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: Umasankar Perunthottathu[Show abstract] [Hide abstract]
ABSTRACT: Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.eLife Sciences 10/2014; 3. DOI:10.7554/eLife.04137 · 8.52 Impact Factor
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ABSTRACT: Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCP) at the plasma membrane that invaginate to form clathrin-coated vesicles; a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCL); these structures remain poorly described and their contribution to cell biology is unclear. We have used quantitative imaging to provide the first comprehensive description of FCL and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and super-resolution microscopy revealed two discrete populations of clathrin structures. CCP were typified by their sphericity, small size and homogeneity. FCL were planar, large and heterogeneous, and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCP are short-lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCL were long-lived with sustained association with dynamin. We investigated the biological relevance of FCL using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCL. Quantitative molecular imaging indicated that FCL partitioned receptors at the cell surface. Our observations suggest that FCL provide stable platforms for the recruitment of endocytic cargo.Molecular Biology of the Cell 08/2014; 25(22). DOI:10.1091/mbc.E14-06-1154 · 4.55 Impact Factor
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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 · 6.59 Impact Factor