Inhibitory Role of Endophilin 3 in Receptor-mediated Endocytosis

Abstract

Endophilin 1 (Endo1) participates in synaptic vesicle biogenesis through interactions of its Src homology 3 domain with the polyphosphoinositide phosphatase Synaptojanin and the GTPase Dynamin. Endo1 has also been reported to affect endocytosis by converting membrane curvature via its lysophosphatidic acid acyltransferase activity. Here we report that a closely related isoform of Endo1, Endo3, inhibits clathrin-mediated endocytosis. Mutational analyses showed that the variable region of Endo3 is important in regulating transferrin endocytosis. In the brain, Endo3 is co-localized with dopamine D2 receptor in olfactory nerve terminals and inhibits its clathrin-mediated endocytosis in COS-7 cells. Furthermore, overexpression of Endo3 in an olfactory epithelium-derived cell line suppressed dopamine D2 receptor-mediated endocytosis and therefore accelerated its dopamine-induced differentiation. These results indicate that Endo3 may act as a negative regulator of clathrin-mediated endocytosis in brain neurons.

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation, Cell Surface Expression, and Secretion of the Amyloid Precursor Protein

“…For TMEM59L no antibody is available that detects the endogenous protein, either by immunoblot or immunofluorescence. The finding that both knockdown and overexpression of TMEM59 and TMEM59L affected APP shedding in a similar manner is reminiscent of what has been reported for other proteins, such as endocytic endophilins (29,52,53), adaptor proteins FE65 (54), and c-Jun N-terminal kinase-interacting proteins (55). Typically, these proteins form hetero-oligomeric complexes.…”

Rheb activation disrupts spine synapse formation through accumulation of syntenin in tuberous sclerosis complex

“…Both cells were plated at 70-80% confluence onto polyethyleneimine-coated dishes and cultured in DMEM supplemented with 10% fetal bovine serum. Cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen) 63 . To quantify the proteasome degradation of Rheb and syntenin proteins, Rheb-and syntenin-coexpressing COS-7 cells were untreated or treated with 20 mM MG132 for 20 h. Cultured hippocampal neurons were treated with 0.5 mM lactacystin for 2 days (DIV [17][18][19] or 100 nM MG132 for 1 day (DIV [18][19].…”

“…Anti-syntenin (goat, Santa Cruz Biotechnology), anti-GFP (rabbit, Invitrogen) or anti-FLAG (mouse, Sigma-Aldrich) antibodies were added to the protein extracts on ice for 1 h, and the mixtures were incubated overnight at 4°C with packed protein G Sepharose (GE Healthcare). The precipitated immune complex was boiled in SDS sample buffer and analysed using western blotting with anti-Rheb (rabbit, CST), anti-FLAG (mouse, Sigma-Aldrich), anti-GFP (rabbit, Invitrogen) or anti-ephrinB3 (rabbit, Invitrogen) antibodies 63 . All representative western blots included in the manuscript are shown as full scans in Supplementary Fig.…”

Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D2Receptor

“…For this reason, most antipsychotics currently in use are DRD2 antagonists. The function of DRD2 is determined by its levels on the plasma membrane (PM) (Lin et al, 2001Binda et al, 2002;Free et al, 2007;Kim et al, 2008a,b;Tirotta et al, 2008;Genedani et al, 2010), and DRD2 endocytosis plays an important role in its desensitization following DA stimulation (Kim et al, 2001;Jeanneteau et al, 2004;Kabbani et al, 2004;Macey et al, 2004;Namkung and Sibley, 2004;Sugiura et al, 2004;Bartlett et al, 2005;Genedani et al, 2005;Torvinen et al, 2005;Paspalas et al, 2006;Iizuka et al, 2007;Kim, 2008;Xiao et al, 2009;Celver et al, 2010;Shimokawa et al, 2010). However, the fate of DRD2 following DA-induced endocytosis remains controversial.…”