Adaptin Research Articles

A Tandem Di-hydrophobic Motif Mediates Clathrin-dependent Endocytosis via Direct Binding to the AP-2 ασ2 Subunits

Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXXΦ, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins. However, some surface proteins, such as the Kir2.3 potassium channel, lack canonical signals but are still targeted for clathrin-dependent endocytosis. Here, we explore the mechanism. We found an unusual endocytic signal in Kir2.3 that is based on two consecutive pairs of hydrophobic residues. Characterized by the sequence ΦΦXΦΦ (a tandem di-hydrophobic (TDH) motif, where Φ is a hydrophobic amino acid), the signal shows no resemblance to other endocytic motifs, yet it directly interacts with AP-2 to target the Kir2.3 potassium channel into the endocytic pathway. We found that the tandem di-hydrophobic motif directly binds to the ασ2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins.

Do GGA Adaptors Bind Internal DXXLL Motifs?

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs.

UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis

The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.

Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef

The HIV-1 protein Nef inhibits antigen presentation by class I major histocompatibility complex (MHC-I). We determined the mechanism of this activity by solving the crystal structure of a protein complex comprising Nef, the MHC-I cytoplasmic domain (MHC-I CD) and the μ1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-μ1 interface, which encompasses the cargo-recognition site of μ1 and the proline-rich strand of Nef. The Nef C terminus induces a previously unobserved conformational change in μ1, whereas the N terminus binds the Nef core to position it optimally for complex formation. Positively charged patches on μ1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity.

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function

The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and μ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats.

Structural Basis of the Intracellular Sorting of the SNARE VAMP7 by the AP3 Adaptor Complex

SummaryVAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.

Regulation of protease‐activated receptor‐4 signaling and trafficking

Protease‐activated receptors (PARs) are G‐protein coupled receptors (GPCRs) that elicit cellular responses to thrombin. PARs play pivotal roles in hemostasis, thrombosis, and inflammatory responses to tissue injury. PAR activation is unique compared to other GPCRs. Thrombin cleaves the receptor's N‐terminus to reveal a “tethered ligand” that binds in cis to the receptor, resulting in G‐protein signaling. Due to the irreversible nature of PAR activation, tight regulatory mechanisms are required to govern their signaling. Thrombin signals predominantly through the activation of PAR1 and PAR4. Most cell types express more than one PAR. Thus, delineating the mechanisms of PAR4 signal regulation is essential to understanding the regulation of thrombin signaling. The goal of this study is to define the mechanisms of PAR4 internalization. To identify the mechanism of PAR4 internalization we depleted cells of certain adaptor proteins by siRNA, which revealed that the clathrin adaptor protein complex‐2 (AP‐2) plays a necessary role in this process. Immunofluorescence microscopy experiments revealed that activated PAR4 colocalizes with clathrin and is trafficked through early endosomes, and late endosomes/lysosomes. This study highlighted several other key aspects of PAR4's signal regulation that will be presented. Supported by NIH R01 HL073328.

Palmitoylation of PAR1 regulates the accessibility of C‐tail YXX φ motifs to AP‐2 and internalization

Protease‐activated receptor‐1 (PAR1), a G‐protein coupled receptor (GPCR) for thrombin, is irreversibly and proteolytically activated. Thus, desensitization and intracellular trafficking of PAR1 are critical for proper cellular responses. Palmitoylation of GPCRs regulates various aspects of signaling and trafficking, however the function of palmitoylation in PAR1 signal regulation is not known. We examined palmitoylation in PAR1 wildtype and in a mutant where C‐tail cysteines were converted to alanines. Here, we show that PAR1 is palmitoylated at these C‐tail cysteine residues. Moreover, the PAR1 palmitoylation mutant displayed enhanced constitutive internalization, whereas agonist‐induced internalization was comparable to wildtype receptor. Constitutive internalization of the PAR1 palmitoylation mutant was dependent on the clathrin adaptor protein complex AP‐2, like wildtype receptor. However, unlike wildtype PAR1, AP‐2‐mediated internalization required the proximal rather than the distal YXXφ motif in this mutant. The PAR1 palmitoylation mutant also displayed defects in phosphorylation and ubiquitination. Thus, palmitoylation stabilizes a conformation of the PAR1 C‐tail that is important for trafficking and other post‐translational modifications. Supported by RO1 HL073328 & UC TRDRP.

