Project Summary / Abstract It has recently been discovered that G-protein coupled receptors (GPCR) can be regulated by extracellular interactions. The mechanisms behind this phenomenon remain poorly understood. One such interaction is the trans-synaptic connection between presynaptic group III metabotropic glutamate receptors (mGluR) and postsynaptic cell-adhesion molecules of the extracellular leucine-rich repeat and fibronectin type III domain-containing (ELFN) family. Group III mGluRs sense glutamate release and inhibit further release in a negative feedback mechanism. A great body of evidence points to the essential role of synaptic glutamate homeostasis in a range of cognitive and motor functions with dysregulation leading to movement disorders and epilepsy. Binding and stabilization of mGluRs by trans-synaptic interactions with ELFN proteins is critical for regulating glutamate homeostasis. Loss of ELFN proteins in mice tremendously augments glutamate release and results in seizures and hyperactivity. Understanding how these proteins interact in the synaptic environment will provide valuable insight into how neurons maintain control over glutamate levels in the synapse and has implications for physiology and disease. Importantly, both mGluRs and ELFN proteins have been shown to form homo- and hetero-dimers. This proposal aims to test the hypothesis that apart from positioning and stabilizing group III mGluRs at the synapses, ELFN proteins act to allosterically modulate mGluR activity in part by influencing the dimerization dynamics of mGluR subunits. To test this hypothesis, I will build a structural model of the ELFN-mGluR interaction using crosslinking coupled with mass spectrometry, hydrogen/deuterium exchange, and cryo-electron microscopy to understand what binding determinants govern the ELFN-mGluR binding interaction. Additionally, I will investigate the mechanisms of mGluR allosteric modulation by ELFN proteins using a variety of cell-based signaling assays. Together, these experiments will provide a clear foundation for understanding how the brain maintains glutamate homeostasis inside the synapse.