The unique distribution of neurotransmitter receptors and their subtypes within a single cell and throughout the brain requires highly selective intracellular targeting mechanisms. 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 the trafficking of NMDA receptors, which are multi-subunit complexes (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. 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). 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 generating a line of genetically-altered mice: a knock-in mouse expressing a point-mutated non-phosphorylatable GluN2B subunit (GluN2B E1479Q). This knock-in mouse will allow us to examine the precise regulation of GluN2B S1480 phosphorylation in neurons, in vivo, and without the requirement of exogenous protein overexpression. It is anticipated that these animals would show an impaired developmental GluN2 subunit switch (Sanz-Clemente et al, 2010). In addition, we are generating a knock-in mouse, GluN2B S1480E, which mimics phosphorylation of that key residue and constitutively blocks binding to PSD-95. Our experiments have shown us this mutation results in very low surface and synaptic expression of GluN2B receptors, thus we expect the mouse to have synaptic deficits as well. We are also exploring the role of tyrosine kinases and phosphatases 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 have discovered that STEP61 binds to PSD-95 but not to other PSD-95 family members. In addition, PSD-95 expression triggers the degradation of STEP61 via ubiquitination and degradation by the proteasome. Surprisingly, we found that STEP61 is not enriched in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95-KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. An important consequence of STEP having low abundance at the PSD is that only extrasynaptic NMDAR expression and currents were increased upon STEP knock-down. Therefore, our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61. More recently, we have characterized the effect of STEP expression on AMPARs. We find that STEP regulates the synaptic expression of GluA2 and GluA3 subunits of AMPARs and we are currently exploring the molecular underpinnings of this effect in detail. 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, the effects are subunit-specific. Over the last few years, we have focused on a new approach to studying structure/function of NMDARs. We are trying 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. Ubiquitination is a post-translational modification that dynamically regulates the synaptic expression of many proteins. Years ago, we performed a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, and identified two candidates. One of these, RNF112, is a brain-specific protein that we have characterized using a variety of approaches. We find that it is a functional GTPase, as well as an E3 ligase. We named it neurolastin (RNF112/Znf179) because it is most closely related to the dynamin superfamily GTPase, atlastin. We generated a knock-out line of mice and in our initial publication, we showed that neurolastin regulates endosome size and spine density in vivo. Neurolastin requires both an intact RING and GTPase domain to maintain spine density. Interestingly, mutations in the RING domain result in mistargeting of neurolastin from a primarily endosomal to primarily mitochondrial localization. We have now identified a mitochondrial targeting sequence on neurolastin. Furthermore, we have analyzed knock-out animals using EM and find mitochondrial defects.

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Lomash, Richa Madan; Sheng, Nengyin; Li, Yan et al. (2017) Phosphorylation of the kainate receptor (KAR) auxiliary subunit Neto2 at serine 409 regulates synaptic targeting of the KAR subunit GluK1. J Biol Chem 292:15369-15377
Liu, Shuxi; Zhou, Liang; Yuan, Hongjie et al. (2017) A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density. J Neurosci 37:4093-4102
Won, Sehoon; Levy, Jon M; Nicoll, Roger A et al. (2017) MAGUKs: multifaceted synaptic organizers. Curr Opin Neurobiol 43:94-101
Chang, Kai; Roche, Katherine W (2016) Structural and molecular determinants regulating mGluR5 surface expression. Neuropharmacology :
Won, Sehoon; Incontro, Salvatore; Nicoll, Roger A et al. (2016) PSD-95 stabilizes NMDA receptors by inducing the degradation of STEP61. Proc Natl Acad Sci U S A 113:E4736-44
Gu, Xinglong; Mao, Xia; Lussier, Marc P et al. (2016) GSG1L suppresses AMPA receptor-mediated synaptic transmission and uniquely modulates AMPA receptor kinetics in hippocampal neurons. Nat Commun 7:10873
Lussier, Marc P; Sanz-Clemente, Antonio; Roche, Katherine W (2015) Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and ?-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications. J Biol Chem 290:28596-603
Lomash, Richa Madan; Gu, Xinglong; Youle, Richard J et al. (2015) Neurolastin, a Dynamin Family GTPase, Regulates Excitatory Synapses and Spine Density. Cell Rep 12:743-51
Sheng, Nengyin; Shi, Yun S; Lomash, Richa Madan et al. (2015) Neto auxiliary proteins control both the trafficking and biophysical properties of the kainate receptor GluK1. Elife 4:
Zheng, Chan-Ying; Chang, Kai; Suh, Young Ho et al. (2015) TARP ?-8 glycosylation regulates the surface expression of AMPA receptors. Biochem J 465:471-7

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