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PubMed2000

Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites.

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.

Adaptor Protein Complex alpha Subunits↗

PubMed2000

Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules.

ADP-ribosylation factor 1 (ARF1) mediates clathrin coat formation on PC12 immature secretory granules (ISGs). We have used two approaches to investigate whether ARF1 interacts directly with the clathrin adaptor protein, AP-1. Using an in vitro recruitment assay and co-immunoprecipitation, we could isolate an AP-1.ARF1 complex. Then we used a site-directed photocross-linking approach to determine the components that act downstream of ARF1 in clathrin coat formation on ISGs. Myristoylated ARF1, with a photolabile phenylalanine analogue incorporated into its putative effector domain (switch 1), showed a specific, GTP-dependent interaction with both the gamma- and beta-adaptin subunits of AP-1 on ISGs. These experiments provide evidence for a direct interaction of ARF1 with AP-1. On mature secretory granules myristoylated ARF1 does not bind, and hence clathrin coat formation cannot be initiated, supporting the hypothesis that molecules involved in coat recruitment are removed during ISG maturation.

ADP-Ribosylation Factor 1↗

PubMed2000

Constitutive endocytosis of GABAA receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons.

Type A GABA receptors (GABA(A)) mediate the majority of fast synaptic inhibition in the brain and are believed to be predominantly composed of alpha, beta, and gamma subunits. Although changes in cell surface GABA(A) receptor number have been postulated to be of importance in modulating inhibitory synaptic transmission, little is currently known on the mechanism used by neurons to modify surface receptor levels at inhibitory synapses. To address this issue, we have studied the cell surface expression and maintenance of GABA(A) receptors. Here we show that constitutive internalization of GABA(A) receptors in hippocampal neurons and recombinant receptors expressed in A293 cells is mediated by clathrin-dependent endocytosis. Furthermore, we identify an interaction between the GABA(A) receptor beta and gamma subunits with the adaptin complex AP2, which is critical for the recruitment of integral membrane proteins into clathrin-coated pits. GABA(A) receptors also colocalize with AP2 in cultured hippocampal neurons. Finally, blocking clathrin-dependant endocytosis with a peptide that disrupts the association between amphiphysin and dynamin causes a large sustained increase in the amplitude of miniature IPSCs in cultured hippocampal neurons. These results suggest that GABA(A) receptors cycle between the synaptic membrane and intracellular sites, and their association with AP2 followed by recruitment into clathrin-coated pits represents an important mechanism in the postsynaptic modulation of inhibitory synaptic transmission.

Adaptor Protein Complex 2↗

PubMed2000

The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase Ialpha-like isoform.

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges.

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca2+ concentration ([Ca2+]i). In a single bovine aortic EC, the [Ca2+]i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca2+ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca2+ wave was unaffected by the removal of extracellular Ca2+, demonstrating its dependence on intracellular Ca2+ release. Microinjection of heparin into the cell inhibited the ATP-induced Ca2+ responses, indicating that the Ca2+ wave is at least partly mediated by the inositol 1,4, 5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca2+ waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca2+]i increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca2+ waves in ECs.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

AP-4, a novel protein complex related to clathrin adaptors.

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis.

During endocytosis, clathrin and the clathrin adaptor protein AP-2, assisted by a variety of accessory factors, help to generate an invaginated bud at the cell membrane. One of these factors is Eps15, a clathrin-coat-associated protein that binds the alpha-adaptin subunit of AP-2. Here we investigate the function of Eps15 by characterizing an important binding partner for its region containing EH domains; this protein, epsin, is closely related to the Xenopus mitotic phosphoprotein MP90 and has a ubiquitous tissue distribution. It is concentrated together with Eps15 in presynaptic nerve terminals, which are sites specialized for the clathrin-mediated endocytosis of synaptic vesicles. The central region of epsin binds AP-2 and its carboxy-terminal region binds Eps15. Epsin is associated with clathrin coats in situ, can be co-precipitated with AP-2 and Eps15 from brain extracts, but does not co-purify with clathrin coat components in a clathrin-coated vesicle fraction. When epsin function is disrupted, clathrin-mediated endocytosis is blocked. We propose that epsin may participate, together with Eps15, in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

The downregulation of CD4 and MHC-I by primate lentiviruses: a paradigm for the modulation of cell surface receptors.

