from the same side as the injury, indicating that this effect is likely specific to the injury paradigm. Next, to begin to test whether extracellular phosphorylation might be important for the behavioral response to injury, we tested whether blockade of extracellular kinase activity with the membrane-impermeant drug K252b might rescue injury-induced mechanical hypersensitivity (Fig 7D). We used intrathecal infusion, a method allowing for local infusion of drugs and widely used clinically to allow for spinal cord—selective treatment [45]. Intrathecal injections of K252b (1 μg) at the time of plantar incision significantly reduced mechanical hypersensitivity for up to 24 hours after incision in adult mice (Fig 7G). These results support a model in which inhibiting extracellular phosphorylation can reduce pain signaling and mechanical hypersensitivity behavior (S8 Fig). To begin to test whether the EphB—NMDAR interaction might play a role in pathological pain, we asked whether inducing the EphB—NMDAR interaction with transduction of EYFP-tagged EphB2 WT or Y504E that results in increased levels of NMDAR in the spinal cord (Fig 6) might result in mechanical hypersensitivity (Fig 7H). Intrathecal injection of lentivirus transducing either EYFP-tagged EphB2 WT or Y504E resulted in a long-lasting enhancement of mechanical sensitivity (Fig 7I and 7J). Consistent with in vivo and in vitro immunostaining and physiological data (Figs 5 and 6), we find that intrathecal injections of the Y504F mutant did not result in a long-lasting increase in mechanical hypersensitivity (Fig 7I). Thus, transduction of EYFP-tagged EphB2 WT or Y504E results in mechanical hypersensitivity and increased levels of GluN1 in regions of the spinal cord linked to pathological pain (Figs 6 and 7I). Injections of lentivirus transducing WT EphB2 and EphB2-Y504E resulted in long-lasting mechanical hypersensitivity, even 2 months after virus injection (Fig 7I and 7J). To test whether these effects are due to phosphorylation of Y504, we asked next whether inhibition of extracellular phosphorylation might block the effects of EphB2 expression. If mechanical hypersensitivity depends on phosphorylation of Y504, then blocking extracellular phosphorylation should only reduce or rescue mechanical hypersensitivity in mice transduced with WT but not in those with Y504E EYFP-tagged EphB2. To avoid potential effects of inhibiting the EphB—NMDAR interaction outside of the spinal cord [45], intrathecal injections of K252b were made near the site of viral injection. Remarkably, intrathecal injection of K252b resulted in rapid (<3 hours) and sustained (>24 hours) reversal of mechanical hypersensitivity compared to control levels when injected into mice overexpressing WT EYFP-tagged EphB2 (p < 0. 01,2-way ANOVA followed by Dunnett’s multiple comparison test; Fig 7J and 7K). Importantly, the rescue of mechanical hypersensitivity to control levels in EphB2 WT—transduced mice (Fig 7K) was found in animals having experienced increased levels of pain for 2 months. We next conducted a series of controls. Although we did not observe evidence of action of K252b outside of the spinal cord, and intrathecal infusion of drugs is used clinically to avoid side effects seen with other injection methods [45], the drug used to block the interaction (K252b) could act elsewhere in the brain. However, injection of K252b had no effects in control EGFP-expressing mice (Fig 7K). Next, to control for effects of K252b injection outside of the spinal cord and to demonstrate the specific action of this compound on EphB2 with an intact extracellular phosphorylation site, we asked whether K252b might alter the mechanical hypersensitivity in mice expressing the constitutively interacting EphB2 Y504E mutant. Importantly, in mice expressing the EYFP-tagged EphB2 Y504E construct, injection of K252b did not block mechanical hypersensitivity (Fig 7K). Thus, K252b, which inhibits extracellular phosphorylation of Y504, is acting selectively within the spinal cord in WT EphB2-overexpressing mice to block mechanical hypersensitivity. Taken together, these findings support a model wherein the EphB—NMDAR interaction mediated by extracellular phosphorylation of EphB cFN3 induces amplification in pain signaling, causing mechanical hypersensitivity (S8 Fig).
