Search

Clathrin heavy chain is the structural component of the clathrin triskelion, but unique functions for the two distinct and highly conserved clathrin light chains (CLCa and CLCb, also known as CLTA and CLTB, respectively) have been elusive. Here, we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60-80% compared to siRNA controls, and wound closure and motility by ∼50%. Surface levels of β1-integrins were unaffected by CLCa depletion. However, CLCa was required for effective targeting of FAK (also known as PTK2) and paxillin to the adherent surface of spreading cells, for integrin-mediated activation of Src, FAK and paxillin, and for maturation of focal adhesions, but not their microtubule-based turnover. Depletion of CLCa also blocked the interaction of clathrin with the nucleation-promoting factor WAVE complex, and altered actin distribution. Furthermore, preferential recruitment of CLCa to budding protrusions was also observed. These results comprise the first identification of CLCa-specific functions, with implications for normal and neoplastic integrin-based signaling and cell migration., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The αvβ3 integrin is known to be highly upregulated during cancer progression and promotes a migratory and metastatic phenotype in many types of tumors. We hypothesized that the αvβ3 integrin is transferred through exosomes and, upon transfer, has the ability to support functional aberrations in recipient cells. Here, for the first time, it is demonstrated that αvβ3 is present in exosomes released from metastatic PC3 and CWR22Pc prostate cancer cells. Exosomal β3 is transferred as a protein from donor to nontumorigenic and tumorigenic cells as β3 protein or mRNA levels remain unaffected upon transcription or translation inhibition in recipient cells. Furthermore, it is shown that upon exosome uptake, de novo expression of an αvβ3 increases adhesion and migration of recipient cells on an αvβ3 ligand, vitronectin. To evaluate the relevance of these findings, exosomes were purified from the blood of TRAMP mice carrying tumors where the expression of αvβ3 is found higher than in exosomes from wild-type mice. In addition, it is demonstrated that αvβ3 is coexpressed with synaptophysin, a biomarker for aggressive neuroendocrine prostate cancer., Implications: Overall this study reveals that the αvβ3 integrin is transferred from tumorigenic to nontumorigenic cells via exosomes, and its de novo expression in recipient cells promotes cell migration on its ligand. The increased expression of αvβ3 in exosomes from mice bearing tumors points to its clinical relevance and potential use as a biomarker. Mol Cancer Res; 14(11); 1136-46. ©2016 AACR., Competing Interests: of Potential Conflicts of Interest The authors declare that they have no conflicts of interest., (©2016 American Association for Cancer Research.)

Integrin engagement on lymphocytes initiates "outside-in" signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the "outside-in" signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC)., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Hedgehog (Hh) receptor Patched-1 (PTCH1) opposes the activation of Gli transcription factors and induces cell death through a Gli-independent pathway. Here, we report that the C-terminal domain (CTD) of PTCH1 interacts with and is ubiquitylated on K1413 by the E3 ubiquitin-protein ligase Itchy homolog (Itch), a Nedd4 family member. Itch induces the ubiquitylation of K1413, the reduction of PTCH1 levels at the plasma membrane, and degradation, activating Gli transcriptional activity in the absence of Hh ligands. Silencing of Itch stabilizes PTCH1 and increases its level of retention at the plasma membrane. Itch is the preferential PTCH1 E3 ligase in the absence of Hh ligands, since of the other seven Nedd4 family members, only WW domain-containing protein 2 (WWP2) showed a minor redundant role. Like Itch depletion, mutation of the ubiquitylation site (K1314R) resulted in the accumulation of PTCH1 at the plasma membrane, prolongation of its half-life, and increased cell death by hyperactivation of caspase-9. Remarkably, Itch is the main determinant of PTCH1 stability under resting conditions but not in response to Sonic Hedgehog. In conclusion, our findings reveal that Itch is a key regulator of ligand-independent Gli activation and noncanonical Hh signaling by the governance of basal PTCH1 internalization and degradation., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of β1-integrin by interference with recycling following normal endocytosis of inactive β1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces β1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin-actin interactions needed for recycling by G-clathrin during migration.

