A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex.

Most transmembrane proteins are selected as transport-vesicle cargo through the recognition of short, linear amino-acid motifs in their cytoplasmic portions by vesicle coat proteins. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits α, β2, μ2 and σ2) recognizes both major endocytic motifs: YxxΦ motifs1 (where Φ can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 2). The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to YxxΦ motifs through μ2. In the ‘inactive’ AP2 core structure3 both motif-binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 amino terminus is displaced and becomes disordered; however, in this structure the YxxΦ-binding site on μ2 remains blocked.D.J.O., B.T.K. and S.E.M. are funded by a Wellcome Trust Senior Research Fellowship to D.J.O. S.H. and K.S. are supported by grants from the Deutsche Forschungsgemeinschaft (SFB635 and SFB670)

Signal binding and membrane interaction of heterotetrameric adaptor protein complexes

Die heterotetrameren Adaptorprotein-Komplexe AP1, AP2, AP3 und AP4 sind an der Sortierung von Transmembranproteinen in der Zelle beteiligt. APs interagieren mit den Sortierungssignalen von Transmembranproteinen und vermitteln dadurch ihre Aufnahme in Transportvesikel. Da jeder AP vermutlich an einem anderen zellulären Transportweg beteiligt ist, kann die Interaktion eines APs mit einem spezifischen Signal entscheidend für die Transportroute des Proteins sein. Im ersten Teil dieser Arbeit wurde deshalb die Spezifität der vier APs für die Tyrosin-haltigen Signale (YXXΦ) der lysosomalen Proteine LAMP-1, LAMP-2A, LAMP-2B, CD63 und des am TGN-lokalisierten Proteins TGN38 mittels Oberflächenplasmonresonanz-Analyse (Biacore) in vitro untersucht. Alle getesteten Signale interagierten kaum mit AP4, aber gut mit AP3. Die Bindung an AP1 und AP2 war deutlich heterogener. Am auffälligsten dabei war, dass die lysosomalen Proteine LAMP-1 und LAMP-2B gut an AP1 banden, wohingegen LAMP-2A und CD63 kaum bzw. gar nicht mit AP1 interagierten. Im Mittelpunkt des zweiten Teils der Arbeit stand der an der Endocytose beteiligte AP2-Komplex, dessen Rekrutierung an die Plasmamembran und Bindung an Sortierungssignale mit Hilfe eines Biacore-basierten Assays für Membranbindung in vitro simuliert wurde. Unsere Daten zeigen eine von ARF6-unabhängige, aber von Phosphatidylinositolen-abhängige Rekrutierung von AP2. Durch den Vergleich von phosphoryliertem und nicht-phosphoryliertem AP2 konnte bestätigt werden, dass die Interaktion mit Tyrosin-haltigen Signalen durch die Phosphorylierung der µ2-Untereinheit unterstützt wird. Die Bindung an Dileucin-haltige Signale erfolgt hingegen unabhängig von der µ2 Phosphorylierung

Crn7 interacts with AP-1 and is required for the maintenance of Golgi morphology and protein export from the Golgi

Crn7 is a novel cytosolic mammalian WD-repeat protein of unknown function that associates with Golgi membranes. Here, we demonstrate that Crn7 knockdown by small interfering RNA results in dramatic changes in the Golgi morphology and function. First, the Golgi ribbon is disorganized in Crn7 KD cells. Second, the Golgi export of several marker proteins including VSV envelope G glycoprotein is greatly reduced but not the retrograde protein import into the Golgi complex. We further establish that Crn7 co-precipitates with clathrin adaptor AP-1 but is not required for AP-1 targeting to Golgi membranes. We identify tyrosine 288-based motif as part of a canonical YXXPhi sorting signal and a major mu1-adaptin binding site in vitro. This study provides the first insight into the function of mammalian Crn7 protein in the Golgi complex.status: publishe

Recombinant Human Erythropoietin: Novel Strategies for Neuroprotective/Neuro-regenerative Treatment of Multiple Sclerosis

Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment