CHAPTER 6 Clathrin Adaptor Proteins in Cargo Endocytosis Linton M. Traub* Abstract ukaryotic cells continuously remodel the protein and lipid composition of the plasma membrane in response to the extracellular milieu. Membrane retrieval typically involves Einward budding of small bilayer-encapsulated vesicles that shutde protein and lipid from the surface to internal endosomal elements. Clathrin-mediated endocytosis is a domi­ nant pathway for internalization in many cell types, and a range of dedicated signals are used to ensure selective sorting in this pathway. Evidence suggests that the clathrin coat uti­ lizes a diverse collection of clathrin-associated sorting proteins (CLASPs) to ensure the effi­ cient and noncompetitive concentration of a wide variety of sorting signals within transport vesicles forming at the cell surface. Introduction The limiting membrane of eukaryotic cells represents the primary interface with the exterior environment. It performs a vital barrier function that, by tighdy controlling the entry of molecules into the cell interior, contributes to long-term cell survival. The plasma membrane is also a platform for first detecting and then responding to extracellular signals, ions and nutrients, as well as potential pathogens. This is because a large array of receptors, ion channels, pumps and transporter proteins are positioned at the cell surface of a typical eukary­ otic cell. Some receptors (mosdy handling nutrient uptake or protein delivery) flux constandy through the plasma membrane while others, primarily involved in signal transduction, only exit the cell surface en masse following ligand stimulation. The process of regulated removal of certain receptors from the surface is perhaps best understood from the point of development,^ where failure to quantitatively clear a particular receptor(s) from the plasma membrane can ultimately result in inappropriate cell fate determination due to erroneous cellular responses to local morphogens or growth factors. Sorting Signals for Selective Internalization In principle, internalization of transmembrane proteins (along with bound ligands) could be either an active or a passive process. There are certainly examples of proteins being retained at the plasma membrane through stabilizing interactions with lipids, lipid rafts, or other proteinaceous components, like scaffolding proteins with PDZ domains for example. Yet, it is generally accepted that endocytic uptake is governed by a positive signal;^ in other words, there •Correspondence Author: Linton M. Traub—Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST Pittsburgh, Pennsylvania 15261, U.S.A. Email: [email protected] Endosomesy edited by Ivan Dikic. ©2006 Landes Bioscience and Springer Science+Business Media. Clathrin Adaptor Proteins in Cargo Endocytosis 63 is a process of preferential sorting of select transmembrane proteins for enrichment within forming carrier vesicles. This is because many proteins/receptors stagnate at the cell surface, redistributing into a diffuse pattern over the plasma membrane, when sorting signals within the cytosolic domain are inactivated either by inherited defects or directed mutation(s), or if the cytosolic region is simply truncated. Indeed, numerous, structurally discrete sorting signals that specify rapid internalization are now known^ (Table 1). Clathrin-Mediated Endocytosis There are several ways in which preferential entry of transmembrane proteins and bound ligands into the cytoplasm can occur. These include the budding of small (--60-100 nm) membrane bound vesicles in caveolin-dependent,^^ clathrin-dependent,^^'^'^ and uncoated caveolin-independent endocytosis, ' as well as through larger forms of membrane in­ ternalization vehicle, as in macropinocytosis and phagocytosis. Of these, probably the best characterized at the mechanistic level is clathrin-mediated endocytosis. Clathrin-coated buds and clathrin-coated vesicles were originally discovered in thin-section electron micrographs on the basis of a highly characteristic brisde-like matrix deposited on the cytosolic aspect of these structures'^ (see Fig. lA). The unusual membrane invaginations also displayed striking concentration of electron dense material within the lumen, located opposite to the bristle coat. These landmark morphological findings accurately mirror the two principal fiinctional activities of clathrin-coated vesicles (and coat-dependent sorting in general); assembly of a polymeric, cytosol-oriented coat while simultaneously gathering select cargo molecules into the invaginating coated vesicle. Table 1, Clathrin •dependent endocytic sorting signals Examples Recognition Signal Type Sequence Protein Protein/Domain References YXX0^ YTRF transferrin receptor AP-2, \a subunit 7, 19 YSKV CI^-MPR YRGV CD^-MPR YQRL TGN38/46 YQTI LAMP-1 YATL LRP1 [DE]XXXL[LI] ERAPLI LIMP-II AP-1,Y/a1 7,47 DKQTLL CD3-Y hemlcomplex EKQPLL tyrosinase AP-2, (x/a2 DQRDLI li hennicomplex? [FY]XNPXY FDNPVY LDL receptor ARH, Dab2,Numb 7, 19 FTNPVY LRP1 PTB domain FENPMY megalin p-arrestin? YTNPAF Sanpodo Phosphorylation - GPCRs P-arrestin 1/2 19,64 Ubiquitin EGFR epsin, epsIS 102, 103 Notch UIM^ domain? Delta ENaC^ ^ Consensus sequences indicated in single letter amino acid notation using PROSITE syntax. 