'Gyrating-' or 'G'-clathrin are coated endocytic structures located near peripheral sorting endosomes (SEs), which exhibit highly dynamic but localized movements when visualized by live-cell microscopy. They have been implicated in recycling of transferrin from the sorting endosome directly to the cell surface, but there is no information about their formation or regulation. We show here that G-clathrin comprise a minority of clathrin-coated structures in the cell periphery and are brefeldin A (BFA)-resistant. Arf6-GTP substantially increases G-clathrin levels, probably by lengthening coated bud lifetimes as suggested by photobleaching and photoactivation results, and an Arf6(Q67L)-GTP mutant bearing an internal GFP tag can be directly visualized in G-clathrin structures in live cells. Upon siRNA-mediated depletion of Arf6 or expression of Arf6(T27N), G-clathrin levels rise and are primarily Arf1-dependent, yet still BFA-resistant. However, BFA-sensitive increased G-clathrin levels are observed upon acute incubation with cytohesin inhibitor SecinH3, indicating a shift in GEF usage. Depletion of both Arf6 and Arf1 abolishes G-clathrin, and results in partial inhibition of fast transferrin recycling consistent with the latter's participation in this pathway. Collectively, these results demonstrate that the dynamics of G-clathrin primarily requires completion of the Arf6 guanine nucleotide cycle, but can be regulated by multiple Arf and GEF proteins, reflecting both overlapping mechanisms operative in their regulation and the complexity of processes involved in endosomal sorting., (© 2012 John Wiley & Sons A/S.)

Cells are dependent on correct sorting of activated receptor tyrosine kinases (RTKs) for the outcome of growth factor signaling. Upon activation, RTKs are coupled through the endocytic machinery for degradation or recycled to the cell surface. However, the molecular mechanisms governing RTK recycling are poorly understood. Here, we show that Golgi-localized gamma ear-containing Arf-binding protein 3 (GGA3) interacts selectively with the Met/hepatocyte growth factor RTK when stimulated, to sort it for recycling in association with "gyrating" clathrin. GGA3 loss abrogates Met recycling from a Rab4 endosomal subdomain, resulting in pronounced trafficking of Met toward degradation. Decreased Met recycling attenuates ERK activation and cell migration. Met recycling, sustained ERK activation, and migration require interaction of GGA3 with Arf6 and an unexpected association with the Crk adaptor. The data show that GGA3 defines an active recycling pathway and support a broader role for GGA3-mediated cargo selection in targeting receptors destined for recycling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report here detection of novel intracellular clathrin-coated structures revealed by continuous high-speed imaging of cells expressing green fluorescent protein fusion proteins. These structures, which we operationally term 'gyrating clathrin' (G-clathrin), are characterized by localized but extremely rapid movement, leading to the hypothesis that they are coated buds on waving membrane tubules. G-clathrin structures have structurally and functionally distinct features. They lack detectable adaptor proteins AP-1 and AP-2 but contain GGA1 [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding protein] as well as the cation-dependent mannose-6-phosphate receptor. While they accumulate internalized transferrin (Tf), they do not contain detectable levels of cargos targeted for the late endosome/lysosome pathway such as EGF and dextran. Pulse-chase studies indicate that Tf appears in G-clathrin structures in the cell periphery after sorting endosomes (SEs), but before filling of the perinuclear endocytic recycling compartment. Furthermore, the inhibitors LY294002 and wortmannin, which inhibit direct recycling of Tf from SEs to the plasma membrane, also block its appearance in G-clathrin. These observations suggest that peripheral G-clathrin contributes to rapid recycling, a kinetically defined compartment that has largely eluded structural identification. More generally, the rapid continuous live cell imaging reported here reveals new aspects of membrane trafficking.

Prostate-specific membrane antigen (PSMA) is an integral membrane glycoprotein expressed in prostatic epithelia and is being evaluated as a therapeutic target in prostate cancer. It undergoes constitutive receptor-mediated endocytosis via clathrin-coated pits, which is enhanced in the presence of monoclonal antibodies directed against it. We describe distinct interactions of PSMA with clathrin and the clathrin adaptor protein-2 (AP-2) complex, two components of clathrin-coated pits. The intracellular N-terminal domain of PSMA interacts with the N-terminal globular domain of clathrin heavy chain. Deletion analysis revealed an important determinant of this interaction residing within the proximal portion of the clathrin heavy chain N-terminal domain (amino acids 1-85) distinct from the clathrin binding sites of other known clathrin-binding proteins. Furthermore, PSMA interacts with the ear domain of alpha-adaptin (an AP-2 subunit), and a glutamic acid residue at position 7 in the cytoplasmic tail of PSMA is essential for this interaction. These data indicate that PSMA exhibits a high affinity, specific association with the clathrin-based endocytic machinery by distinct interactions with both clathrin and AP-2. Thus, although PSMA is a new member of the dual AP and clathrin binding proteins, its alpha-adaptin and clathrin heavy chain binding determinants are distinct from those of other members.