The human and simian immunodeficiency viruses (HIV and SIV) downregulate the cell surface expression of CD4, their primary receptor, and of class I histocompatibility complex (MHC-I), a critical mediator of immune recognition. While the first of these effects seems important to preserve viral infectivity, the second likely promotes immune evasion. Three HIV-1 proteins, Nef, Env and Vpu, contribute to downregulate CD4, Env forms a complex with CD4 in the endoplasmic reticulum, thereby retaining the receptor in this compartment. Nef and Vpu, on the other hand, act as connectors between CD4 and specific intracellular trafficking pathways, targeting the receptor for degradation in the lysosome and the proteasome, respectively. Some of the downstream partners of the viral proteins in these events have been identified, and include the adaptor complex of clathrin-coated pits, the beta subunit of COP-I coatomer, and the ubiquitin pathway-related h-beta TrCP protein. HIV-induced MHC-I downregulation, mostly the effect of Nef, also reflects a redistribution of this receptor, with its accumulation in the Golgi. The modalities of this process, however, are as yet imperfectly understood. New evidence indicates that the mechanisms employed by primate lentiviruses to downmodulate CD4 and MHC-I are also exploited by a number of cellular regulatory processes.

Adaptor Protein Complex alpha Subunits↗

PubMed1993

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

Adaptor Protein Complex 2↗

PubMed2001

Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery.

Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Here, we report the identification of two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery. The human stonins are homologous to the Drosophila melanogaster stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Constitutive internalization and association with adaptor protein-2 of the interleukin-6 signal transducer gp130.

The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin-6 (IL-6)-type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal-independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL-6/IL-6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL-6/IL-6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP-2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

A beta-turn endocytic code is required for optimal internalization of the growth hormone receptor but not for alpha-adaptin association.

Intracellular trafficking of GH and its receptor, more particularly the chicken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduced as compared with the rat GHR. On the contrary, activation of gene transcription through Janus kinase-2 was similar for both species. Secondary structures of the cytoplasmic domain of chicken and rat GHR were compared, since beta-turns were reported as internalization signals. The substitution of Pro335-Asp335, present in mammalian GH receptors, with Thr307-Gln308 in the cGHR leads to the loss of a beta-turn within a conserved cytoplasmic region. Mutational analysis indicated that the lower rate of internalization of cGHR, as compared with mammalian GHR, was due to this motif. Our data further show that alpha-adaptin, a subunit of adaptor protein AP-2, associates with the GHR upon hormone stimulation. The clathrin-coated pit pathway therefore seems to be involved in the endocytosis of cGHR, as AP-2 is known to intervene in the recruitment of receptors to these pits. Interaction with alpha-adaptin may occur through a common epitope of the chicken and mammalian GHR, since receptors from both species bind similar amounts of alpha-adaptin; alternatively, two different epitopes with similar affinity may be involved. Therefore, not alpha-adaptin but an uncharacterized factor, presumably interacting with the identified beta-turn endocytic code, is responsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be derived from comparative studies.

Adaptor Protein Complex alpha Subunits↗

PubMed2000

Genomic structure of the mouse Ap3b1 gene in normal and pearl mice.

The mouse hypopigmentation mutant pearl is an established model for Hermansky-Pudlak syndrome (HPS), a genetically heterogenous disease with misregulation of the biogenesis/function of melanosomes, lysosomes, and platelet dense granules. The pearl (Ap3b1) gene encodes the beta3A subunit of the AP-3 adaptor complex, which regulates vesicular trafficking. The genomic structure of the normal Ap3b1 gene includes 25 introns and a putative promoter sequence. The original pearl (pe) mutation, which has an unusually high reversion rate on certain strain backgrounds, has been postulated to be caused by insertion of a transposable element. Indeed, the mutation contains a 215-bp partial mouse transposon at the junction point of a large tandem genomic duplication of 6 exons and associated introns. At the cDNA level, three pearl mutations (pearl, pearl-8J, and pearl-9J) are caused by deletions or duplications of a complete exon(s).

Adaptor Protein Complex alpha Subunits↗

PubMed2000

Distinct requirements for the AP-3 adaptor complex in pigment granule and synaptic vesicle biogenesis in Drosophila melanogaster.

