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. In collaboration with the NIMH Transgenic Core Facility 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 be a valuable tool for understanding how this process contributes to the refinement of neuronal connections. We are also exploring the role of tyrosine kinases on phosphatases on the regulation of NMDA receptor surface and synaptic expression. We find robust effects with the tyrosine phosphatase STEP and also specificity in STEP binding to certain PSD-95 family members. We are currently investigating the molecular basis of STEP binding to PSD-95 and the functional role of this interaction on STEP, PSD-95 and ultimately synaptic NMDA receptors. We have also investigated the role of posttranslational modifications, such as ubiquitination, phosphorylation and glycosylation on AMPA receptor trafficking. We have 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 have systematically evaluated the phosphorylation of the loop region of the AMPA receptor subunits. Site-directed mutagenesis combined with an in vitro kinase assays revealed the presence of two CK2-phosphorylated serine residues in the GluA1 intracellular loop1 region, including S567 and a more robust substrate for CK2, S579. To investigate a role for CK2 in AMPAR trafficking, we reduced the endogenous expression of CK2 using an shRNA against the regulatory subunit CK2 beta, and we detected a reduction of GluA1 surface expression, whereas GluA2 was unchanged. Importantly, the expression of GluA1 phosphodeficient mutant (S579A) in hippocampal neurons displayed reduced surface expression. Therefore, our study identifies CK2 as a regulator of GluA1 surface expression by phosphorylating the intracellular loop1 region. We are following up on these studies to understand the subunit specific regulation in the loop region and how it affect endogenous AMPARS. 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 and RNF112. Previously, we have demonstrated that RNF167 regulates excitatory synaptic transmission. Interestingly, we now find that RNF112 is a brain-specific functional GTPase, as well as E3 ligase. We have now named it neurolastin (RNF112/Znf179) because it is most closely related to the dynamin superfamily GTPase, atlastin. Neurolastin is the first identified protein with a unique domain organization harboring both GTPase and RING domains. We have demonstrated that neurolastin has the ability to hydrolyze GTP to mono-phosphate (GMP) and the GTPase activity is involved in the maintenance of dendritic spine density. In addition neurolastin leads to an increase in the density of dendritic spines on hippocampal neurons, whereas expression of the GTPase activity mutant did not affect the spine density. Subsequently, we also observed a significant decrease in the frequency of mEPSCs in hippocampal slices from knockout mice indicating a marked reduction in the number of functional synapses in the absence of neurolastin. These results indicate that neurolastin affects synaptic function by regulating synaptogenesis and spine maintenance.
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