Phosphorylation of protein ectodomains can regulate Drosophila wing development, neurite outgrowth, osteoclast migration, and aggregation of amyloid beta [46–49]; however, these events appear to occur largely constitutively and are not known to be regulated by cell—cell signaling. In contrast, phosphorylation of Y504 on EphB2 is induced by ephrin-B and results in the recruitment of NMDAR to synaptic sites. The activity that mediates extracellular phosphorylation of EphB2 is present in ACSF of cultured neurons after eprhin-B2 stimulation and requires ATP. Importantly, ample ATP is found at synaptic terminals [50] and ATP can be released into the extracellular space by synaptic or secretory vesicles [51,52]. Together with recent work demonstrating that a wide variety of proteins are constitutively extracellularly phosphorylated [4,5] our data suggest a model in which, similar to intracellular tyrosine phosphorylation, inducible extracellular tyrosine phosphorylation of specific amino acids can regulate extracellular domain—mediated protein—protein interactions. Intracellular phosphorylation sites enable protein—protein interactions with specific amino acid motifs. Similarly, the conserved region surrounding the Y504 residue on EphB2 appears to define a new extracellular interaction domain. Based on homology, we could accurately predict that EphA8 would also interact with the NMDAR. In contrast, the corresponding regions of EphA3 and EphA4 that do not co-IP with the NMDAR are less homologous to EphB2 [11], although we have not ruled out the possiblity that they may also become phosphorylated. In addition, mutation of the homologous amino acid on EphB1 (Y502) can drive increased or reduced interactions with the NMDAR. These data suggest that the motif XpYVXQVR may be important for interactions between the extracellular domains of proteins. Phosphoprotein databases provide additional examples of phosphorylated tyrosine residues with unknown function in homologous regions of FN3 domains of proteins linked to synaptogenesis, axon guidance, and target recognition, such as Sidekicks and DSCAML1 (S1F Fig). It will be important to determine whether phosphorylation of the FN3 domain of other proteins also regulates protein—protein interactions and whether extracellular phosphorylation is an underappreciated mechanism contributing to the development and function of the nervous system and synapse. How might extracellular phosphorylation enable protein—protein interaction? In the intracellular space, changes in the charge of tyrosine residues in specific amino acid motifs control the binding of specific SH2 domain—containing molecules [1]. Interestingly, the EphB2 Y504F constructs fail to interact with the NMDAR but are likely not functioning in a dominant negative manner. These findings are consistent with a model in which the EphB—NMDAR interaction is mediated by charge because phenylalanine is not a positively charged amino acid. The role of charge is difficult to test, as positively charged amino acids are bulky and may affect protein—protein interactions in other ways, such as steric hindrance. However, a dominant negative mutant should be able to be designed using surface charge analysis of the region surrounding Y504. It is likely that phosphorylation of the Y504 residue would alter the secondary structure or interaction surface of the FN3 domain. Supporting this idea, analysis of the crystal structures of Eph family members indicates that Y504 of EphB2 is exposed and suggests that the cFN3 of Eph family members, but not the N-terminal FN3 (nFN3) domain, might have a pocket for binding to other molecules [53]. Indeed, the crystal structures of ligand-unbound EphA4 and ligand-bound EphA4 indicate that ligand binding results in changes to the structure of cFN3 [54]. These data suggest that phosphorylation of Y504 could modify FN3 structure. Consistent with this model, phosphorylation of Y504 in EphB2 is necessary and sufficient to induce binding to the NMDAR, and we find that homologous FN3 domains and tyrosine residues can be phosphorylated. However, detailed structural analysis will be required to resolve these questions. FN3 domains of other synaptic proteins contain a homologous residue, suggesting that extracellular phosphorylation is a novel mechanism that may mediate many events related to synaptic function and behavior. Extracellular phosphorylation of EphB2 at Y504 stabilizes GluN2B-containing NMDARs on the cell surface and drives the accumulation of GluN2B-containing NMDARs at synaptic sites in young neurons. During development, GluN2B-containing NMDARs predominate at synaptic sites, and the presence of GluN2B-containing NMDARs at synapses can enhance synaptic plasticity [55]. Interestingly, phosphorylation of EphB2 at Y504 is down-regulated as synapses mature, and the proportion of GluN2B-containing NMDARs is reduced. EphB2 regulates a number of other aspects of GluN2B-containing NMDAR function. EphB2 activation can drive the Src kinase—dependent phosphorylation of GluN2B on specific tyrosine residues that prevent AP2-mediated internalization [56]. The kinase-active EphB2 reduces calcium-dependent inactivation of GluN2B-containing (but not GluN2A-containing) NMDAR currents [14]. However, in adult animals, EphBs are