SummaryPhosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown if phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. Here, we show that PKC-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both these events are necessary for the internalization of GluK2 containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation leads to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Thus, we describe a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.

The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitinlike modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKCdependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.

Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression.

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABA B receptor (GABA B R) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABA B1 subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABA B1 subunit via a coiled-coil domain. GISP co-fractionates with GABA B R and with the postsynaptic density and co-immunoprecipitates with GABA B1 and GABA B2 from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABA B Rs. When co-expressed with GABA B Rs in human embryonic kidney cells, GISP promotes GABA B R surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABA B Rs.

Background: Heterodimerization of GABAB1 and GABAB2 subunits is required for functional GABABRs.Results: GABABR subunits are differentially regulated by activation of synaptic or extrasynaptic NMDARs.Conclusion: GABABR trafficking and function is regulated by NMDARs.Significance: GABABRs are potential targets for treating diseases such as stroke and cerebral ischemia.

1Both these authors contributed equally to this study.Abbreviations used: EGFR, epidermal growth factor receptor; ESCRT, endosomal sorting complex required for transport; GISP, G proteincoupled receptor interacting scaffold protein; GST, glutathione S-transferase; HEK293, human embryonic kidney cells. AbstractThe neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GA-BA B receptor subunit GABA B1 . Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA B2 subunit, their co-expression in HEK293 cells increases levels of GABA B2 . GISP also inhibits TSG101-dependent GABA B2 down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA B2 degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.

Brain function depends on co-ordinated transmission of signals from both excitatory and inhibitory neurotransmitters acting upon target neurons. NMDA, AMPA and mGluR receptors are the major subclasses of glutamate receptors that are involved in excitatory transmission at synapses, mechanisms of activity dependent synaptic plasticity, brain development and many neurological diseases. In addition to canonical role of regulating presynaptic release and activating postsynaptic potassium channels, GABAB receptors also regulate glutamate receptors. There is increasing evidence that metabotropic GABAB receptors are now known to play an important role in modulating the excitability of circuits throughout the brain by directly influencing different types of postsynaptic glutamate receptors. Specifically, GABAB receptors affect the expression, activity and signaling of glutamate receptors under physiological and pathological conditions. Conversely, NMDA receptor activity differentially regulates GABAB receptor subunit expression, signaling and function. In this review I will describe how GABAB receptor activity influence glutamate receptor function and vice versa. Such a modulation has widespread implications for the control of neurotransmission, calcium-dependent neuronal function, pain pathways and in various psychiatric and neurodegenerative diseases.

Group II metabotropic receptors (mGluRs) regulate central synaptic transmission by modulating neurotransmitter release. However, the lack of pharmacological tools differentiating between mGlu2 and mGlu3 receptors has hampered identification of the roles of these two receptor subtypes. We have used LY395756 [(1SR,2SR,4RS,5RS,6SR)-2-amino-4-methylbicyclo[3.1.0]-hexane2,6-dicarboxylic], an agonist at mGlu2 receptors and an antagonist at mGlu3 receptors in cell lines, to investigate the roles of these receptors in the temporo-ammonic path from entorhinal cortex to CA1-stratum lacunosum moleculare in rat hippocampal slices. Surprisingly, the degree of inhibition of the field EPSP induced by LY395756 fell into two distinct groups, with EC 50 values of Ͻ1 M and Ͼ100 M. In "sensitive" slices, LY395756 had additive actions with a mixed mGlu2/mGlu3 agonist, DCG-IV [(2S,2ЈR,3ЈR)-2-(2Ј,3Ј-dicarboxycyclopropyl)glycine], whereas in "insensitive" slices, LY395756 reduced the effect of DCG-IV, with an IC 50 of ϳ1 M. This separation into sensitive and insensitive slices could be explained by LY395756 acting as an mGlu2 agonist and mGlu3 antagonist, respectively, a finding supported by data from mice lacking these receptors. The heterogeneity was correlated with differences in expression levels of mGlu2 receptors within our Wistar colony and other Wistar substrains. The initial search for a behavioral correlate indicated that rats lacking mGlu2 receptors showed anxiety-like behavior in open-field and elevated plus maze assays. These findings have implications for rat models of psychiatric disease and are especially pertinent given that mGlu2 receptors are targets for compounds under development for anxiety.