Molecular Mechanisms of the EphB-NMD Receptor Extracellular Interaction

Proper function in the mammalian central nervous system relies on the precise communication between billions of neurons. In order to develop distinct neural circuits, neurons must rely on tightly coordinated cell-cell interactions. These molecular interactions form the trillions of synapses in the mammalian brain. The trans-synaptic connection between neurons is essential for neuronal function. While the intracellular interactions and signaling regulating neuronal communication have been extensively studied, much less literature focuses on the role of extracellular interactions between receptors on the same cell. Interactions occurring outside of the cell are crucial to ensuring proper trans-synaptic communication. Additionally, extracellular interactions have been shown to regulate synaptic localization. Here I will discuss the facets of extracellular protein-protein interactions, their role in synaptic molecular organization, and the role of extracellular kinases in facilitating these interactions. In the first study, we describe a single extracellular tyrosine whose inducible phosphorylation may represent a new class of mechanism mediating protein-protein interaction and regulating protein function. We demonstrate that the interaction between EphB2 receptor tyrosine kinase, 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. Next, we describe a mechanism for extracellular protein-protein interactions mediated by the charge of extracellular domains. We show that the positive surface charge of the hinge region of the N-terminal domain in the GluN1 subunit of the NMDAR is required to maintain NMDARs at dendritic spine synapses and mediates the direct extracellular interaction with the negatively charged phospho-tyrosine on EphB2. Localization of NMDARs to dendritic spines is essential for excitatory synaptic transmission and plasticity and our work illustrates that receptor movement is constrained by protein-protein interactions with the extracellular domain of the NMDAR. We further demonstrate that loss of the EphB-NMDAR interaction by either mutating GluN1 or knocking down endogenous EphB2 increases NMDAR mobility. Lastly, I explore the possible mechanisms of EphB2 Y504 phosphorylation. I identify a secreted extracellular tyrosine kinase, vertebrate lonesome kinase (VLK), that is capable of phosphorylating EphB2 at Y504 and induces EphB2 interaction with the NMDA receptor. My findings also show that VLK fractionates with synaptic vesicles suggesting a mechanism of pre-synaptic secretion into the synaptic cleft. Together, these studies impact our understanding of the role and importance of extracellular protein-protein interactions in synaptic development and function