required for normal synaptic levels of both GluN2A and GluN2B, suggesting a role for additional developmentally regulated mechanisms. It remains to be determined whether the ability of EphB2 to interact with GluN2A-containing NMDAR relies on the same Y504 site or a different mechanism. Further indication of the importance of extracellular modification of the EphB2 receptor is suggested by findings in human disease models. Disruption of the EphB—NMDAR interaction in models of Alzheimer disease [19] or in NMDAR encephalitis [18] that results in reduced surface levels of NMDAR may involve the FN3 domain of the EphB2 protein, further supporting the importance of understanding phosphorylation of these domains. The kinase regulating the phosphorylation of Y504 on EphB2 remains to be identified. Interestingly, Y504 is not phosphorylated on the kinase-dead version of EphB2 (EphB2 KD) implicating the kinase domain of EphB2. It is not likely, though, that the ephrin-B—induced extracellular phosphorylation of EphB2 Y504 is phosphorylated directly by the EphB2 kinase because (1) p*Y504 requires extracellular ATP, (2) p*Y504 occurs on the cell surface, (3) p*Y504 is blocked by a membrane-impermeable kinase inhibitor K252b, (4) extracellular treatment with phosphatases augments the interaction, (5) an activity that can phosphorylate Y504 is secreted following ephrin-B treatment, and (6) although K252b effectively inhibited p*Y504, it failed to block EphB2 kinase activity. However, while the EphB2 kinase is unlikely directly responsible for phosphorylating Y504, given that p*Y504 requires ephrin-B activation of EphB2, it is likely that the EphB2 kinase participates in the signal transduction pathway necessary for p*Y504. Further work will be needed to identify the genuine Y504 kinase. One interesting set of candidates is the recently identified family of extracellular kinases (such as Fam20C) that function in the secretion pathway [46]. A second class of candidates is represented by the tyrosine kinase VLK, which can be secreted from platelets and is an essential gene [6,7]. The mechanism of action of this family is unknown, but since these kinases can function extracellularly, they might be good candidates for the genuine p*Y504 kinase, which appears to be secreted. While disorders like Alzheimer disease and NMDAR-encephalitis may be linked to pathological reduction of the EphB—NMDAR interaction [18,21], induction of hypersensitivity and pain can occur by enhancing EphB-dependent effects on NMDAR function [39]. Indeed, increases in the amount of ephrin-B ligand have been reported in the spinal cord in pain models. Consistent with a role for the EphB—NMDAR interaction are the following: (1) MK801 blocks the effect of ephrin-B1 on pathological pain [40], (2) local intrathecal application of EphB-Fc blocks pathological pain [24,42], and (3) Nav1. 8+ nociceptive sensory neuron (DRG) -specific ephrin-B2 knockout regulates inflammatory and neuropathic pain [57]. These findings suggest that in the spinal cord and periphery, EphB-dependent modulation of NMDAR function drives NMDAR-dependent hyperexcitability of spinal circuits, causing pathological pain associated with traumatic nerve injury and cancer-induced nerve damage [25,41,58,59]. Consistent with these previous findings, we show that a single intrathecal injection of activated ephrin-B2 results in hypersensitivity. In addition to functions in the induction of pathological pain, we find that the EphB—NMDAR interaction also appears to play an important role in maintaining long-lasting mechanical hypersensitivity. Virally induced expression of WT EphB2 in dorsal horn neurons results in sustained mechanical hypersensitivity. Importantly, injection of K252b 2 months after induction by an EphB2-expressing virus rapidly alleviates this mechanical hypersensitivity. These findings suggest that inhibition of extracellular phosphorylation might provide a target suitable to alleviate more chronic pain states. In animals injected with virus transducing the EphB2 Y504E point mutant that induces a constitutive, ligand-independent interaction with the NMDARs, injection of K252b did not influence this established mechanical hypersensitivity. This underscores the specific, local effect of extracellular EphB2 phosphorylation. Consistent with clinical practices [45], our findings indicate that intrathecally injected K252b is acting selectively within the spinal cord on WT EphB2 overexpressing cells and not likely targeting regions outside of the spinal cord. In addition to blocking EphB2-EYFP—induced mechanical hypersensitivity, K252b reduced incision-induced mechanical hypersensitivity, suggesting a key role for EphB—NMDAR interactions in this context, a notion supported by biochemical findings in our models. Although K252b is not itself a suitable compound for further development, these data suggest that extracellular phosphorylation and inhibition of the EphB—NMDAR interaction may provide potential therapeutic targets for the mitigation of pathological pain. In summary, extracellular phosphorylation of EphB mediates the direct extracellular EphB—NMDAR interaction to direct NMDARs to synaptic sites in a variety of neuronal types and regulates behaviorally relevant events such as pathological pain.