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2alpha inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2alpha and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5-20 microm/s). The movement is micro-tubule-based as revealed by use of inhibitors, and PI3K-C2alpha accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2alpha structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2alpha and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2alpha and the microtubule motor machinery, with implications for membrane trafficking in intact cells.

Remodeling of cell membranes takes place during motile processes such as cell migration and cell division. Defects of proteins involved in membrane dynamics, including clathrin and dynamin, disrupt cytokinesis. To understand the function of clathrin-containing structures (CCS) in cytokinesis, we have expressed a green fluorescent protein (GFP) fusion protein of clathrin light chain a (GFP-clathrin) in NRK epithelial cells and recorded images of dividing cells near the ventral surface with a spinning disk confocal microscope. Punctate GFP-CCS underwent dynamic appearance and disappearance throughout the ventral surface. Following anaphase onset, GFP-CCS between separated chromosomes migrated toward the equator and subsequently disappeared in the equatorial region. Movements outside separating chromosomes were mostly random, similar to what was observed in interphase cells. Directional movements toward the furrow were dependent on both actin filaments and microtubules, while the appearance/disappearance of CCS was dependent on actin filaments but not on microtubules. These results suggest that CCS are involved in remodeling the plasma membrane along the equator during cytokinesis. Clathrin-containing structures may also play a role in transporting signaling or structural components into the cleavage furrow.

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2alpha was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2alpha, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2alpha in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2alpha can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2alpha employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.

The adaptor complex AP-2 plays an important role in cargo selection and clathrin lattice formation during clathrin-mediated endocytosis. In a recent issue of Molecular Cell, Honing et al. demonstrate that high-affinity AP-2 membrane association is achieved through a combination of low-affinity interactions with membrane phosphoinositides and cargo proteins, regulated by phosphorylation.

Polarization and segregation of the T-cell receptor (TCR) and integrins upon productive cytotoxic T-lymphocyte (CTL) target cell encounters are well documented. Much less is known about the redistribution of major histocompatibility complex class I (MHC-I) and intercellular adhesion molecule-1 (ICAM-1) proteins on target cells interacting with CTLs. Here we show that human leucocyte antigen-A2 (HLA-A2) MHC-I and ICAM-1 are physically associated and recovered from both the raft fraction and the fraction of soluble membranes of target cells. Conjugation of target cells with surrogate CTLs, i.e. polystyrene beads loaded with antibodies specific for HLA-A2 and ICAM-1, induced the accumulation of membrane rafts, and beads loaded with ICAM-1-specific antibodies caused the selective recruitment of HLA-A2 MHC-I at the contact area of the target cells. Disruption of raft integrity on target cells led to a release of HLA-A2 and ICAM-1 from the raft fraction, abatement of HLA-A2 polarization, and diminished the ability of target cells bearing viral peptides to induce a Ca(2+) flux in virus-specific CTLs. These data suggest that productive engagement of ICAM-1 on target cells facilitates the polarization of MHC-I at the CTL-target cell interface, augmenting presentation of cognate peptide-MHC (pMHC) complexes to CTLs. We propose that ICAM-1-MHC-I association on the cell membrane is a mechanism that enhances the linkage between antigen recognition and early immunological synapse formation.

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.

Nonvisual arrestins (arr) modulate G protein-coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing beta2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3-GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16 degrees C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R-YFP and DsRed-clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of 'unroofed' cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

Phosphoinositides play key regulatory roles in vesicular transport pathways in eukaryotic cells. Clathrin-mediated membrane trafficking has been shown to require phosphoinositides, but little is known about the enzyme(s) responsible for their formation. Here we report that clathrin functions as an adaptor for the class II PI 3-kinase C2alpha (PI3K-C2alpha), binding to its N-terminal region and stimulating its catalytic activity, especially toward phosphorylated inositide substrates. Further, we show that endogenous PI3K-C2alpha is localized in coated pits and that exogenous expression affects clathrin-mediated endocytosis and sorting in the trans-Golgi network. These findings provide a mechanistic basis for localized inositide generation at sites of clathrin-coated bud formation, which, with recruitment of inositide binding proteins and subsequent synaptojanin-mediated phosphoinositide hydrolysis, may regulate coated vesicle formation and uncoating.

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2alpha could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2alpha closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2alpha was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH(2)-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922-20929). Here we used deletion and site- directed mutagenesis to determine that alpha residues 21-80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55-57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated alpha subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871-881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits.

