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Abstract: SUMOylation is emerging as an important mechanism for modulating protein function in many cell types. A large variety of proteins have been proposed as SUMO targets based on the presence of a consensus SUMOylation core motif (Ψ-K-x-D/E). In neurons these include multiple synaptic proteins but it has not been established whether proteins carrying this motif are SUMOylated either in vitro or in vivo. Here we use a bacterial SUMOylation assay to systematically test for SUMO-1 modification of a selection of neuronal proteins containing one or more amino acid sequences predicted as high-probability SUMOylation sites in computer-based searches. Of the 39 proteins analysed only 14 sites were posttranslationally modified by SUMO-1, including the group III metabotropic glutamate receptors and the kainate receptor subunit GluR7. These results identify new candidate proteins that may be involved in the SUMO regulation of synaptic activity and also demonstrate that the presence of the Ψ-K-x-D/E motif is not sufficient to indicate that a protein can be SUMOylated in this bacterial system. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Kainate receptors (KARs) are crucial for the regulation of both excitatory and inhibitory neurotransmission, but little is known regarding the mechanisms controlling KAR surface expression. We used super ecliptic pHluorin (SEP)-tagged KAR subunit GluR6a to investigate real-time changes in KAR surface expression in hippocampal neurons. Sindbis virus-expressed SEP-G1uR6 subunits efficiently co-assembled with native KAR subunits to form heteromeric receptors. Diffuse surface-expressed dendritic SEP-GIuR6 is rapidly internalized following either N-methyl-D-aspartate or kainate application. Sustained kainate or transient N-methyl-D-aspartate application resulted in a slow decrease of base-line surface KAR levels. Surprisingly, however, following the initial loss of surface receptors, a short kainate application caused a long lasting increase in surfaceexpressed KARs to levels significantly greater than those prior to the agonist challenge. These data suggest that after initial endocytosis, transient agonist activation evokes increased KAR exocytosis and reveal that KAR surface expression is bidirectionally regulated. This process may provide a mechanism for hippocampal neurons to differentially adapt their physiological responses to changes in synaptic activation and extrasynaptic glutamate concentration. [ABSTRACT FROM AUTHOR]