Faculty Bibliography

Phosphorylation of S880 within the GluR2 C-terminus has been reported to promote endocytosis of AMPA receptors (AMPARs) by preventing GluR2 interaction with the putative synaptic anchoring proteins GRIP and ABP. It is not yet established however, whether S880 phosphorylation induces removal of AMPARs from synaptic sites, and the trafficking of phosphorylated GluR2 subunits with surface and endocytosed GluR2 has not been directly compared within the same intact neurons. Here we show that phosphorylation of GluR2 subunits by PKC activated with phorbol esters is compartmentally restricted to receptors located at the cell surface. Endogenous AMPARs containing S880-phosphorylated GluR2 remained highly synaptic and colocalized with postsynaptic markers to the same extent as AMPARs which did not contain S880-phosphorylated GluR2. Moreover, following S880 phosphorylation, exogenous GluR2 homomers were found specifically at the cell surface and did not co-traffic with the internalized endosomal GluR2 population. We also show that GluR2 is endogenously phosphorylated by a constitutively active kinase pharmacologically related to PKC, and this phosphorylation is opposed by the protein phosphatase PP1. Our results demonstrate a population of hippocampal AMPARs which do not require interaction with GRIP/ABP for synaptic anchorage

Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions of the subunit intracellular C-terminal domains (CTDs) with other proteins, but the mechanisms involved in this process are still unclear. We have found that the GluR1 CTD binds to cGMP-dependent protein kinase II (cGKII) adjacent to the kinase catalytic site. Binding of GluR1 is increased when cGKII is activated by cGMP. cGKII and GluR1 form a complex in the brain, and cGKII in this complex phosphorylates GluR1 at S845, a site also phosphorylated by PKA. Activation of cGKII by cGMP increases the surface expression of AMPARs at extrasynaptic sites. Inhibition of cGKII activity blocks the surface increase of GluR1 during chemLTP and reduces LTP in the hippocampal slice. This work identifies a pathway, downstream from the NMDA receptor (NMDAR) and nitric oxide (NO), which stimulates GluR1 accumulation in the plasma membrane and plays an important role in synaptic plasticity

Cadherins function in the adhesion of presynaptic and postsynaptic membranes at excitatory synapses. Here we show that the cadherin-associated protein neural plakophilin-related arm protein (NPRAP; also called delta-catenin) binds via a postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 (PDZ) interaction to AMPA receptor (AMPAR)-binding protein (ABP) and the related glutamate receptor (GluR)-interacting protein (GRIP), two multi-PDZ proteins that bind the GluR2 and GluR3 AMPAR subunits. The resulting cadherin-NPRAP-ABP/GRIP complexes serve as anchorages for AMPARs. Exogenous NPRAP that was bound to cadherins at adherens junctions of Madin-Darby canine kidney cells recruited ABP from the cytosol to form cadherin-NPRAP-ABP complexes, dependent on NPRAP interaction with the ABP PDZ domain 2. The cadherin-NPRAP-ABP complexes also bound GluR2. In cultured hippocampal neurons, dominant-negative mutants of NPRAP designed to disrupt tethering of ABP to NPRAP-cadherin complexes reduced surface levels of endogenous GluR2, indicating that interaction with cadherin-NPRAP-ABP complexes stabilized GluR2 at the neuronal plasma membrane. Cadherins, NPRAP, GRIP, and GluR2 copurified in the fractionation of synaptosomes and the postsynaptic density, two fractions enriched in synaptic proteins. Furthermore, synaptosomes contain NPRAP-GRIP complexes, and NPRAP localizes with the postsynaptic marker PSD-95 and with AMPARs and GRIP at spines of hippocampal neurons. Thus, tethering is likely to take place at synaptic or perisynaptic sites. NPRAP also binds PSD-95, which is a scaffold for NMDA receptors, for AMPARs in complexes with auxiliary subunits, the TARPs (transmembrane AMPA receptor regulator proteins), and for adhesion molecules. Thus, the interaction of scaffolding proteins with cadherin-NPRAP complexes may anchor diverse signaling and adhesion molecules at cadherins

AMPA-type (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review

RNA editing modifies the GluR2 AMPA receptor subunit pore loop at the Q/R site and limits receptor Ca(2+) permeability. Editing failure is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis. We show that channels with unedited GluR2 are highly toxic in cultured hippocampal neurons. Toxicity exceeds that of other Ca(2+)-permeable AMPA receptor types and is influenced by agonist binding site mutations, ability to desensitize, and extracellular Ca(2+) levels. Significantly, toxicity also depends on GluR2's constitutive surface trafficking, a function dependent on GluR2 C-terminal domain interaction with NSF, a specialized chaperone. We have exploited the interaction between unedited GluR2 and NSF to reduce GluR2 surface levels. We show that a peptide that blocks the GluR2-NSF interaction reduces unedited GluR2 toxicity by reducing receptor surface expression. Peptide block of trafficking provides a model for design of drugs to reduce unedited GluR2 excitotoxicity in neurodegenerative diseases that result from editing failure

Neuronal development, plasticity and survival require activity-dependent synapse-to-nucleus signaling. Most studies implicate an activity-dependent regulation of gene expression in this phenomenon. However, little is known about other nuclear functions that are regulated by synaptic activity. Here we show that a newly identified component of rat postsynaptic densities (PSDs), AIDA-1d, can regulate global protein synthesis by altering nucleolar numbers. AIDA-1d binds to the first two postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domains of the scaffolding protein PSD-95 via its C-terminal three amino acids. Stimulation of NMDA receptors (NMDARs), which are also bound to PSD-95, results in a Ca(2+)-independent translocation of AIDA-1d to the nucleus, where it couples to Cajal bodies and induces Cajal body-nucleolar association. Long-term neuronal stimulation results in an AIDA-1-dependent increase in nucleolar numbers and protein synthesis. We propose that AIDA-1d mediates a link between synaptic activity and control of protein biosynthetic capacity by regulating nucleolar assembly

Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS

AMPA receptors (AMPARs) conduct fast, excitatory currents that depolarize neurons and trigger action potentials. AMPARs took on new importance when it was shown that AMPAR transport can increase or decrease the number of AMPARs at synapses and give rise to synapse plasticity, including long-term potentiation (LTP) and long-term depression (LTD). This review considers how transmembrane AMPAR regulatory proteins (TARPs), a novel family of AMPAR auxiliary subunits, have changed our view of AMPAR transport and raised some perplexing questions

The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner

Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain insights into the transcriptional mechanisms that regulate NPFF gene expression, we cloned and sequenced 9.8 and 1.5 kb of the mouse and rat NPFF 5'-flanking region, respectively. Regions with high sequence homology between mouse, rat and human were expected to have high probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with the homeobox proteins Oct-1, PDX1, Pit-1 and MEIS and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The distribution of NPFF gene expression was examined in the mouse using in situ hybridization and RT-PCR. NPFF expression was also evident during mouse embryogenesis. A fixed transcription initiation site for the mouse NPFF gene was found. A novel splice variant with a retained intron of the NPFF gene was characterized. Chimeric luciferase reporter gene constructs for the mouse NPFF gene revealed a minimal promoter region and a region with transcriptional suppressor features. An NGF responsive area was found using mouse NPFF reporter gene constructs. We postulate that Oct-1, PDX1, Pit-1, MEIS and DREAM are likely transcriptional regulators of NPFF gene expression