Adaptor Protein Complex‐2 and epsin‐1 mediate Protease‐activated Receptor‐1 internalization via phosphorylation‐ and ubiquitination‐dependent sorting signals

Signaling by protease‐activated receptor‐1 (PAR1), a G protein‐coupled receptor (GPCR) for thrombin, is tightly regulated. PAR1 desensitization is mediated by β‐arrestins. However, internalization of PAR1 occurs through a clathrin‐ and dynamin‐dependent pathway independent of β‐arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex‐2 (AP‐2). However, AP‐2 depletion only partially inhibits agonist‐induced internalization of PAR1, suggesting a function for other clathrin adaptors. We now report that AP‐2 and epsin‐1 are both critical mediators of agonist‐stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin‐interacting motifs of epsin‐1 are required for epsin‐1‐dependent internalization of activated PAR1. AP‐2 regulates activated PAR1 internalization via recognition of distal C‐tail phosphorylation sites rather than the canonical tyrosine‐based motif. We further demonstrate a new function for AP‐2 in modulating the signaling responses of activated PAR1. Thus, AP‐2 and epsin‐1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This work was supported by National Institutes of Health Grant and a University of California Tobacco‐related Disease Research Program Exploratory Award.

AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster

Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

Pik1-ing clathrin adaptors

Clathrin adaptor proteins are essential for clathrin-coated vesicle biogenesis, yet the mechanisms governing their recruitment and interactions remain incompletely defined. The clathrin adaptors Gga and AP-1 are now shown to be recruited sequentially to the trans-Golgi network in two waves of clathrin coat assembly, coupled by Pik1-mediated phosphatidylinositol-4-phosphate synthesis. These findings reveal mechanistic insights into the functional and regulatory relationships between these clathrin adaptors.

Endocytosis Genes Facilitate Protein and Membrane Transport in C. elegans Sensory Cilia

Multiple intracellular transport pathways drive the formation, maintenance, and function of cilia, a compartmentalized organelle associated with motility, chemo-/mechano-/photosensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the etiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In thisstudy, we employ the genetically tractable nematode Caenorhabditis elegans to investigate whether endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins. Here we show that localization of the clathrin light chain, AP-2 clathrin adaptor, dynamin, and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume. These data implicate C.elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume and suggest that membrane retrieval from these compartments is counterbalanced by BBS-8 and RAB-8-mediated membrane delivery.

AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development inDrosophila melanogaster

Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts.

Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.

NMDA Receptor-Mediated PIP5K Activation to Produce PI(4,5)P 2 Is Essential for AMPA Receptor Endocytosis during LTD

NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca²⁺ influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses.

Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway

Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs), which encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki-signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs, and place Drosophila endocytic nTSGs into a network that involving two major signaling pathways implicated in oncogenesis.

Dynamic redistribution of calcium sensitive potassium channels (hKCa3.1) in migrating cells

Calcium-sensitive potassium channels (K(Ca)3.1) are expressed in virtually all migrating cells. Their activity is required for optimal cell migration so that their blockade leads to slowing down. K(Ca)3.1 channels must be inserted into the plasma membrane in order to elicit their physiological function. However, the plasma membrane of migrating cells is subject to rapid recycling by means of endo- and exocytosis. Here, we focussed on the endocytic internalization and the intracellular transport of the human isoform hK(Ca)3.1. A hK(Ca)3.1 channel construct with an HA-tag in the extracellularly located S3-S4 linker was transfected into migrating transformed renal epithelial MDCK-F cells. Channel internalization was visualized and quantified with immunofluorescence and a cell-based ELISA. Movement of hK(Ca)3.1 channel containing vesicles as well as migration of MDCK-F cells were monitored by means of time lapse video microscopy. hK(Ca)3.1 channels are endocytosed during migration. Most of the hK(Ca)3.1 channel containing vesicles are moving at a speed of up to 2 µm/sec in a microtubule-dependent manner towards the front of MDCK-F cells. Our experiments indicate that endocytosis of hK(Ca)3.1 channels is clathrin-dependent since they colocalize with clathrin adaptor proteins and since it is impaired when a C-terminal dileucine motif is mutated. The C-terminal dileucine motif is also important for the subcellular localization of hK(Ca)3.1 channels in migrating cells. Mutated channels are no longer concentrated at the leading edge. We therefore propose that recycling of hK(Ca)3.1 channels contributes to their characteristic subcellular distribution in migrating cells.

Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.