| N-Methyl-D-aspartate receptors (NMDARs) are a principal subtype of excitatory ligandgated ion channel with prominent roles in physiology and disease in the mammalian central nervous system (CNS). Activation of NMDARs requires binding of both glutamate and glycine. Apart from its co-agonist action, glycine can also prime NMDARs for subsequent internalization upon binding of both glutamate and glycine. However, the molecular basis responsible for mediating and regulating glycine priming and NMDAR endocytosis is largely unknown. In my thesis, I discovered the principle that although NMDAR gating and priming share a common requirement for glycine binding, the molecular constraints for gating are distinct from those for priming through two mutations of the glycine binding site in GluN1 subunit of the NMDAR that, while maintaining gating of NMDARs, eliminate glycine priming of the receptors. One of the molecular signatures of glycine priming is recruitment of the endocytic adaptor protein AP-2. I have characterized the two regions in GluN2 subunits required for enhanced AP-2 association. This unexpected result suggests binding of glycine initiates a conformational change transmitted from GluN1 to GluN2 allowing for docking of endocytic machinery. Furthermore, I have discovered that naturally occurring splice variants of GluN1 subunit, containing a 21 amino acid sequence in the N-terminus domain (N1) cassette, abrogate glycine stimulated AP-2 recruitment and glycine-primed NMDAR internalization. These findings imply that there are distinct populations of native NMDARs in the CNS -- those lacking N1 that show glycine-primed internalization and those containing N1 that are not primable. Collectively, my thesis work demonstrates a dramatic all-or-none priming effect with splice variants of NMDARs, a highly unexpected discovery providing novel insight into the molecular mechanisms and physiological role of glycine priming. Ultimately, elucidating principles and mechanisms of glycine priming lay the foundation for new types therapeutic approaches for CNS disorders, approaches without the deleterious consequences of directly blocking NMDARs. |
