Molecular Switches Involving the AP-2 β2 Appendage Regulate Endocytic Cargo Selection and Clathrin Coat Assembly
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.
- SourceAvailable from: Pau Gorostiza
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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. "
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 · 6.59 Impact Factor
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- "GST-TACC3-His 6 was phosphorylated with Aurora A, incubated with purified clathrin triskelia, and then affinity purified on glutathione beads. The appendage+hinge fragment of the 2 subunit of AP-2 was used as a positive control for clathrin binding (Edeling et al., 2006). Wild-type TACC3 coprecipitated clathrin, only after phosphorylation, but this binding was greatly reduced by either S558A or LL(566,567)AA mutations (Fig. 3 C). "
ABSTRACT: Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3-clathrin binding. A dileucine motif and Aurora A-phosphorylated serine 558 on TACC3 bound to the "ankle" of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3-clathrin.The Journal of Cell Biology 08/2013; 202(3):463-78. DOI:10.1083/jcb.201211127 · 9.69 Impact Factor
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- "This putative endosomal distribution of LRP-1::GFP is consistent with AP1 functioning in receptor sorting decisions within the endosome (Reusch et al., 2002; Gravotta et al., 2012; Shafaq-Zadah et al., 2012; Xu et al., 2012; Zhang et al., 2012). Given that C. elegans encodes a stabilize the complex (Robinson, 2004; Edeling et al., 2006). AP-2 serves a general requirement in the formation of endocytic clathrin coats and is essential to internalization of myriad receptors, including LDLR, epidermal growth factor receptor (EGFR), and the transferrin receptor (Motley et al., 2003; Boucrot et al., 2010). "
ABSTRACT: Low density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNAi screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to that observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR.Molecular biology of the cell 12/2012; 24(3). DOI:10.1091/mbc.E12-02-0163 · 5.98 Impact Factor