Subunit-Specific Regulation Of Glutamate Receptors

我的实验室利用生物化学和分子技术的结合研究谷氨酸受体运输和定位的调节。谷氨酸受体是哺乳动物大脑中主要的兴奋性神经递质受体，是一个多样化的家族，有许多不同的亚型。嗜电性谷氨酸受体包括AMPA、NMDA和盐酸盐受体亚型，每一种受体都是由不同的亚基形成的。代谢性谷氨酸受体（mGluR1-8）是G蛋白偶联受体（gpcr），它们以同型二聚体的形式组装。我们专注于定义调节谷氨酸受体和突触支架蛋白的突触定位和功能调节的亚单位特异性机制。这些机制包括翻译后修饰，如磷酸化和泛素化，以及蛋白质-蛋白质相互作用。

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. We 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. Over the last decade, we have shifted to a new approach to studying structure/function of NMDARs using human genetics to inform our research. 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 GRIN genes (that encode NMDA receptor subunits. Specifically, we are examining variants causing mutations in the intracellular C-terminal domain of the GluN2 NMDAR subunits (GluN2A and GluN2B). As the human genetics data have accumulated, it has become clear that many de novo mutations in NMDA receptor subunits are highly associated with neurodevelopmental disorders including autism spectrum disorder, intellectual disability and epilepsy. Therefore, our goal is to better understand the synaptic dysfunction caused by these disease-associated rare variants with an eye towards developing therapeutics. Because of our expertise in studying receptor trafficking and protein interactions, we primarily focus on rare variants identified in the intracellular C-termini of NMDA receptor subunits, although we are also embarking on studies using mice with GRIN2B haploinsufficiency. In a recently published study, we characterized a rare variant in GluN2A (S1459G) identified in an epilepsy cohort (Mota Vieira et al., 2020). 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 phosphorylation of this residue dictates the receptor interactions with PSD-95 and sorting nexin 27 (SNX27). Thus we identified a regulatory site that determines the trafficking and synaptic expression of GluN2A-containing NMDA receptors. We are currently finishing up a related study on a GRIN2A rare variant (identified in a patient with neurodevelopmental disorders) encoding a frameshift that results in a truncation of half of the C-terminal domain as well as encoding a unique stretch of amino acids. We find that the receptor has increased surface expression, but specifically enriched at extrasynaptic sites. There are defects in protein interactions, spine density, and synapse number. Again, our approach reveals new findings in NMDA receptor structure/function. We hope to use the information from the analyses of different pathogenic rare variants to better understand NMDA receptor trafficking and localization and help test for more precise therapeutics.