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

Abstract

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.

Author summary

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.