0 indicates a bulky hydrophobic amino acid; Leu, Met, lie, Phe, Val, '^CI-MPR is cation-independent mannose 6-phosphate receptor. ^ CD-MPR is cation-dependent mannose 6-phosphate receptor. "ENaC is epithelial sodium channel. ^UIM is ubiquitin interaction motif. 64 Endosi Figure 1. Clathrin-coat morphology. A) Thin-section electron micrograph of a purified rat brain nerve ending (synaptosome) revealing the characteristic cytosol-oriented 'bristle' matrix (arrows) enveloping several cIathrin-coated vesicles budding from the presynaptic plasma membrane (PM). At the nerve ending, coated vesicles retrieve synaptic vesicle membrane components to replenish the pool of vesicles (arrowheads) required for sustained neurotransmission. A mito­ chondrion (asterisk) is positioned in close proximity to the synaptic region. B) Freeze-etch electron micrograph of the cytoplasmic face of the ventral plasma membrane of a cultured cell revealing the typical polyhedral clathrin lattice. The progressive invagination of coated mem­ brane is clearly seen in different intermediates of the budding process. This image was graciously provided by John Heuser. The 200 A bristle-like structures radiating oflF clathrin-coated membranes correspond to the regular coat assemblage. The major constituent is, of course, clathrin, a tri-legged protein complex composed of three 192-kDa heavy chains and three '-25-kDa regulatory light chains, one bound to each heavy chain (Fig. 2). Clathrin trimers can self-assemble onto spherical polyhedral assemblies in vitro, which are highly reminiscent of coated vesicles in vivo. It is the geometric nature of the triskelion that dictates the formation of the hexagon/pentagon-containing lattice, the most recognizable feature of the clathrin coat (Fig. IB). The orientation and rigidity of the fibrous heavy chains imparts an inherent sidedness to the triskelion; when assembled upon biological membranes, the amino-terminal region of the heavy chain, which folds into a 7-bladed P-propeller structure, ^^ is oriented closest to the bilayer. The carboxyl termini, which bundle together at the central vertex in a helical tripod arrangement, are positioned furthest from the membrane surface. However, clathrin does not have the capacity to associate with phospholipid bilayers direcdy. Instead, adaptor proteins couple the clathrin lattice with the underlying membrane by synchronously binding to the clathrin terminal domain, to phospholipids, and to cargo sorting signals. The AP-2 Adaptor Complex A second major protein constituent of the endocytic clathrin coat is the AP-2 adaptor, ^^ composed of four distinct subunits; two large --lOO-kDa subunits (a and p2), a 50-kDa medium subunit (|Ll2), and a 17-kDa small subunit (a2).^^ The functional complex has a characteristic architecture of two small appendages projecting oflFa larger globular core through Clathrin Adaptor Proteins in Cargo Endocytosis 65 HIP1 4I~ CALM AP-2 ANTH a subunir appendage AP180 If adaptor wmm MS^W • nSsubunit core epsin [52 subunit J (i2 subunit appendage Numb Dab2 heavy chain terminal domain ARH (3-arrestJn / clathrin Ptdlns(4,5)P2 binding Figure 2. The clathrin-AP-2-CLASP interaction network. Schematic depiction of the protein- protein interactions between clathrin, AP-2 and selected CLASPs is shown. Images are modeled on the known molecular structures of the coat components, but are not drawn to scale. The clathrin terminal domain and the AP-2 appendages serve as platforms to coordinate the protein- protein interaction networksestablishedduringcoatassembly. The location of interaction motifs used to engage these binding platforms are indicated by color-coded vertical lines. In AP180 and CALM, where the interaction motifs have not been defined precisely, interaction is generally signified by a horizontal line. The location of the three tandem UlMs in epsin 1 (ovals) is shown. pGPCR indicates phosphorylated (ligand-activated) G protein-coupled receptor, while l/LWEQ is a modular F-actin bindingfold that is also conserved in the S. cerevisiae F4IP1 orthologue Sla2p. protease-sensitive stalks of apparendy variable length.^ Structural studies show the core is a heterotetramer comprised of the amino-terminal