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. 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. 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. Because it is anticipated that these animals will show an impaired developmental GluN2 subunit switch (Sanz-Clemente et al, 2010), they will provide a valuable tool for understanding how this process contributes to the refinement of neuronal connections. 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. Our laboratory has studied the regulation of Group I metabotropic trafficking for many years. Primarily we have focused on the effects of protein phosphorylation on mGluR5, which include trafficking and signaling. We have now systematically examined the role of different regions of mGluR5 in receptor trafficking to the plasma membrane. We generated a series of mGluR5 truncations and ligand binding mutants and used a surface-binding assay to evaluate dimerization and surface expression. Interestingly, the C terminus is not essential for mGluR5 surface expression. In contrast, the 7th transmembrane domain (TM7) plays a critical role in its surface expression in both heterologous cells and neurons. Furthermore, a ligand binding mutation within the extracellular domain of mGluR5 (Y64A/T174A) that blocks ligand binding impairs both surface expression and dimerization of mGluR5 in neurons. The integrity of both the whole 7TM domain and the C- terminal tail of mGluR5 are also important for stabilizing dimerization with the extracellular domain. Our findings reveal that there are multiple domains that regulate dimerization and trafficking of mGluR5. 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 continue to study the mechanisms by which neurolastin affects membrane dynamics in the nervous system. We continue to have fruitful collaborations with several laboratories investigating the role of auxiliary subunits in regulating AMPARs and kainate receptors. For AMPARs, there are quite a few proteins that have been show to co-assemble including TARPs, CNIHs and CKAMP44, which are important for AMPAR forward trafficking to synapses. We recently worked with Dr. Wei Lus laboratory (NINDS) to investigate GSG1L, another AMPAR binding protein. We found that it negatively regulates AMPAR-mediated synaptic transmission. Overexpression of GSG1L strongly suppresses, and GSG1L knockout (KO) enhances, AMPAR-mediated synaptic transmission. In collaboration with Dr. Roger Nicolls laboratory (UCSF) we studied the kainate receptor auxiliary subunits, Neto1 and Neto2. Using the hippocampal CA1 pyramidal neuron as a null background system, we find that surface expression of the kainate receptor GluK1 is minimal, but both Neto1 and Neto2 profoundly increase GluK1 surface and synaptic expression. However, the Neto regulation GluK1 synaptic targeting was distinct from the Neto regulation of forward trafficking to the surface.
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