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. We focus on defining subunit-specific mechanisms that regulate the synaptic localization and functional regulation 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 (GluN1;NR2A-D;NR3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of NR2A and NR2B 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 (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. 
However, how synaptic activity drives this process remains unclear because CK2 is a constitutively active kinase, which is not directly regulated by calcium. We recently demonstrated that activated CaMKII couples GluN2B and CK2 to form a tri-molecular complex and increase CK2-mediated phosphorylation of GluN2B S1480. In addition, a GluN2B mutant, which contains an insert to mimic the GluN2A sequence and cannot bind to CaMKII, displays reduced S1480 phosphorylation and increased surface-expression. Importantly, we find that although disrupting GluN2B/CaMKII binding reduces synapse number, it increases synaptic-GluN2B content. Therefore, the GluN2B/CaMKII association controls synapse density and PSD composition in an activity-dependent manner, including recruitment of CK2 to remove GluN2B from synapses. In addition to characterizing the receptor subunits, 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. We have also identified two critical residues on GluN2B responsible for the non-PDZ binding to SAP102. Strikingly, either mutation of these critical residues or knockdown of endogenous SAP102 can rescue the defective surface expression and synaptic localization of PDZ binding-deficient GluN2B. These data reveal an unexpected, nonscaffolding role for SAP102 in the synaptic clearance of GluN2B-containing NMDARs. 

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. Because this S567 is a relatively weak CaMKII substrate in contrast to its substrate residue in the GluA1 C-terminus (Ser831), and that the first half of the region is moderately conserved between subunits, we sought to identify other putative kinases. We performed a bioinformatics analysis of AMPARs and found that CK2 was a good candidate to phosphorylate the intracellular loop1 region of AMPAR subunits GluA1 and GluA2. Using in vitro kinase assays, we determined that CK2 phosphorylates the GluA1 and GluA2 intracellular loop1 region, but not their C-termini. Site-directed mutagenesis combined with an in vitro kinase assays suggests the presence of two CK2-phosphorylated serine residues in the GluA1 intracellular loop1 region, including S567. 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. AMPA receptors (AMPARs) mediate the majority of fast excitatory neurotransmission and their density at post-synaptic sites determines synaptic strength. Ubiquitination is a post-translational modification that dynamically regulates the synaptic expression of many proteins. However, very few of the ubiquitinating enzymes implicated in the process have been identified. In a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, we identified RNF167. Predominantly lysosomal, a sub-population of RNF167 is located on the surface of cultured neurons. Using a RING mutant RNF167 or a specific shRNA to eliminate endogenous RNF167, we demonstrated that AMPAR surface expression increases in hippocampal neurons with disrupted RNF167 activity and that RNF167 is involved in activity-dependent ubiquitination of AMPARs. In addition, RNF167 regulates synaptic AMPAR currents, whereas synaptic NMDAR currents are unaffected. Therefore, our study identifies RNF167 as a selective regulator of AMPAR-mediated neurotransmission. In a slightly new direction, we have investigated the activity-dependent regulation of neuroligins, which are postsynaptic cell adhesion molecules important for synaptic function through their transsynaptic interaction with neurexins. The localization and synaptic effects of neuroligin-1 are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca2+/CaM Kinase II. We recently found that Ca2+/CaM Kinase II robustly phosphorylates the intracellular domain of neuroligin-1 but not neuroligin-3. We show that NL-1 has a single dominant CaMKII site, T739, which is phosphorylated in response to synaptic activity in neurons. Furthermore, a phospho-deficient mutant reduces the basal and activity-driven surface expression of neuroligin-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. Our findings are the first to show a direct functional interplay between CaMKII and neuroligin-1, two primary components of excitatory synapses.

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Coultrap, Steven J; Freund, Ronald K; O'Leary, Heather et al. (2014) Autonomous CaMKII mediates both LTP and LTD using a mechanism for differential substrate site selection. Cell Rep 6:431-7
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