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.

The dynamic regulation of actin polymerisation plays crucial roles in cell morphology and endocytosis. The mechanistic details of these processes and the proteins involved are not fully understood, especially in neurons. PICK1 is a PDZ-BAR-domain protein involved in regulated AMPAR endocytosis in neurons. Here, we demonstrate that PICK1 binds F-actin and the actinnucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerisation. RNAi knockdown of PICK1 in neurons induces a reorganisation of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1 (W413A) that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalisation. This study identifies PICK1 as a new negative regulator of Arp2/3-mediated actin polymerisation that is critical for a specific form of vesicle trafficking and also for the development of neuronal architecture.The dynamic actin cytoskeleton plays multiple roles in eukaryotic cells to regulate cell morphology, cell motility and vesicle trafficking by exerting mechanical forces that alter the shape of the plasma membrane. The Arp2/3 complex is the major catalyst for the formation of branched actin networks that mediate changes in membrane geometry. Proteins such as N-WASP, WAVE and cortactin bind and activate the Arp2/3 complex so that changes in cell morphology or vesicle trafficking occur at appropriate times and subcellular locations1-3.Regulation of the actin cytoskeleton is crucial for neuronal morphogenesis1,4,5. Recent reports have suggested a role for the Arp2/3 activator N-WASP in neurite elongation and branching in neuronal development6,7. However, negative regulators of the Arp2/3 complex in these processes have not yet been identified. A role for Arp2/3-mediated actin dynamics in the regulation of endocytosis in mammalian cell lines is becoming increasingly well established. Localised alterations in actin turnover are proposed to provide mechanical forces that contribute to plasma membrane curvature, vesicle scission, and propulsion of nascent vesicles away from the membrane2,8-10. The specific molecular mechanisms for regulating actin to control endocytosis in neurons are unknown. Dendritic spines, the sites of excitatory synapses in neurons, are particularly enriched in filamentous (F-) actin11-13, which is extremely dynamic, rapidly cycling between globular (G-) actin and F-actin14. Since designated endocytic sites are found in dendritic spines15,16, it seems likely that the densely packed, dynamic actin filaments influence endocytosis. PICK1 is a PDZ and BAR-* correspondence: e-mail: jon.hanley@bristol.ac.uk, tel: +44 (0)117 331 1944, fax: +44 (0)117 929 1687. AUTHOR CONTRIBUTIONS D.L.R. planned and performed the biochemistry and some imaging experiments. S.M. supervised generation of shRNA, planned and performed some imaging experiments. E.L.J. generated shRNA constructs. J.G.H planned and performed imaging experiments, mutagenesis/cl...

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. The modification of proteins by the covalent attachment of chemical groups, lipids, sugars or other proteins is important for the spatial and temporal regulation of their function. Of the protein-based modifications, ubiquitylation has been most extensively studied. However, the importance of an analogous modification, SUMOylation, is becoming increasingly apparent. SUMOylation involves the reversible covalent attachment of a member of the SUMO family to lysine residues in target proteins. In the ten years since its discovery, SUMOylation has predominantly been regarded as a nuclear process; however, multiple cytosolic and plasmamembrane targets of SUMOylation have recently been reported, many of which are integral to neuronal function. Furthermore, there is accumulating evidence that perturbations in neuronal SUMOylation contribute to numerous pathological conditions. Here we give an overview of recent studies that demonstrate the growing number of extranuclear functions of neuronal SUMOylation and the implications of SUMOylation in neurological disorders. The SUMO familySUMOylation was first reported as a modification that affects the localization of the nuclear pore component Ran GTPase activating protein (RanGAP) 1,2 . Since then, over 100 targets for SUMOylation have been reported. The fundamental importance of SUMOylation to eukaryotic cells has been established by the discovery that knocking down or deleting the SUMO genes or components of the SUMO pathway is fatal for mammalian cells 3 and causes cell-cycle arrest in yeast 4,5 . (TABLE 1).SUMO isoforms are synthesized as inactive precursors that must be matured by a family of SUMO-specific proteases (SENPs) (FIG. 3). SUMO proteins are then activated by an ATPdependent heterodimer of SUMO1 activating enzyme subunit 1 (SAE1) and SAE2 (REF.18), which passes the activated SUMO protein onto the specific and unique 'conjugating' enzyme, ubiquitin conjugating enzyme 9 (UBC9), through a transesterification reaction 8,19 . UBC9, usually acting in conjunction with an E3 'ligating' enzyme, then catalyses SUMO conjugation to the substrate. SUMOylation and ubiquitylation are mechanistically similar, but there are fundamental differences between the two pathways. Although an E3 enzyme is generally essential for ubiquitylation (for exce...

BackgroundThe stress hormone corticosterone has the ability both to enhance and suppress synaptic plasticity and learning and memory processes. However, until today there is very little known about the molecular mechanism that underlies the bidirectional effects of stress and corticosteroid hormones on synaptic efficacy and learning and memory processes. In this study we investigate the relationship between corticosterone and AMPA receptors which play a critical role in activity-dependent plasticity and hippocampal-dependent learning.Methodology/Principal FindingsUsing immunocytochemistry and live cell imaging techniques we show that corticosterone selectively increases surface expression of the AMPAR subunit GluR2 in primary hippocampal cultures via a glucocorticoid receptor and protein synthesis dependent mechanism. In agreement, we report that corticosterone also dramatically increases the fraction of surface expressed GluR2 that undergo lateral diffusion. Furthermore, our data indicate that corticosterone facilitates NMDAR-invoked endocytosis of both synaptic and extra-synaptic GluR2 under conditions that weaken synaptic transmission.Conclusion/SignificanceOur results reveal that corticosterone increases mobile GluR2 containing AMPARs. The enhanced lateral diffusion properties can both facilitate the recruitment of AMPARs but under appropriate conditions facilitate the loss of synaptic AMPARs (LTD). These actions may underlie both the facilitating and suppressive effects of corticosteroid hormones on synaptic plasticity and learning and memory and suggest that these hormones accentuate synaptic efficacy.

Summary The calcium-sensing receptor (CaSR) is a class III G-protein-coupled receptor (GPCR) that responds to changes in extracellular calcium concentration and plays a crucial role in calcium homeostasis. The mechanisms controlling CaSR trafficking and surface expression are largely unknown. Using a CaSR tagged with the pH-sensitive GFP super-ecliptic pHluorin (SEP-CaSR), we show that delivery of the GPCR to the cell surface is dependent on receptor-activity-modifying proteins (RAMPs). We demonstrate that SEP-CaSRs are retained in the endoplasmic reticulum (ER) in COS7 cells that do not contain endogenous RAMPs whereas they are delivered to the plasma membrane in HEK 293 cells that do express RAMP1. Coexpression of RAMP1 or RAMP3, but not RAMP2, in COS7 cells was sufficient to target the CaSR to the cell surface. RAMP1 and RAMP3 colocalised and coimmunoprecipitated with the CaSR suggesting that these proteins associate within the cell. Our results indicate that RAMP expression promotes the forward trafficking of the GPCR from the ER to the Golgi apparatus and results in mature CaSR glycosylation, which is not observed in RAMP-deficient cells. Finally, silencing of RAMP1 in the endogenously expressing HEK293 cells using siRNA resulted in altered CaSR traffic. Taken together, our results show that the association with RAMPs is necessary and sufficient to transfer the immature CaSR retained in the ER towards the Golgi where it becomes fully glycosylated prior to delivery to the plasma membrane and demonstrate a role for RAMPs in the trafficking of a class III GPCR.