Molecular Switches Involving the AP-2 β2 Appendage Regulate Endocytic Cargo Selection and Clathrin Coat Assembly

Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, United Kingdom.
Developmental Cell (Impact Factor: 9.71). 04/2006; 10(3):329-42. DOI: 10.1016/j.devcel.2006.01.016
Source: PubMed

ABSTRACT Clathrin-associated sorting proteins (CLASPs) expand the repertoire of endocytic cargo sorted into clathrin-coated vesicles beyond the transmembrane proteins that bind physically to the AP-2 adaptor. LDL and GPCRs are internalized by ARH and beta-arrestin, respectively. We show that these two CLASPs bind selectively to the AP-2 beta2 appendage platform via an alpha-helical [DE](n)X(1-2)FXX[FL]XXXR motif, and that this motif also occurs and is functional in the epsins. In beta-arrestin, this motif maintains the endocytosis-incompetent state by binding back on the folded core of the protein in a beta strand conformation. Triggered via a beta-arrestin/GPCR interaction, the motif must be displaced and must undergo a strand to helix transition to enable the beta2 appendage binding that drives GPCR-beta-arrestin complexes into clathrin coats. Another interaction surface on the beta2 appendage sandwich is identified for proteins such as eps15 and clathrin, suggesting a mechanism by which clathrin displaces eps15 to lattice edges during assembly.

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    • "(B) Schematic of the BAP-long/b-adaptin interaction. Conserved residues critical for the interaction are shown in red (Edeling et al., 2006; Schmid et al., 2006). Crosslinking positions tested in the peptide panel are shown as yellow cysteine side chains. "
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    ABSTRACT: Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a β-arrestin P-long 20-mer peptide (BAP-long) sequence to assess the relevance of secondary structure in their interaction with β-adaptin 2 and to identify the design requirements for photoswitchable inhibitors of PPI (PIPPIs). We observe that flexible structures show a greater inhibitory capacity and enhanced photoswitching ability and that the absence of helical structures in free inhibitor peptide is not a limitation for PIPPI candidates. Therefore, our PIPPIs expand the field of potential inhibitors of PPIs to the wide group of flexible peptides, and we argue against using a stable secondary structure as a sole criterion when designing PIPPI candidates. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & Biology 01/2015; 22(1):31-37. DOI:10.1016/j.chembiol.2014.10.022 · 6.65 Impact Factor
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    • "Eps15 is localized at the rims of clathrin-coated pits [11-13], and is thought to participate in formation of the clathrin lattice. Consistent with this idea, dominant-negative Eps15 constructs inhibit endocytosis of diverse cargoes, including transferrin receptor and the epidermal growth factor receptor (EGFR) [14]. "
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    ABSTRACT: Background Eps15 is an endocytic adaptor protein that stimulates clathrin-mediated endocytosis. Among other interactions, Eps15 binds ubiquitin via UIM domains, recruiting ubiquitinated cargo into clathrin-coated vesicles. In EGF-treated cells, Eps15 also localizes to endosomes. The basis of this localization is not known. Results We show that accumulation of ubiquitinated cargo can recruit Eps15 to endosomes via UIM domain interactions. First, treatment of SK-Br-3 breast cancer cells, which overexpress the EGFR family member ErbB2, with geldanamycin to promote receptor ubiquitination and endosomal transport, recruited FLAG-Eps15 to endosomes. Two in-frame ubiquitin constructs, PM-GFP-Ub (retained in endosomes after endocytosis), and GFP-FYVE-UbΔGG (targeted directly to endosomes) also recruited Eps15 to endosomes, as did slowing endosome maturation with constitutively-active Rab5-Q79L. Endosomal recruitment required the UIM domains, but not the N-terminal EH domains or central coiled-coil domains, of Eps15. Silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes. In fact, Hrs silencing itself modestly recruited Eps15 to endosomes, probably by accumulating endogenous ubiquitinated cargo. Eps15 silencing did not affect lysosomal degradation of ubiquitinated ErbB2; however, GFP-FYVE-UbΔGG overexpression inhibited internalization of EGFR and transferrin receptor. Conclusions We show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.
    BMC Cell Biology 09/2014; 15(1):34. DOI:10.1186/1471-2121-15-34 · 2.34 Impact Factor
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    • "Second, it was demonstrated that multiple sites are used for clathrin function in yeast and human cells (Collette et al., 2009; Willox and Royle, 2012). Third, interaction sites on the leg of CHC may play a role in adaptor engagement (Edeling et al., 2006; Knuehl et al., 2006). Much of this information was known when the pitstops were first described. "
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    ABSTRACT: Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. "Pitstop" compounds have been developed that block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin's N-terminal domain.
    Biology Open 04/2014; 3(5). DOI:10.1242/bio.20147955 · 2.42 Impact Factor
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