All animal procedures were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University, the University of Pennsylvania, the University of Texas at Dallas, and the University of Arizona and were in accordance with International Association for the Study of Pain guidelines (protocol numbers: 01286 and 01797 TJU, 14–04 UTD, 802645 UPenn and 09–115 UA). Embryonic day 17 (E17) to E18 Long—Evans rats (Charles River) were used for preparing dissociated cortical neural culture as previously described [13,14]. Postnatal day 30 wild-type CD1 mice (Charles River) were used for synaptosome preparation as previously described [14]. Male ICR mice (Harlan) were used for all behavioral studies. Mice were used for behavioral experiments starting at 8–12 weeks of age. For LC-MS/MS analysis, NG108 cells were transfected with FLAG-tagged EphB2 receptor, treated with ephrin-B1, and lysed. Samples immunoprecipitated with α-FLAG antibody were separated by SDS-PAGE, the gels were stained with Coomassie Blue, and the EphB2 band was excised and digested with trypsin in-gel. After enrichment of phosphopeptides using TiO2, LC-MS/MS analysis was conducted (Thermo Fisher Scientific LTQ-Orbitrap). Generation of FLAG-tagged full-length EphB2, truncated EphB2 (fEphB2 Tr), and kinase-dead (KD; K663R) EphB2 were previously described [11]. Generation of EphB2-EYFP was previously described [60]. Single amino acid point mutations to Y481 and Y504 were introduced using sequence-specific primers and site-directed mutagenesis (Stratagene, La Jolla, CA). EphB2 expression constructs were generated based on published sequences. Expression of each EphB2 version used was validated using RT-PCR from mouse brain cDNA. In some cases, the RT-PCR products were validated by sequencing. FLAG- and EYFP-tagged EphB2 expression constructs gave similar results in assays tested. Lentiviruses were produced and purified by the Gene Therapy Program at the Penn Vector Core facility of the University of Pennsylvania. HEK293T cells were transfected using the calcium phosphate method. Dissociated cortical neurons were prepared from embryonic day 17 (E17) to E18 rats. Dissociated spinal cord neurons were prepared from E14. 5 rats. Cultured neurons were transfected at DIV 3 or 14 using Lipofecatmine 2000. Immunoprecipitations and western blot analysis were performed as previously described with small changes [11]. Cell-surface biotinylations were performed as previously described [14,38]. For phosphotyrosine surface staining, live neurons were incubated for 10 minutes at room temperature in PBS with α-phosphotyrosine, then washed and fixed. For synaptic NMDAR staining, cells were fixed and permeabilized with 0. 1% saponin at room temperature. For immunohistochemical analysis, spinal cord tissues were frozen in OCT compound and cut into sections and then fixed and stained. Images were obtained using Leica TCS SP5 confocal scanning microscopy and Leica TCS SP8 STED microscopy. Analysis was done using NIH ImageJ. All data shown as mean ± SEM. Phospho-specific antibodies against α-EphB2 Y504 were generated against a phosphopeptide of the sequence Ac-CKGLKAGAI-pY-VGQVRA-NH2 (Covance, Denver, PA). Synaptosomes were prepared as previously described [14]. Electrophysiological recordings from DIV 21–23 cultured rat cortical neurons were performed using whole-cell patch methods as previously described [14]. Evoked currents were generated by light stimulation (470 nanometers) of neurons expressing channelrhodopsin-2. Behavioral testing and drug administration were performed as described previously [35].
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.
phosphorylation
medicine and health sciences
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nervous system
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tyrosine
immunoprecipitation
amino acids
hypersensitivity
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aromatic amino acids
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post-translational modification
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Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain