Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.     

CVAK104 is a novel regulator of clathrin-mediated SNARE sorting

Clathrin-coated vesicles (CCVs) mediate transport between the plasma membrane, endosomes and the trans Golgi network. Using comparative proteomics, we have identified coated-vesicle-associated kinase of 104 kDa (CVAK104) as a candidate accessory protein for CCV-mediated trafficking. Here, we demonstrate that the protein colocalizes with clathrin and adaptor protein-1 (AP-1), and that it is associated with a transferrin-positive endosomal compartment. Consistent with these observations, clathrin as well as the cargo adaptors AP-1 and epsinR can be coimmunoprecipitated with CVAK104. Small interfering RNA (siRNA) knockdown of CVAK104 in HeLa cells results in selective loss of the SNARE proteins syntaxin 8 and vti1b from CCVs. Morpholino-mediated knockdown of CVAK104 in Xenopus tropicalis causes severe developmental defects, including a bent body axis and ventral oedema. Thus, CVAK104 is an evolutionarily conserved protein involved in SNARE sorting that is essential for normal embryonic development.     

FCH domain only-2 organizes clathrin-coated structures and interacts with Disabled-2 for low-density lipoprotein receptor endocytosis

Clathrin-mediated endocytosis regulates the internalization of many nutrient and signaling receptors. Clathrin and endocytic accessory proteins are recruited to receptors by specific adaptors. The adaptor Disabled-2 (Dab2) recruits its cargoes, including the low-density lipoprotein receptor (LDLR), and mediates endocytosis, even when the major adaptor protein AP2 is depleted. We hypothesized that the accessory proteins normally recruited by AP2 may be recruited by Dab2 if AP2 is absent. We identified one such accessory protein, the F-BAR protein FCH domain only-2 (FCHO2), as a major Dab2-interacting protein. The μ-homology domain (μHD) of FCHO2 binds directly to DPF sequences in Dab2 that also bind AP2. Disrupting the Dab2-FCHO2 interaction inhibited Dab2-mediated LDLR endocytosis in AP2-depleted cells. Depleting FCHO2 reduced the number but increased the size of clathrin structures on the adherent surface of HeLa cells and inhibited LDLR and transferrin receptor clustering. However, LDLR was internalized efficiently by FCHO2-deficient cells when additional time was provided for LDLR to enter the enlarged structures before budding, suggesting that later steps of endocytosis are normal under these conditions. These results indicate FCHO2 regulates the size of clathrin structures, and its interaction with Dab2 is needed for LDLR endocytosis under conditions of low AP2.     

The beta2-adaptin clathrin adaptor interacts with the mitotic checkpoint kinase BubR1

The adaptor AP2 is a heterotetrameric complex that associates with clathrin and regulatory proteins to mediate rapid endocytosis from the plasma membrane. Here, we report the identification of the mitotic checkpoint kinase BubR1 as a novel binding partner of beta2-adaptin, one of the AP2 large subunits. Using two-hybrid experiments and in vitro binding assays, we show that beta2-adaptin binds to BubR1 through its amino-terminal beta2-'trunk' domain, while the beta2-binding region of BubR1 maps to the carboxy-terminal kinase domain. Subcellular immunolocalization studies suggest that the interaction between BubR1 and beta2-adaptin could take place in the cytosol at any time during the cell cycle. In addition, we found that BubR1 and the BubR1-related kinase, Bub1, also bind to beta-adaptins of other AP complexes. Together, these results support a model in which the mitotic checkpoint kinases BubR1 and BuB1, by binding to beta-adaptins, may play novel roles in the regulation of vesicular intracellular traffic.     

HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain

Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis.     

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.     

A Coated Vesicle-associated Kinase of 104 kDa (CVAK104) Induces Lysosomal Degradation of Frizzled 5 (Fzd5)

Receptor internalization is recognized as an important mechanism for controlling numerous cell surface receptors. This event contributes not only to regulate signal transduction but also to adjust the amount of cell surface receptors. Frizzleds (Fzds) are seven-pass transmembrane receptor family proteins for Wnt ligands. Recent studies indicated that Fzd5 is internalized in response to Wnt stimulation to activate downstream signaling pathways. After internalization, it appears that Fzd5 is recycled back to the plasma membrane. However, whether internalized Fzd5 is sorted to lysosomes for protein degradation remains unclear. We here report that a coated vesicle-associated kinase of 104 kDa (CVAK104) selectively induces lysosomal degradation of Fzd5. We identify CVAK104 as a novel binding partner of Dishevelled (Dvl), a scaffold protein in the Wnt signaling pathway. Interestingly, we find that CVAK104 also interacts with Fzd5 but not with Fzd1 or Fzd4. CVAK104 selectively induces intracellular accumulation of Fzd5 via the clathrin-mediated pathway, which is suppressed by coexpression of a dominant negative form of Rab5. Fzd5 is subsequently degraded by a lysosomal pathway. Indeed, knockdown of endogenous CVAK104 by RNA interference results in an increase in the amount of Fzd5. In contrast, Wnt treatment induces Fzd5 internalization but does not stimulate its degradation. Overexpression or knockdown of CVAK104 results in a significant suppression or activation of the Wnt/β-catenin pathway, respectively. These results suggest that CVAK104 regulates the amount of Fzd5 by inducing lysosomal degradation, which probably contributes to the suppression of the Wnt signaling pathway.Internalization of cell surface receptors is an important event to regulate signal transduction from the extracellular environment (1, 2). This event contributes to control the amount of receptors at the plasma membrane. Internalization mainly occurs via the clathrin-dependent pathway. It is characterized by the recruitment of adaptor protein (AP),² such as AP-2, and the assembly of a clathrin coat, which helps the inward budding of clathrin-coated vesicles (3). Internalized receptors are transported to early endosomes, from where they are either recycled back to the plasma membrane or directed to degradative components, such as lysosomes. Rab5, a member of the Rab family GTPase proteins that exert regulatory functions in the endocytic and exocytic trafficking, regulates the fusion of plasma membrane-derived vesicles with early endosomes and homotypic fusion among early endosomes (4).Accumulating data indicate that numerous regulatory proteins also play important roles in endocytic processes. Coated vesicle-associated kinase of 104 kDa (CVAK104) is one of these accessory proteins, which was recently discovered by mass spectroscopy analysis of AP preparations form bovine brain (5). Several groups reported that CVAK104 interacts with clathrin (5–7). In addition, CVAK104 binds to AP-2 and phosphorylates the β subunit of AP-2 in vitro, suggesting a role in the clathrin-mediated endocytosis (5). Furthermore, it was recently demonstrated that CVAK104 also functions in trafficking between the trans-Golgi network and endosomes. For example, knockdown of CVAK104 by small interfering RNAs (siRNAs) results in missorting of the lysosomal enzyme cathepsin D (6). CVAK104 also regulates sorting of t-SNARE proteins from the trans-Golgi network to late endosomes in which they function as an adaptor for docking and fusion of vesicles (7). These reports suggest an importance of CVAK104 in intracellular trafficking that occurs after endocytosis. The Wnt signaling pathway is evolutionarily conserved from nematodes to mammals and is involved in embryonic development and various human diseases, including cancer (8–10). In this signaling pathway, Dishevelled (Dvl) functions as an essential signal transducer from the Wnt receptors to downstream components. Dvl is composed of three conserved domains: an N-terminal Dishevelled-Axin (DIX) domain, a PSD95/Dlg/ZD1 (PDZ) domain in the middle, and a C-terminal Dishevelled-Egl10-pleckstrin (DEP) domain. It is well known that these three domains are required for protein-protein interaction to transduce signals to downstream targets. Dvl also possesses a region harboring positively charged (basic) amino acid residues (termed the basic region) (11–14). It is reported that the basic region is also required for interaction with several downstream signaling components. Indeed, Frat1 and NRX (nucleoredoxin) interact with Dvl through the basic region and the PDZ domain (15, 16). Furthermore, Par1 binds only to the basic region (17). These results suggest that the basic region plays a critical role in the function of Dvl.Frizzled (Fzd) receptors are seven-pass transmembrane proteins. The Fz genes were first identified in Drosophila in a screen for mutations that disrupt the polarity of epidermal cells in the adult fly (18). Ten genes encoding Fzds have been identified in the human genome (19), and the overall structure of Fzd receptors is well conserved among the 10 proteins and also throughout evolution (20, 21). Accumulating evidence indicates that Fzd receptors are internalized in response to their Wnt ligands. Wnt5a induces the internalization of Fzd4 (22). Wnt3a induces the internalization of Fzd5 via the clathrin-dependent pathway (23). In addition, Wnt11 cooperates with atypical receptor-related tyrosine kinase to promote the internalization of Fzd7 via the β-arrestin-2-dependent pathway (24). These ligand-dependent internalizations of Fzd receptors are required for activating signaling pathways. Recent studies also demonstrate that Dvl not only functions as a signal transducer but also plays important roles in internalization of the Fzd receptor. It has been reported that Dvl recruits β-arrestin-2 to internalize Fzd4 in response to Wnt5a treatment (22) and that interaction between Dvl and AP-2 is needed to stimulate internalization of Fzd4 (25). After internalization, cell surface receptors are generally recycled back to the plasma membrane or sorted to lysosomes for protein degradation. It has also been reported that Fzd5 internalized in a ligand-dependent manner appears to be recycled back to the plasma membrane, because internalized Fzd5 co-localizes with Rab11, which plays an important role in the recycling process (23). However, whether receptor degradation, another common consequence after receptor internalization, occurs in the case of Fzd5 still remains unknown.In this study, we search for in vivo Dvl binding partners and identify CVAK104 as a novel Dvl-interacting protein. We also find that CVAK104 interacts with Fzd5 and that expression of CVAK104 induces intracellular accumulation of Fzd5 through the clathrin-dependent pathway. Interestingly, CVAK104 selectively interacts with and induces accumulation of Fzd5 but not Fzd1 or Fzd4. In addition, we find that Fzd5 internalized in the presence of CVAK104 is subsequently degraded by a lysosomal pathway, suggesting a novel mechanism for regulating the turnover of a specific subclass of Fzd receptors.     

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.     

CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles

Several peripheral membrane proteins associated with clathrin-coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV-associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (μ2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G-associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, μ2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the 'stripped' CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.     

Functional analysis of interaction sites on the N-terminal domain of clathrin heavy chain

In clathrin-mediated membrane traffic, clathrin does not bind directly to cargo and instead binds to adaptors that mediate this function. For endocytosis, the main adaptor is the adaptor protein (AP)-2 complex, but it is uncertain how clathrin contacts AP-2. Here we tested in human cells the importance of the three binding sites that have been identified so far on the N-terminal domain (NTD) of clathrin. We find that mutation of each of the three sites on the NTD, alone or in combination, does not block clathrin/AP-2-mediated endocytosis in the same way as deletion of the NTD. We report here the fourth and final site on the NTD that is required for clathrin/AP-2-mediated endocytic function. Each of the four interaction sites can operate alone to mediate endocytosis. The observed functional redundancy between interaction sites on the NTD explains how productivity of clathrin-coated vesicle formation is ensured.     

HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276-335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.     

Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.     

The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.     

The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors

One target for the small GTPase Cdc42 is the nonreceptor tyrosine kinase activated Cdc42-associated kinase (ACK), which binds selectively to Cdc42.GTP. We report that ACK1 can associate directly with the heavy chain of clathrin. A central region in ACK1 containing a conserved motif behaves as a clathrin adaptor and competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain. Overexpressed ACK1 perturbs clathrin distribution, an activity dependent on the presence of C-terminal "adaptor" sequences that are also present in the related nonkinase gene 33. ACK1 interacts with the adaptor Nck via SH3 interactions but does not form a trimeric complex with p21-activated serine/threonine kinase, which also binds Nck. Stable low level expression of green fluorescent protein-ACK1 in NIH 3T3 cells has been used to localize ACK1 to clathrin-containing vesicles. The co-localization of ACK1 in vivo with clathrin and AP-2 indicates that it participates in trafficking, underlying an ability to increase receptor-mediated transferrin uptake.     

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.     

EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.     

Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line‐derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin‐associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin‐mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.      

AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin.

Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.     

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.