Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals

During receptor-mediated endocytosis, AP2 complexes act as a bridge between the cargo membrane proteins and the clathrin coat by binding to sorting signals via the mu 2 subunit and to clathrin via the beta subunit. Here we show that binding of AP2 to sorting signals in vitro is regulated by phosphorylation of the mu 2 subunit of AP2. Phosphorylation of mu 2 enhances the binding affinity of AP2 for sorting motifs as much as 25-fold compared with dephosphorylated AP2. The recognition of sorting signals was not affected by the phosphorylation status of the alpha or beta 2 subunit, suggesting that phosphorylation of mu 2 is critical for regulation of AP2 binding to sorting signals. Phosphorylation of mu 2 occurs at a single threonine residue (Thr-156) and is mediated by the newly discovered adaptor-associated kinase, AAK1, which copurifies with AP2. We propose that phosphorylation of the AP2 mu 2 subunit by AAK1 ensures high affinity binding of AP2 to sorting signals of cargo membrane proteins during the initial steps of receptor-mediated endocytosis.     

Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of mu2 phosphorylation demonstrated that clathrin is a specific activator of the mu2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of m2 phosphorylation. Furthermore, phosphorylated mu2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-mu2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.     

Molecular architecture and functional model of the endocytic AP2 complex

AP2 is the best-characterized member of the family of heterotetrameric clathrin adaptor complexes that play pivotal roles in many vesicle trafficking pathways within the cell. AP2 functions in clathrin-mediated endocytosis, the process whereby cargo enters the endosomal system from the plasma membrane. We describe the structure of the 200 kDa AP2 "core" (alpha trunk, beta2 trunk, mu2, and sigma2) complexed with the polyphosphatidylinositol headgroup mimic inositolhexakisphosphate at 2.6 A resolution. Two potential polyphosphatidylinositide binding sites are observed, one on alpha and one on mu2. The binding site for Yxxphi endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered mu2 linker, is necessary to allow Yxxphi motif binding. A model for AP2 recruitment and activation is proposed.     

Binding of AP2 to sorting signals is modulated by AP2 phosphorylation

The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15-33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis.     

Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2

The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.     

AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin

AAK1, the adaptor-associated kinase 1, phosphorylates the μ2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the μ2 subunit in vitro , μ2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates μ2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization.     

Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor

Clathrin-coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the micro 2 subunit of the AP-2 adaptor complex, which recognizes tyrosine-based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled-2 (Dab2) recognizes the FXNPXY internalization motif in LDL-receptor family members via an N-terminal phosphotyrosine-binding (PTB) domain. Here, we show that in addition to binding AP-2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP-2 and clathrin-binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo-specific adaptor proteins, including beta-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.     

Conformational regulation of AP1 and AP2 clathrin adaptor complexes

Heterotetrameric clathrin adaptor protein complexes (APs) orchestrate the formation of coated vesicles for transport among organelles of the cell periphery. AP1 binds membranes enriched for phosphatidylinositol 4‐phosphate, such as the trans Golgi network, while AP2 associates with phosphatidylinositol 4,5‐bisphosphate of the plasma membrane. At their respective membranes, AP1 and AP2 bind the cytoplasmic tails of transmembrane protein cargo and clathrin triskelions, thereby coupling cargo recruitment to coat polymerization. Structural, biochemical and genetic studies have revealed that APs undergo conformational rearrangements and reversible phosphorylation to cycle between different activity states. While membrane, cargo and clathrin have been demonstrated to promote AP activation, growing evidence supports that membrane‐associated proteins such as Arf1 and FCHo also stimulate this transition. APs may be returned to the inactive state via a regulated process involving phosphorylation and a protein called NECAP. Finally, because antiviral mechanisms often rely on appropriate trafficking of membrane proteins, viruses have evolved novel strategies to evade host defenses by influencing the conformation of APs. This review will cover recent advances in our understanding of the molecular inputs that stimulate AP1 and AP2 to adopt structurally and functionally distinct configurations.     

Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis

The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.     

Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.     

Disruption of adaptor protein 2μ (AP‐2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP‐2μ) is required for release site replenishment and hearing. We show that hair cell‐specific disruption of AP‐2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane‐proximal vesicles and intact endocytic membrane retrieval. Sound‐driven postsynaptic spiking was reduced in a use‐dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome‐like vacuoles, fewer clathrin‐coated endocytic intermediates, and vesicle depletion of the membrane‐distal synaptic ribbon in AP‐2μ‐deficient IHCs, indicating a further role of AP‐2μ in clathrin‐dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP‐2 sorts its IHC‐cargo otoferlin. We propose that binding of AP‐2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP‐2 in synaptic vesicle reformation.     

Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.     

Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.     

Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.     

The structure and function of the beta 2-adaptin appendage domain

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.     

In vivo phosphorylation of adaptors regulates their interaction with clathrin

The coat proteins of clathrin-coated vesicles (CCV) spontaneously self- assemble in vitro, but, in vivo, their self-assembly must be regulated. To determine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual components of the CCV coat were isolated by immunoprecipitation from Madin-Darby bovine kidney cells, labeled with [32P]orthophosphate under normal culture conditions. The predominant phosphoproteins identified were subunits of the AP1 and AP2 adaptors. These included three of the four 100-kD adaptor subunits, alpha and beta 2 of AP2 and beta 1 of AP1, but not the gamma subunit of AP1. In addition, the mu 1 and mu 2 subunits of AP1 and AP2 were phosphorylated under these conditions. Lower levels of in vivo phosphorylation were detected for the clathrin heavy and light chains. Analysis of phosphorylation sites of the 100-kD adaptor subunits indicated they were phosphorylated on serines in their hinge regions, domains that have been implicated in clathrin binding. In vitro clathrin-binding assays revealed that, upon phosphorylation, adaptors no longer bind to clathrin. In vivo analysis further revealed that adaptors with phosphorylated 100-kD subunits predominated in the cytosol, in comparison with adaptors associated with cellular membranes, and that phosphorylated beta 2 subunits of AP2 were exclusively cytosolic. Kinase activity, which converts adaptors to a phosphorylated state in which they no longer bind clathrin, was found associated with the CCV coat. These results suggest that adaptor phosphorylation influences adaptor-clathrin interactions in vivo and could have a role in controlling coat disassembly and reassembly.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

Yeast Endocytic Adaptor AP‐2 Binds the Stress Sensor Mid2 and Functions in Polarized Cell Responses

The AP‐2 complex is a heterotetrameric endocytic cargo‐binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin‐mediated endocytosis. While budding yeast has clear homologues of all four AP‐2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP‐2 has remained enigmatic. Here, we demonstrate that AP‐2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae. Deletion of APM4, the cargo‐binding mu subunit of AP‐2, causes defects in pseudohyphal growth, generation of a mating projection and the cell wall damage response. In an apm4 null mutant, the cell wall stress sensor Mid2 is unable to relocalize to the tip of a mating projection following pheromone addition, or to the mother bud neck in response to cell wall damage. A direct binding interaction between Mid2 and the mu homology domain of Apm4 further supports a model in which AP‐2 binds Mid2 to facilitate its internalization and relocalization in response to specific signals. Thus, Mid2 is the first cargo for AP‐2 identified in yeast. We propose that endocytic recycling of Mid2 and other components is required for polarized cell responses ensuring cell wall deposition and is tightly monitored during cell growth.      

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.