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. 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) totally 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 mutants have such profound (and inverse) effects on synaptic targeting of GluN2B-containing NMDARs, we hope to be able to study the consequences in vivo. 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. In particular, STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs via direct binding to the C-terminal PDZ ligand (-ESDV). We previous studies, we discovered that STEP61 binds to PSD-95 but not to other PSD-95 family members, and that PSD-95 expression triggers the degradation of STEP61 via ubiquitination and degradation by the proteasome. More recently, we used a proteomics approach to define the STEP interactome. These experiments led us to characterize the effect of STEP expression on AMPARs because GluA2 was identified in the STEP interactome. We find that STEP regulates the synaptic expression of GluA2 and GluA3 subunits of AMPARs. Our data support a model in which STEP regulates the synaptic and extrasynaptic organization of AMPA and NMDA receptors. It is intriguing that removing STEP from neurons by knock-down or knock-out strategies results in increased extrasynaptic NMDA receptors, but increased synaptic AMPA receptors, and these effects are subunit-specific. We also identified the mGlu receptor, 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 few years, we have focused on a new approach to studying structure/function of NMDARs. We have exploited a "bedside-to-bench" approach to help guide us in testing receptor domains that are important for synaptic function. In particular, 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. We found that one mutation in particular, identified in a patient with autism, reduced the surface expression of GluN2B as well as the binding to PSD-95. This variant, GluN2B S1415L (S1413L in mouse), showed a deficit in rescue of synaptic NMDAR currents and fewer dendritic spines. This phenotype is interesting because there are many examples in the literature of spine abnormalities being associated with autism. More broadly, this research shows that using patient data is an effective approach to probing the structure/function relationship of NMDARs. We have now generated a knock-in mouse of GluN2B S1413L and we are characterizing its phenotype. Our biochemical studies already reveal a region-specific effect, with hippocampus showing deficits in synaptic proteins. We are conducting behavioral analyses as well. Our lab has continued to pivot to primarily use human genetics to inform our studies. We now focus on characterizing rare variants identified in synaptic proteins in patients with neurodevelopmental disorders. Our first endeavor was studying GluN2B S1415L (described above). We have now 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 find 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.