Subunit-Specific Regulation Of Glutamate Receptors

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

My laboratory studies the regulation of glutamate receptor trafficking and localization using a combination of biochemical and molecular techniques. Glutamate receptors are the major excitatory neurotransmitter receptors in the mammalian brain and are a diverse family with many different subtypes. The ionotropic glutamate receptors include AMPA, NMDA, and kainate receptor subtypes, each of which are formed from a variety of subunits. The metabotropic glutamate receptors (mGluR1-8) are G protein-coupled receptors (GPCRs), which are assembled as homodimers. We focus on defining subunit-specific mechanisms that regulate the synaptic localization and functional regulation of glutamate receptors as well as synaptic scaffolding proteins. These mechanisms include posttranslational modifications such as phosphorylation and ubiquitination, as well as protein-protein interactions. A major focus of the lab is the study of the molecular mechanisms regulating NMDA receptors, and the goal is to better understand NMDA receptor function under normal circumstances and the specific dysfunction underlying some neurodevelopmental disorders. NMDA receptors are multi-subunit complexes (tetramers) composed of homologous subunits (GluN1; GluN2A-D; GluN3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of GluN2A and GluN2B in receptor trafficking and synaptic expression. We primarily focus on GluN2A and GluN2B because these subunits are highly expressed in hippocampus and cortex and are known to undergo activity- and developmentally-regulated trafficking events. Specifically, NMDA receptors are removed from synapses in an activity- and calcium-dependent manner via casein kinase 2 (CK2) phosphorylation of the PDZ-ligand of the GluN2B subunit (S1480). Based on our work over the last decade, we find that the NR2B subunit, and not NR2A, is specifically phosphorylated by CK2 and phosphorylation of NR2B increases in the second postnatal week and is important in the subunit switch (GluN2B to GluN2A), which takes place in many cortical regions during development and in response to activity. These data support unique contributions of the individual NMDA receptor subunits to NMDA receptor trafficking and localization. Our studies have shown that a single point mutation in the GluN2B C-terminus (E1479Q) completely blocks CK2 phosphorylation of S1480 and results in significant increases in synaptic GluN2B. We are currently characterizing two lines of genetically-altered mice. First, we have made a knock-in mouse expressing a point-mutant in GluN2B to render it non-phosphorylatable by CK2 (GluN2B E1479Q). This knock-in mouse allows us to examine the precise regulation of GluN2B S1480 phosphorylation in neurons, in vivo, and without the requirement of exogenous protein overexpression. In addition, we have also generated a knock-in mouse, GluN2B S1480E, which mimics phosphorylation of that key residue and constitutively blocks binding to PSD-95, which results in very low surface expression in our previous studies expressing exogenous subunits in neuronal cultures. Because these two point mutations have such profound (and inverse) effects on synaptic targeting of GluN2B-containing NMDARs, we hope to be able to study the consequences in vivo. This project is a multi-year initiative that has experienced some delays during the Covid 19 pandemic slow down and downsizing of animal colonies, but it is now again in progress. We also continue to explore the role of tyrosine phosphorylation on the regulation of synaptic NMDARs. GluN2B contains a classic tyrosine-based endocytic motif (-YEKL) that is a strong regulator of NMDAR surface expression. Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) target Y1472, which affects endocytosis and synaptic expression of receptors. We recently used a proteomics approach to define the STEP interactome. These experiments led us to characterize the effect of STEP expression on AMPARs. We also identified one of the metabotropic glutamate receptors, mGlu5, in the STEP interactome and we are characterizing the effect of that interaction as well. Clearly, STEP acts as a master regulator of glutamate receptor expression by driving dephosphorylation of key tyrosine residues within a variety of synaptic proteins (e.g. Fyn) and the glutamate receptors themselves. Over the last decade, we have shifted to a new approach to studying structure/function of NMDARs. We began with this bedside-to-bench strategy to help guide us in testing receptor domains that are important for synaptic function. We used information from published papers and public databases that report variants identified by deep sequencing of patients with neurological or psychiatric disorders. We then began conducting experiments on missense variants identified in the intracellular C-terminal domain of the GluN2B NMDAR subunit. As the human genetics data have accumulated, it has become clear that many de novo mutation in NMDA receptor subunits, encoded by GRIN genes, are highly associated with neurodevelopmental disorders including autism spectrum disorder, intellectual disability and epilepsy. Therefore, our studies are aimed to try to better understand the synaptic dysfunction with these disease-associated rare variants with an eye towards developing therapeutics. Because of our expertise in studying receptor trafficking and protein interactions, we focus on rare variants identified in the intracellular C-termini of NMDA receptor subunits. Our most recent study characterized a rare variant in GluN2A (S1459G) identified in an epilepsy cohort (the patient also was diagnosed with intellectual disability). This de novo mutation is within the extreme C-terminal domain near the PDZ ligand. We found that this serine is a CaMKII site and dictates the proteins interactions with PSD-95 and sorting nexin 27 (SNX27), thus regulating the trafficking and synaptic expression of GluN2A-containing NMDA receptors. We are currently studying a collection of truncating mutations identified in both GRIN2A and GRIN2B genes that are associated with neurodevelopmental disorders.