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. We focus on defining subunit-specific mechanisms that regulate different subtypes of glutamate receptors. 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 (NR1;NR2A-D;NR3A-B). Over the last year, we have made significant progress in the detailed characterization of NR2A vs. NR2B trafficking and synaptic expression. We find that the NR2B subunit, and not NR2A, is specifically phosphorylated by casein kinase 2 (CK2) on a critical residue in the NR2B C-terminal domain. CK2 phosphorylation of NR2B increases in the second postnatal week and is important in the subunit switch (NR2B to NR2A), 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. We are also studying the specific regulation of NR2A and NR2B by the PSD-95 family of proteins (PSD-95, PSD-93, SAP97, SAP102) Our results support a unique role for SAP102 in regulating NR2B-containing NMDA receptors. SAP102 is highly expressed early in development and mediates the trafficking of both NMDA receptors and AMPA receptors during synaptogenesis. We find that NR2B interacts with SAP102, not PSD-95, via a secondary PDZ-independent binding domain. The NR2B binding site is located within the SAP102 N-terminal domain and is regulated by alternative splicing of SAP102. We find that SAP102 that possesses an N-terminal insert is developmentally regulated at both mRNA and protein levels. In addition the alternative splicing of SAP102 regulates dendritic spine morphology. Expression of SAP102 that contains the N-terminal insert promotes lengthening of dendritic spines, whereas a short hairpin RNA knockdown of the same SAP102 splice variant causes spine shrinkage. In addition, blocking NMDA receptor activity prevents the spine lengthening induced by the N-terminal splice variant of SAP102. It has been reported that mutations in human SAP102 cause mental retardation, which is often accompanied by abnormalities in dendritic spines. However, little is known about the role of SAP102 in regulating synapse formation or spine morphology. Our findings provide the first evidence that SAP102 links NMDA receptor activation to alterations in spine morphology. We have also examined the postsynaptic machinery that mediates NMDA receptor surface expression and trafficking, including the postsynaptic SNARE, SNAP23. We found that SNAP-23 regulated the surface expression and membrane recycling of NMDA receptors. We generated Snap23-null mice by homologous recombination. Attesting to the importance of SNAP-23 function in mouse development, we found the SNAP-23 KO mice were not viable. We were unable to obtain newborn SNAP-23-deficient mice, and analysis of pre-implantation embryos from Snap23+/- matings revealed that Snap23-null blastocysts were dying prior to implantation at embryonic day E3.5. These data reveal a critical role for SNAP-23 during embryogenesis. We have also investigated the role of posttranslational modifications, such as ubiquitination and phosphorylation, on AMPA receptor trafficking. We found that the first intracellular loop domain (Loop1) of GluA1, a previously overlooked region within AMPA receptors, is critical for receptor targeting to synapses, but not for delivery of receptors to the plasma membrane. We identified a CaMKII phosphorylation site (S567) in the GluA1 Loop1, which is phosphorylated in vitro and in vivo. Furthermore, we show that S567 is a key residue that regulates Loop1-mediated AMPA receptor trafficking, revealing a unique mechanism for targeting AMPA receptors to synapses to mediate synaptic transmission. In addition, we have described activity-dependent ubiquitination of AMPA receptors and are currently investigating specific E3 ligases that regulate AMPA receptor ubiquitination and trafficking.

Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2011
Total Cost
$1,296,870
Indirect Cost
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State
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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
Won, Sehoon; Levy, Jon M; Nicoll, Roger A et al. (2017) MAGUKs: multifaceted synaptic organizers. Curr Opin Neurobiol 43:94-101
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
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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