A. Simonds

The Conserved Isoleucine-Valine-Phenylalanine Motif Couples Activation State and Endocytic Functions of β-Arrestins

Traffic, 2007

Beta-arrestins (barrs) play a central role in the regulation of G-protein-coupled receptors (GPCR... more Beta-arrestins (barrs) play a central role in the regulation of G-protein-coupled receptors (GPCRs). Their binding to phosphorylated activated GPCRs induces a conformational transition to an active state resulting in the release of their flexible C-terminal tail. Binding sites for clathrin and the adaptor protein (AP)-2 clathrin adaptor complex are then unmasked, which drive the recruitment of barrs-GPCR complexes into clathrin-coated pits (CCPs). A conserved isoleucine-valine-phenylalanine (IVF) motif of the C-terminal tail controls barr activation through intramolecular interactions. Here, we provide structural, biochemical and functional evidence in living cells that the IVF motif also controls binding to AP-2. While the F residue is directly involved in AP-2 binding, substitutions of I and V residues, markedly enhanced affinity for AP-2 resulting in active barr mutants, which are constitutively targeted to CCPs in the absence of any GPCR activation. Conformational change and endocytic functions of barrs thus appear to be coordinated via the complex molecular interactions established by the IVF motif.

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Dynamic Interaction of HIV-1 Nef with the Clathrin-Mediated Endocytic Pathway at the Plasma Membrane

Traffic, 2006

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Understanding Living Clathrin-Coated Pits

Traffic, 2004

Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and ... more Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and in vitro assays. Recently, the study of endocytosis has extended into the living cell. The tracking of individual clathrincoated pits and vesicles (CCPs and CCVs) has provided new insight into understanding the dynamic nature of CCPs. The use of total internal reflection fluorescence microscopy (TIR-FM), also termed evanescent field microscopy, has enabled the direct observation of events occurring within a restricted area of the cell adjacent to and including the adherent plasma membrane. TIR-FM is now actively being pursued in the study of endocytic processes. The direct observation of CCP-associated proteins including clathrin itself, dynamin and, most recently, AP-2 has considerably challenged old models, confirming some points but raising very interesting new questions.

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APP intracellular domain-WAVE1 pathway reduces amyloid-β production

Nature medicine, Jan 17, 2015

An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheime... more An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aβ production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aβ. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aβ levels and restored memory deficits in ...

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Dynamic Interaction of HIV-1 Nef with the Clathrin-Mediated Endocytic Pathway at the Plasma Membrane

by Alexandre Benmerah, Jérôme Bouchet, and A. Simonds

Traffic, 2007

Because of the discovery of coated pits and vesicles more than 40 years ago and the identificatio... more Because of the discovery of coated pits and vesicles more than 40 years ago and the identification of clathrin as a major component of the coat, it has been assumed that clathrin-coated pits (CCPs) are responsible for the uptake of most plasma membrane receptors undergoing internalization. The recent molecular characterization of clathrin-independent routes of endocytosis confirms that several alternative endocytic pathways operate at the plasma membrane of mammalian cells. This heterogeneous view of endocytosis has been expanded still further by recent studies, suggesting that different subpopulations of CCPs responsible for the internalization of specific sets of cargo may coexist. In the present review, we have discussed the experimental evidence in favor or against the existence of distinct parallel clathrindependent pathways at the plasma membrane.

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Dynamics of clathrin and adaptor proteins during endocytosis

by Alexandre Benmerah, S. Kemal, and A. Simonds

AJP: Cell Physiology, 2006

The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at th... more The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for Ն30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis. adaptor complex; epsin; total internal reflection fluorescence microscopy

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Understanding Living Clathrin-Coated Pits

by Alexandre Benmerah and A. Simonds

Traffic, 2004

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The Conserved Isoleucine-Valine-Phenylalanine Motif Couples Activation State and Endocytic Functions of β-Arrestins

by Alexandre Benmerah, Harvey McMahon, A. Simonds, and Stefano Marullo

Traffic, 2007

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Analysis of the AP-2 Adaptor Complex and Cargo During Clathrin-Mediated Endocytosis

by Alexandre Benmerah and A. Simonds

Traffic, 2005

Previously, we reported that the hetero-tetrameric adaptor complex AP-2 co-localizes with the sta... more Previously, we reported that the hetero-tetrameric adaptor complex AP-2 co-localizes with the static population of clathrin spots, whereas it is excluded from clathrin spots that disappear from the plasma membrane (forming clathrin-coated vesicles). More recently however, another group provided evidence that AP-2 markers could be observed coincident with disappearing clathrin spots. Thus, we tested several possible explanations for the apparent discrepancies in these two studies. We evaluated the potential contribution of nonred emission of clathrin-dsRed (used in both studies) in the simultaneous measurement of AP-2 and clathrin at various times. Additionally, we directly compared two different green fluorescent protein-tagged AP-2 constructs (similar to those used in the previous reports). These studies demonstrated that the duration of expression time greatly influences the subcellular localization of the AP-2 markers. Furthermore, we quantitatively evaluated the AP-2 fluorescence at the sites of numerous static and disappearing clathrin spots (at least 80 per group) and confirmed our initial observation that while AP-2 is present in nearly all static clathrin spots, it is excluded from the disappearing population of clathrin spots. Finally, in order to verify that clathrin spot disappearance represents clathrin-coated vesicle internalization, we simultaneously imaged clathrin and the cargo molecule transferrin at the cell surface.

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APP intracellular domain-WAVE1 pathway reduces amyloid-β production

by Jung-hyuck Ahn and A. Simonds

Nature medicine, Jan 17, 2015

An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheime... more An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aβ production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aβ. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aβ levels and restored memory deficits in ...

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