Many biologically important macromolecules are internalized into cells by clathrin-coated pit endocytosis. The mechanism of clathrin-coated pit budding has been investigated intensively, and considerable progress has been made in characterizing the proteins involved in internalization. Membrane lipid composition and the lateral organization of lipids and proteins within membranes are believed to play an important role in the regulation of membrane-trafficking processes. Here we report that membrane cholesterol plays a critical role in clathrin-coated pit internalization. We show that acute cholesterol depletion, using beta-methyl-cyclodextrin, specifically reduces the rate of internalization of transferrin receptor by more than 85%, without affecting intracellular receptor trafficking back to the cell surface. The effect on endocytosis is attributable to a failure of coated pits to detach from the plasma membrane, as visualized by using a green fluorescent protein-clathrin conjugate in living cells. Ultrastructural studies indicate that acute cholesterol depletion causes accumulation of flat-coated membranes and a corresponding decrease in deep-coated pits, consistent with the possibility that flat clathrin lattices are direct precursors of indented pits and endocytic vesicles in intact cells. We conclude that clathrin is unable to induce curvature in the membrane depleted of cholesterol.

Here we visualize new aspects of the dynamics of endocytotic clathrin-coated pits and vesicles in mammalian cells by using a fusion protein consisting of green fluorescent protein and clathrin light chain a. Clathrin-coated pits invaginating from the plasma membrane show definite, but highly limited, mobility within the membrane that is relaxed upon treatment with latrunculin B, an inhibitor of actin assembly, indicating that an actin-based framework may be involved in the mobility of these pits. Transient, motile coated vesicles that originate from coated pits can be detected, with multiple vesicles occasionally appearing to emanate from a single pit. Despite their seemingly random distribution, coated pits tend to form repeatedly at defined sites while excluding other regions. This spatial regulation of coated-pit assembly and function is attributable to the attachment of the coated pits to the membrane skeleton.

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote beta2-adrenergic receptor (beta2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) to function as a dominant-negative with respect to beta2AR internalization and compared its properties with those of beta-arrestin and beta-arrestin-V53D, a previously reported dominant-negative mutant. In vitro studies demonstrated that beta-arrestin-V53D bound better to clathrin than beta-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas beta-arrestin (319-418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the beta2AR in HEK293 cells, beta-arrestin (319-418) effectively inhibited agonist-promoted receptor internalization, whereas beta-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of beta2AR internalization by beta-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both beta-arrestin (319-418) and beta-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of beta-arrestin (319-418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking.

We have recently demonstrated that the nonvisual arrestins, beta-arrestin and arrestin3, interact with high affinity and stoichiometrically with clathrin, and we postulated that this interaction mediates internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this study, we localized the clathrin binding domain of arrestin3 using a variety of approaches. Truncation mutagenesis demonstrated that the COOH-terminal half of arrestin3 is required for clathrin interaction. Assessment of the clathrin binding properties of various glutathione S-transferase-arrestin3 fusion proteins indicated that the predominant clathrin binding domain is contained within residues 367-385. Alanine scanning mutagenesis further localized this domain to residues 371-379, and site-directed mutagenesis demonstrated the critical importance of both hydrophobic (Leu-373, Ile-374, and Phe-376) and acidic (Glu-375 and Glu-377) residues in the arrestin3/clathrin interaction. These results are complementary to the observation that hydrophobic and basic residues in clathrin are critical for its interaction with nonvisual arrestins (Goodman, O. B. , Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997) J. Biol. Chem. 272, 15017-15022). Lastly, an arrestin3 mutant in which Leu-373, Ile-374, and Phe-376 were mutated to Ala was significantly defective in its ability to promote beta2-adrenergic receptor internalization in COS-1 cells when compared with wild-type arrestin3. Taken together, these results implicate a discrete region of arrestin3 in high affinity binding to clathrin, an interaction critical for agonist-promoted internalization of the beta2-adrenergic receptor.

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans approximately 80 amino acids. At least three molecular determinants, located at residues 650-660, 680-690, and 720-730, are involved in the binding. AP-2 binds to eps15 through its alpha subunit (alpha-adaptin); in particular, the COOH-terminal region of alpha-adaptin, the so-called alpha-ear, contains the eps15 binding region.

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4, 5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble -myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 alpha subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 alpha sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian alpha, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4, 5-trisphosphate to AP-2 inhibits the protein's clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors.

