Identification of Mechanism Involved in the Maintenance of Pain Behaviors, Mechanical Hypersensitivity

A study recently published in PLoS Biology shows that in vivo extracellular phosphorylation of EphB2 by Ephrin-B upon injury promotes the binding of EphB2 to N-methyl-D-aspartate (NMDA) receptors on spinal and cortical neurons, and the subsequent upregulation of NMDA receptors at synaptic sites.¹ This extracellular phosphorylation of EphB2, a posttranslational modification often indicative of protein activation, was shown to maintain pain behaviors and mechanical hypersensitivity following injury.

The recruitment and stabilization of glutamate receptors, including NMDA receptors, to excitatory synapses are regulated by intracellular proteins that include EphB receptor tyrosine kinases. In addition, binding of presynaptic Ephrin-B to postsynaptic EphB in the extracellular space mediates the interaction between EphB and NMDA receptors in a manner independent of EphB kinase activity, a mechanism thought to be critical for synaptic development and function.² In the developed brain, the interaction between EphB and NMDA receptors modulates the synaptic expression of these glutamate receptors.³ Extracellular interaction between Ephrin-B and EphB has been implicated in allodynia associated with bone cancer,⁴ neuropathic pain,⁵ and chronic constriction injury.⁶ The extracellular localization of this regulatory mechanism makes it an attractive pharmacologic target.

Exogenous expression of EphB2 in a neuroblastoma cell line exposed to Ephrin-B1 led to the identification by mass spectrometry analysis of 2 phospho-tyrosine residues on the extracellular portion of EphB2, 1 of which (tyrosine [Y] 504) is well-conserved across species and found to be enriched in mouse spinal and cortical synaptosomes, the isolated synaptic terminals of these neuronal populations. In addition, the phosphorylation of this tyrosine residue was found to be induced by Ephrin-B2 in cultures of cortical neurons, an event shown to be both necessary and sufficient for the interaction between EphB2 and NMDA receptors.

In addition, NMDA receptor-dependent synaptic currents (evoked and miniature excitatory postsynaptic currents) and the accumulation of these receptors at the synapse, both in vitro and in vivo, were shown to both be dependent on Y504 phosphorylation of EphB2.

The intrathecal administration of Ephrin-B2 (known to induce the interaction between EphB and NMDA receptors) produced mechanical hypersensitivity in adult mice, as indicated by the reduction of the withdrawal threshold (n=6; P <.0001). This and other results indicate that the extracellular phosphorylation of EphB2 on Y504 is a mechanism that contributes to pathological pain and mechanical hypersensitivity.

This very comprehensive and thorough study shows that the phosphorylation of a single tyrosine residue on EphB2 in the synaptic cleft mediates the recruitment of NMDA receptors to the synapse and is a mechanism that contributes to the maintenance of mechanical hypersensitivity in response to injury. “Taken together, our findings suggest that extracellular phosphorylation of EphB…is a critical regulator of synaptic NMDA [receptor] function and has implications for understanding synaptic plasticity and disease, especially pathological pain,” concluded the researchers.

“[E]xtracellular phosphorylation of EphB mediates the direct extracellular EphB—NMDA [receptor] interaction to direct NMDA [receptors] to synaptic sites in a variety of neuronal types and regulates behaviorally relevant events such as pathological pain,” they added. 

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References

1. Hanamura K, Washburn HR, Sheffler-collins SI, et al. Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain. PLoS Biol. 2017;15(7):e2002457. doi: 10.1371/journal.pbio.2002457
2. Dalva MB, Takasu MA, Lin MZ, et al. EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell. 2000;103(6):945-956. doi: 10.1016/S0092-8674(00)00197-5
3. Nolt MJ, Lin Y, Hruska M, et al. EphB controls NMDA receptor function and synaptic targeting in a subunit-specific manner. J Neurosci. 2011;31(14):5353-5364. doi: 10.1523/JNEUROSCI.0282-11.2011
4. Dong Y, Mao-ying QL, Chen JW, Yang CJ, Wang YQ, Tan ZM. Involvement of EphB1 receptor/ephrinB1 ligand in bone cancer pain. Neurosci Lett. 2011;496(3):163-167. doi: 10.1016/j.neulet.2011.04.008
5. Kobayashi H, Kitamura T, Sekiguchi M, et al. Involvement of EphB1 receptor/EphrinB2 ligand in neuropathic pain. Spine. 2007;32(15):1592-1598. doi: 10.1097/BRS.0b013e318074d46a
6. Song XJ, Cao JL, Li HC, Zheng JH, Song XS, Xiong LZ. Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy. Eur J Pain. 2008;12(8):1031-1039. doi: 10.1016/j.ejpain.2008.01.011