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.     

Binding of AP2 to sorting signals is modulated by AP2 phosphorylation

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

Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation

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

AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.     

Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.     

Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane

Clathrin in coated vesicles is linked to transmembrane receptors by adaptor protein complexes. The Golgi-associated adaptor complex HA1 is a tetramer, made up of beta', gamma, 47-kDa, and 20-kDa subunits, whereas the tetrameric plasma membrane adaptor, HA2, contains alpha, beta, 50-kDa, and 16-kDa subunits (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929). Here we report on the structural organization of adaptor subunits as revealed by proteolytic dissection. We show that the beta' and gamma subunits of HA1 are cleaved into 60-67-kDa "trunk" and 32-44-kDa "head" fragments. Interactions between adaptor subunits involve the trunk domains only. In overall organization of their domains, the Golgi and plasma membrane adaptors are very similar. The similarity encompasses also the location of phosphorylated serine residues in the alpha a, beta, beta', and gamma subunits, which are found in the head domains in all cases. In the alpha a and beta subunits they probably occur in the proline- and glycine-rich hinge region, which connects the head to the trunk. Identical adaptor fragments were obtained by controlled digestion of clathrin-coated vesicles. Under conditions that did not affect the integrity of the clathrin heavy chain, the adaptor head fragments were always quantitatively released from coated vesicles. The release of the bulk of the adaptors occurred concomitantly with the cleavage of their beta-type subunits (beta and beta') and under buffer conditions that prevent aggregation of adaptors. These observations taken together with the results of reconstitution experiments confirm and extend previous data (Ahle, S., and Ungewickell, E. (1989) J. Biol. Chem. 264, 20089-20093) which suggested that adaptors attach to clathrin through their beta-type (beta and beta') subunits. Moreover, high affinity interaction between adaptors and clathrin requires the participation of regions from both the head and trunk domains of the beta-type subunits.     

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.     

The gamma/sigma1 and alpha/sigma2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma1 or alpha/sigma2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta1/mu1 and beta2/mu2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma1 and alpha/sigma2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma1 hemicomplex, whereas minimally affecting binding to alpha/sigma2. There was an excellent correlation between binding to the alpha/sigma2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.     

Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation

Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32)P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs.     

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

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

Adaptor complex-independent clathrin function in yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.     

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

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

Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex

Coatomer is a cytosolic protein complex that forms the coat of COP I- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.     

The beta 1 and beta 2 subunits of the AP complexes are the clathrin coat assembly components.

The beta 1 and beta 2 subunits are the closely-related large chains of the trans-Golgi network AP-1 and the plasma membrane AP-2 clathrin-associated protein complexes, respectively. Recombinant beta 1 and beta 2 subunits have been generated in Escherichia coli. It was found that, in the absence of all the other AP subunits, beta 1 and beta 2 interact with clathrin and drive the efficient assembly of clathrin coats. In addition, beta 2 subunits and AP complexes compete for the same clathrin binding site. The appearance of the clathrin/beta coats is the same as the barrel-shaped structures formed with native AP complexes. It is proposed that the principal function of the beta subunits is to initiate coat formation, while the remaining subunits of the AP complexes have other roles in coated pit and coated vesicle function.     

Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo-AP2 interactions occur via the mu2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for mu2 phosphorylation is in cargo recruitment because mu2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of mu2 phosphorylation. We identify clathrin as a specific activator of the mu2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of mu2 phosphorylation. Furthermore, we show that AP2 containing phospho-mu2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-mu2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-mu2 levels.     

Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site.

In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXXphi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated by phosphoinositide lipids that are phosphorylated at the D-3 position of the inositol ring, and is also enhanced by the formation of clathrin-AP-2 coats. Here, we describe the detection of a specific interaction between peptides containing a second sorting motif, the dileucine motif, and AP-1, the clathrin adaptor complex responsible for sorting proteins at the trans-Golgi network (TGN). Surprisingly, the site of dileucine binding is the beta1 subunit, not mu 1. A YXXphi-containing peptide from a protein trafficked within the TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphate and 3,4, 5-triphosphate did not activate the interaction between dileucine-containing peptides and AP-1 but instead inhibited it, and clathrin-AP-1 coat formation did not alter the interaction. Thus, there are at least two physically separate binding sites for sorting signals on APs, which are also regulated independently.     

ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.     

The phosphorylation of coated membrane proteins in intact neurons

To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two-dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non-equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have previously described, as well as a novel 155-kD polypeptide reported here, have structural and functional counterparts in cultured neurons. They also indicate that phosphorylation of the 100-110-kD AP may be involved in the regulation of coated membrane structure and function. The extent of phosphorylation of the AP50 in intact cells and in isolated coated vesicles is strikingly different: it has been suggested that the latter process reflects an autophosphorylation reaction (Campbell C., J. Squicciarini, M. Shia, P. F. Pilch, and R. E. Fine, 1984, Biochemistry, 23:4420-4426).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding

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.