The AP-3 adaptor protein complex has been implicated in the biogenesis of lysosome-related organelles, such as pigment granules/melanosomes, and synaptic vesicles. Here we compare the relative importance of AP-3 in the biogenesis of these organelles in Drosophila melanogaster. We report that the Drosophila pigmentation mutants orange and ruby carry genetic lesions in the sigma3 and beta3-adaptin subunits of the AP-3 complex, respectively. Electron microscopy reveals dramatic reductions in the numbers of electron-dense pigment granules in the eyes of these AP-3 mutants. Mutant flies also display greatly reduced levels of pigments housed in these granules. In contrast, electron microscopy of retinula cells reveals numerous synaptic vesicles in both AP-3 mutant and wild-type flies, while behavioral assays show apparently normal locomotor ability of AP-3 mutant larvae. Together, these results demonstrate that Drosophila AP-3 is critical for the biogenesis of pigment granules, but is apparently not essential for formation of a major population of synaptic vesicles in vivo.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

The leucine-based sorting motifs in the cytoplasmic domain of the invariant chain are recognized by the clathrin adaptors AP1 and AP2 and their medium chains.

Recognition of sorting signals within the cytoplasmic tail of membrane proteins by adaptor protein complexes is a crucial step in membrane protein sorting. The three known adaptor complexes, AP1, AP2, and AP3, have all been shown to recognize tyrosine- and leucine-based sorting signals, which are the most common sorting signals within membrane protein cytoplasmic tails. Although tyrosine-based signals are recognized by the micro-chains of adaptor complexes, the subunit recognizing leucine-based sorting signals is less clear. In this report we show by surface plasmon resonance that the two leucine-based sorting signals within the cytoplasmic tail of the invariant chain bind independently from each other to AP1 and AP2 but not to AP3. We also show that both motifs can be recognized by the micro-chains of AP1 and AP2. Moreover, by using monomeric as well as trimeric invariant chain constructs, we show that adaptor binding does not require trimerization of the invariant chain.

Adaptor Protein Complex 1↗

PubMed1999

Recycling of furin from the plasma membrane. Functional importance of the cytoplasmic tail sorting signals and interaction with the AP-2 adaptor medium chain subunit.

The predominant intracellular localization of the eukaryotic subtilisin-like endoprotease furin is the trans-Golgi network (TGN), but a small fraction is also found on the cell surface. Furin on the cell surface is internalized and delivered to the TGN. The identification of three endocytosis motifs, a tyrosine (YKGL(765)) motif, a leucine-isoleucine (LI(760)) motif, and a phenylalanine (Phe(790)) signal, in the furin cytoplasmic domain suggested that endocytosis of furin occurs via an AP-2/clathrin-dependent pathway. Since little is known about proteins containing multiple sorting components in their cytoplasmic domain, the combination of diverse internalization signals in the furin tail raised the question of their individual role. Here we present data showing that the furin tail interacts with the medium (micro2) subunit of the AP-2 plasma membrane-specific adaptor complex in vitro and that this interaction primarily depends on recognition of the tyrosine-based sorting signal and to less extent on the leucine-isoleucine motif. We further provide evidence that the three endocytosis signals are of different functional importance for furin internalization and retrieval to the TGN in vivo, with the tyrosine-based motif being the major determinant, followed by the phenylalanine signal, whereas the leucine-isoleucine motif is only a minor component. Finally, we report that phosphorylation of the furin tail by casein kinase II is not only important for efficient interaction with micro2 and internalization from the plasma membrane but also determines fast retrieval of the protein from the plasma membrane to the TGN.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors.

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulation-dependent dephosphorylation in nerve terminals.

Clathrin-mediated endocytosis was shown to be arrested in mitosis due to a block in the invagination of clathrin-coated pits. A Xenopus mitotic phosphoprotein, MP90, is very similar to an abundant mammalian nerve terminal protein, epsin, which binds the Eps15 homology (EH) domain of Eps15 and the alpha-adaptin subunit of the clathrin adaptor AP-2. We show here that both rat epsin and Eps15 are mitotic phosphoproteins and that their mitotic phosphorylation inhibits binding to the appendage domain of alpha-adaptin. Both epsin and Eps15, like other cytosolic components of the synaptic vesicle endocytic machinery, undergo constitutive phosphorylation and depolarization-dependent dephosphorylation in nerve terminals. Furthermore, their binding to AP-2 in brain extracts is enhanced by dephosphorylation. Epsin together with Eps15 was proposed to assist the clathrin coat in its dynamic rearrangements during the invagination/fission reactions. Their mitotic phosphorylation may be one of the mechanisms by which the invagination of clathrin-coated pits is blocked in mitosis and their stimulation-dependent dephosphorylation at synapses may contribute to the compensatory burst of endocytosis after a secretory stimulus.

Adaptor Protein Complex alpha Subunits↗