Atsushi Nishimune

SUMOylation regulates kainate-receptor-mediated synaptic transmission.

The small ubiquitin-like modifier protein (SUMO) regulates transcriptional activity and the translocation of proteins across the nuclear membrane. The identification of SUMO substrates outside the nucleus is progressing but little is yet known about the wider cellular role of protein SUMOylation. Here we report that in rat hippocampal neurons multiple SUMOylation targets are present at synapses and we show that the kainate receptor subunit GluR6 is a SUMO substrate. SUMOylation of GluR6 regulates endocytosis of the kainate receptor and modifies synaptic transmission. GluR6 exhibits low levels of SUMOylation under resting conditions and is rapidly SUMOylated in response to a kainate but not an N-methyl-D-aspartate (NMDA) treatment. Reducing GluR6 SUMOylation using the SUMO-specific isopeptidase SENP-1 prevents kainate-evoked endocytosis of the kainate receptor. Furthermore, a mutated non-SUMOylatable form of GluR6 is not endocytosed in response to kainate in COS-7 cells. Consistent with this, electrophysiological recordings in hippocampal slices demonstrate that kainate-receptor-mediated excitatory postsynaptic currents are decreased by SUMOylation and enhanced by deSUMOylation. These data reveal a previously unsuspected role for SUMO in the regulation of synaptic function.

Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction

Post-translational protein modifications are integral components of signalling cascades that enable cells to efficiently, rapidly and reversibly respond to extracellular stimuli. These modifications have crucial roles in the CNS, where the communication between neurons is particularly complex. SUMOylation is a post-translational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in target proteins. It is well established that SUMOylation controls many aspects of nuclear function, but it is now clear that it is also a key determinant in many extranuclear neuronal processes, and it has also been implicated in a wide range of neuropathological conditions.

Protein SUMOylation modulates calcium influx and glutamate release from presynaptic terminals

Posttranslational modification by small ubiquitin‐like modifier (SUMO) proteins is emerging as an important regulatory mechanism for neuronal function and dysfunction. Although multiple potential presynaptic SUMOylation substrate proteins have been proposed from sequence analysis the functional consequences of presynaptic SUMOylation have not been determined. Here we show that SUMOylation of presynaptic proteins modulates neurotransmitter release. Increasing protein SUMOylation by entrapping recombinant SUMO‐1 in synaptosomes decreased glutamate release evoked by KCl whereas decreasing SUMOylation with the SUMO‐specific protease SENP‐1 enhanced KCl‐evoked release. In contrast, SUMO increased and SENP‐1 decreased synaptosomal glutamate release evoked by kainate stimulation. Consistent with these results, SENP‐1 increased Ca2+ influx into synaptosomes evoked by KCl whereas it decreased kainate‐induced Ca2+ influx. These results demonstrate that, in addition to postsynaptic effects, protein SUMOylation acts to modulate neurotransmitter release and thereby regulate synaptic function.

Analysis of SUMO-1 modification of neuronal proteins containing consensus SUMOylation motifs

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 (Psi-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 Psi-K-x-D/E motif is not sufficient to indicate that a protein can be SUMOylated in this bacterial system.

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.

Bidirectional regulation of kainate receptor surface expression in hippocampal neurons

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-GluR6 subunits efficiently co-assembled with native KAR subunits to form heteromeric receptors. Diffuse surface-expressed dendritic SEP-GluR6 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 surface-expressed 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.

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.

Regional quantification of muscarinic acetylcholine receptors and β‐adrenoceptors in human airways

BACKGROUND AND PURPOSE Muscarinic acetylcholine receptors (mAChRs) and β‐adrenoceptors in the airways and lungs are clinically important in chronic obstructive pulmonary disease (COPD) and asthma. However, the quantitative and qualitative estimation of these receptors by radioligand binding approaches in human airways has not yet been reported because of tissue limitations.

Development of GABAB subunits and functional GABAB receptors in rat cultured hippocampal neurons.

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) play a critical role in inhibitory synaptic transmission in the hippocampus but the ontogeny of their subunit synthesis and synaptic localisation has not been determined. Here we report the distributions and developmental profiles of GABA(B1) and GABA(B2) subunits in cultured rat embryonic hippocampal neurons. Limited levels of GABA(B1) and GABA(B2) immunoreactivity were present at 3 days in vitro (DIV). At 7 DIV, when baclofen-evoked inwardly rectifying K(+) channel-mediated responses first appear in the cells, there was a more widespread expression within the soma and proximal dendrites. Levels of the K(+) channel GIRK 1 were relatively constant at all time points suggesting channel availability does not limit the appearance of functional GABA(B)Rs. At 14 DIV the staining displayed a punctate dendritic distribution and near maximal GABA(B)R-mediated electrophysiological responses were obtained. About half of the puncta for each GABA(B)R subunit in dendrites co-localised with the synaptic marker SV2a suggesting that these subunits are at or very near to synapses. Interestingly, at all ages strong GABA(B)R immunoreactivity was also present in the nuclei of neurons. These results provide an important developmental baseline for future studies aimed at investigating, for example, the trafficking and functional regulation of these receptors.

Visualization and Tissue Distribution of α1L-Adrenoceptor in Human Prostate by the Fluorescently Labeled Ligand Alexa-488-Silodosin

PURPOSE Although alpha(1L)-adrenoceptor is recognized as a target of alpha(1) antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine alpha(1L)-AR vs alpha(1A)-AR tissue distribution because alpha(1L)-AR is now considered another phenotype sharing the alpha(1A)-AR gene and protein molecule. We labeled the alpha(1A) and alpha(1L)-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor(R) 488 (Alexa-488-silodosin) to visualize alpha(1L)-AR expression. MATERIALS AND METHODS Radioligand binding and functional bioassay experiments were done to assess alpha(1)-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed. RESULTS Although Alexa-488-silodosin had about 10 times lower affinity for all alpha(1)-AR subtypes than silodosin in binding and functional studies, it had high selectivity to alpha(1A) and alpha(1L)-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing alpha(1A)-AR but not alpha(1B)-and alpha(1D)-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin. CONCLUSIONS Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to alpha(1A) and alpha(1L)-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of alpha(1L)-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile.