Sriharsha Kantamneni

SUMOylation and phosphorylation of GluK2 regulate kainate receptor trafficking and synaptic plasticity

Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown whether phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. We found that protein kinase C–mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both of 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 led to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Our results suggest a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.

Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis.

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 ubiquitin-like 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 PKC-dependent 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.

Ischaemia differentially regulates GABAB receptor subunits in organotypic hippocampal slice cultures

Reduced synaptic inhibition due to dysfunction of ionotropic GABA(A) receptors has been proposed as one factor in cerebral ischaemia-induced excitotoxic cell death. However, the participation of the inhibitory metabotropic GABA(B) receptors in these pathological processes has not been extensively investigated. We used oxygen-glucose deprivation (OGD) and NMDA-induced excitotoxicity as models to investigate whether ischaemia-like challenges alter the protein levels of GABA(B1) and GABA(B2) receptor subunits in rat organotypic hippocampal slice cultures. Twenty-four hours after the insult both OGD and NMDA produced a marked decrease in the total levels of GABA(B2) (approximately 75%), while there was no significant change in the levels of GABA(B1) after OGD, but an increase after NMDA treatment (approximately 100%). The GABA(B) receptor agonist baclofen (100 microM) was neuroprotective following OGD or NMDA treatment if added before or during the insult. GABA(B) receptors comprise heterodimers of GABA(B1) and GABA(B2) subunits and our results suggest that the separate subunits are independently regulated in response to extreme neuronal stress. However, because GABA(B2) is required for functional surface expression, down-regulation of this subunit removes an important inhibitory feedback mechanism under pathological conditions.

GISP: a novel brain-specific protein that promotes surface expression and function of GABAB receptors

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 GABAB receptor (GABABR) trafficking, we screened for proteins that bind to the carboxy‐terminus of the GABAB1 subunit. We report the identification and characterization of a novel 130‐kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABAB1 subunit via a coiled‐coil domain. GISP co‐fractionates with GABABR and with the postsynaptic density and co‐immunoprecipitates with GABAB1 and GABAB2 from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABABRs. When co‐expressed with GABABRs in human embryonic kidney cells, GISP promotes GABABR 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 GABABRs.

SUMOylation is required for glycine-induced increases in AMPA receptor surface expression (ChemLTP) in hippocampal neurons.

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.

Study of Novel Selective mGlu2 Agonist in the Temporo-Ammonic Input to CA1 Neurons Reveals Reduced mGlu2 Receptor Expression in a Wistar Substrain with an Anxiety-Like Phenotype

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 EC50 values of <1 μm and >100 μm. In “sensitive” slices, LY395756 had additive actions with a mixed mGlu2/mGlu3 agonist, DCG-IV [(2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine], whereas in “insensitive” slices, LY395756 reduced the effect of DCG-IV, with an IC50 of ∼1 μm. 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.

GISP binding to TSG101 increases GABAB receptor stability by down-regulating ESCRT-mediated lysosomal degradation

The 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 GABAB receptor subunit GABAB1. 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 GABAB2 subunit, their co‐expression in HEK293 cells increases levels of GABAB2. GISP also inhibits TSG101‐dependent GABAB2 down‐regulation in human embryonic kidney 293 cells whereas over‐expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABAB2 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.

Activity-dependent SUMOylation of the brain-specific scaffolding protein GISP

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

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. Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.

Cross-talk and regulation between glutamate and GABAB receptors.

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.