We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane 'blebs', detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The 'blebs' are distinguishable from 'ruffles' or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5'[gamma-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(gamma) and p85/PI 3-K, regulated by G beta gamma subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stiumlated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 approximately 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 microM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.

The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we studied the relationship between activation of IgE-bound high affinity Fc receptors (FCepsilonRI) and coated pit formation. Because these receptors are rapidly internalized via clathrin-coated pits only when cross-linked by the binding of multivalent antigens, we were able to separate activation from internalization by using an immobilized antigen. The FCepsilonRIs, initially uniformly distributed over the cell surface. relocalized and aggregated on the antigen-exposed membrane. The process was specific for the antigen, and temperature- and time-dependent. This stimulation initiated a cascade of cellular responses typical of FCepsilonRI signaling including membrane ruffling, cytoskeletal rearrangements, and, in the presence of Ca2+, exocytosis. Despite these responses, no change in coated pit disposition or in the distribution of clathrin and assembly protein AP2 was detected, as monitored by immunoblotting and by quantitative (vertical sectioning) confocal microscopy analysis of immunofluorescently stained cells. Specifically, there was no decrease in the density of clathrin-coated pits in regions of the cell membrane not in contact with the antigen, and there was no apparent increase in clathrin-coated pits in proximity to stimulated FCepsilonRI receptors as would have been expected if the receptors were inducing formation of new pits by active recruitment. These results indicate that de novo formation of clathrin-coated pits is not a prerequisite for rapid internalization or a direct response to stimulation of FCepsilonRI receptors. Therefore, increases in coated pits reported to occur in response to activation of some signaling receptors must be consequences of the signal transduction processes, rather than strictly serving the purpose of the internalization of the receptors.

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed "assembly proteins," or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2 alpha and AP2 beta, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2 beta (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with APs. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34.

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated beta 2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated alpha a exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of alpha a bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a approximately 29-kDa alpha a clathrin-binding fragment that remained tightly cage-associated. Translated alpha a also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated beta 2 subunit. Clathrin binding by both the alpha and beta subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H. (1987) Cell Biol. 105, 1989). It also raises the possibility that the alpha-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other alpha functions.

AP-3 (AP180) in rat sympathetic neurons maintained in culture was analyzed by pulse-chase labeling with [35S]methionine to look for post-translational modifications. At early times, two lower molecular weight precursors of the mature species were detected. By 10 min, all of the AP-3 was found in the mature form which is stable for at least 9 h. We show here that at least one of these processing events is due to the addition of O-linked N-acetylglucosamine (GlcNAc) which is present on the mature form of the protein. Wheat germ agglutinin, a GlcNAc-specific probe, bound to AP-3 and the binding was blocked by excess GlcNAc but not by excess mannose. Purified AP-3, and AP-3 in coated vesicles derived from bovine brain, served as substrates for beta-D-galactosyltransferase which is specific for terminal GlcNAc residues. Analysis of the disaccharide released by beta-elimination indicated that single GlcNAc residues are attached to AP-3 through an O-glycosidic linkage to threonine or serine residues. In vivo 32P-labeled AP-3, the result of serine phosphorylation (Keen, J. H., and Black, M.M. (1986) J. Cell Biol. 102, 1325-1333), bound to wheat germ agglutinin-Sepharose indicating that phosphorylation and glycosylation can occur simultaneously on the same molecule. Both modifications have been mapped to the central 50-kDa structural domain that is responsible for the anomalous migration of AP-3. Consistent with localization to the nonclathrin binding domain, the O-GlcNAc modification does not play a discernible role in the interaction of AP-3 with clathrin.

Clathrin is the structural protein of coated membranes involved in receptor-mediated endocytosis and aspects of Golgi sorting in eukaryotic cells. We have now detected a stoichiometric complex of clathrin with a novel protein of M(r) approximately 100,000 (100K) in lysates of different mammalian cells. Formation of the complex, which also includes the 70K heat-shock protein Hsc70, occurs within 15 min of synthesis. The 100K protein has been identified as valosin-containing protein (VCP; ref. 1), an early substrate for tyrosine phosphorylation on T-cell receptor activation. Further, VCP is the mammalian homologue of yeast Cdc48p (ref. 3) and is a member of a larger gene family that includes putative ATP-binding proteins involved in vesicle transport and fusion, 26S proteasome function, regulation of the expression of human immunodeficiency virus, and assembly of peroxisomes. The association with clathrin and the morphological and catalytic similarity to the chaperonin proteins indicate that VCP may modulate protein-protein interactions in membrane transport processes.

We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.