The epidermal growth factor receptor (EGFR) is targeted for lysosomal degradation by ubiquitin-mediated interactions with the ESCRTs (endosomal-sorting complexes required for transport) in multivesicular bodies (MVBs). We show that secretory carrier membrane protein, SCAMP3, localizes in part to early endosomes and negatively regulates EGFR degradation through processes that involve its ubiquitylation and interactions with ESCRTs. SCAMP3 is multimonoubiquitylated and is able to associate with Nedd4 HECT ubiquitin ligases and the ESCRT-I subunit Tsg101 via its PY and PSAP motifs, respectively. SCAMP3 also associates with the ESCRT-0 subunit Hrs. Depletion of SCAMP3 in HeLa cells by inhibitory RNA accelerated degradation of EGFR and EGF while inhibiting recycling. Conversely, overexpression enhanced EGFR recycling unless ubiquitylatable lysines, PY or PSAP motifs in SCAMP3 were mutated. Notably, dual depletions of SCAMP3 and ESCRT subunits suggest that SCAMP3 has a distinct function in parallel with the ESCRTs that regulates receptor degradation. This function may affect trafficking of receptors from prelysosomal compartments as SCAMP3 depletion appeared to sustain the incidence of EGFR-containing MVBs detected by immunoelectron microscopy. Together, our results suggest that SCAMP3, its modification with ubiquitin, and its interactions with ESCRTs coordinately regulate endosomal pathways and affect the efficiency of receptor down-regulation. INTRODUCTIONThe internalization of cell surface receptors and transporters coupled to degradation or recycling is critical for nutrient uptake and regulating cell signaling. The epidermal growth factor receptor (EGFR) has been studied extensively as a prototypical receptor that is targeted for lysosomal degradation after ligand-stimulated internalization. Disrupted degradation of the EGFR and other receptors has been linked to the pathogenesis of many diseases including several cancers. An essential element in the down-regulation process is ubiquitin conjugation to EGFR by Cbl, an E3 ubiquitin ligase (reviewed in Marmor and Yarden, 2004). It is thought that the ubiquitin enables successive passage of the receptor to the endosomal-sorting complexes required for transport: ESCRT-0, I, II, and III. These complexes are evolutionarily conserved and specialize in targeting and packaging of membrane proteins into intraluminal vesicles (ILVs) upstream of lysosomal degradation. The ESCRT-0 complex is composed of Hrs, STAM, and Eps15b and is enriched in flat clathrin patches on early endosomes via interactions involving Hrs' FYVE and coiled-coiled domains (Raiborg et al., 2001a(Raiborg et al., , 2002Bache et al., 2003b;Roxrud et al., 2008). Ubiquitin-interacting motifs (UIMs) in ESCRT-0 subunits bind ubiquitylated ligands such as EGFR; interaction of Hrs with the UEV domain of ESCRT-I protein Tsg101 is thought to facilitate passage of the receptor to ESCRT-I (Polo et al., 2002;Bache et al., 2003a;Lu et al., 2003). ESCRT-I in turn recruits ESCRT-II, which may function in sequential liga...

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcγRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcγR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcγRs resulted in a marked increase in the tyrosine phosphorylation of FcγRIIa, p58lyn, and p120c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcγRs and tyrosine-phosphorylated proteins including p120c-cbl were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcγRs as well as p120c-cbl with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-β-cyclodextrin, the cross-linking did not induce the association of FcγRs as well as p120c-cbl with DRMs. In addition, although the physical association between FcγRIIa and p58lyn was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-β-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcγRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcγRII to lipid rafts is important for the activation of FcγRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Chemical synapses are specialized sites of the communication between neurons where information is processed and integrated. Electron microscopy has allowed morphological Received November 24, 2010; revised manuscript received December 8, 2010; accepted December 9, 2010. Address correspondence and reprint requests to Hiroyuki Sakagami, MD, PhD, Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan. E-mail: sakagami@med.kitasato-u.ac.jp Abbreviations used: Arf, ADP ribosylation factor; BRAG, brefeldin A-resistant Arf-GEF; DGC, dystrophin-associated glycoprotein complex; GABA A R, GABA A receptor; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GGA1, Golgi-localizing, c-adaptin ear homology domain, Arf-binding protein 1; GST, glutathione S-transferase; HA, hemagglutinin; IRSP, insulin receptor tyrosine kinase substrate of 53 kDa; MAGI, membrane-associated guanylate kinase with inverted orientation; PDZ, PSD-95/Discs large/Zona occludens 1; PSD, post-synaptic density; SDS, sodium dodecyl sulfate; SDS-PAGE, SDSpolyacrylamide gel electrophoresis; S-SCAM, synaptic scaffolding molecule; synArfGEF(Po), potential synaptic Arf-GEF; VGAT, vesicular c-aminobutyric acid transporter. AbstractSynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/ MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both b-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.

Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25 is a neuronal SNARE protein essential for neurotransmitter release from presynaptic terminals. Three palmitoylated SNAP-25 family proteins: SNAP-25a, SNAP-25b, and SNAP-23, are expressed in the brain, but little is known about their distributions and functions. In the present study, we generated specific antibodies to distinguish these three homologous proteins. Immunoblot and immunohistochemical analyses revealed that SNAP-25b was distributed in synapse-enriched regions throughout almost the entire brain, whereas SNAP-25a and SNAP-23 were expressed in relatively specific brain regions with partially complementary expression patterns. SNAP-25a and SNAP-25b, but not SNAP-23, were also present in the axoplasm of nerve fibers. The intracellular localization was also different, and although SNAP-25b and SNAP-23 were found primarily in membrane and lipid raft-enriched fractions of mouse brain homogenates, a substantial amount of SNAP-25a was recovered in soluble fractions. In PC12 cells, SNAP-25b was localized to the plasma membrane, but SNAP-25a and SNAP-23 were distributed throughout the cytoplasm. The expression and distribution of these three proteins were also differentially regulated in the early postnatal period. These results indicate that the three SNAP-25 family proteins display a differential distribution in the brain as well as in neuronal cells, and possibly play distinct roles.

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