Phosphoinositide 3-kinase regulates β2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/β-arrestin complex

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

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

Journal of Cell Science ◽

10.1242/jcs.112.3.317 ◽

1999 ◽

Vol 112 (3) ◽

pp. 317-327 ◽

Cited By ~ 3

Author(s):

T. Sorkina ◽

A. Bild ◽

F. Tebar ◽

A. Sorkin

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Adaptor Protein ◽

Receptor Internalization ◽

Image Deconvolution ◽

Protein Markers ◽

Coated Pits ◽

Early Endosomes ◽

Epidermal Growth ◽

Clathrin Adaptor

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.

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Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

The Journal of Cell Biology ◽

10.1083/jcb.200610154 ◽

2007 ◽

Vol 177 (5) ◽

pp. 905-916 ◽

Cited By ~ 76

Author(s):

Breann L. Wolfe ◽

Adriano Marchese ◽

JoAnn Trejo

Keyword(s):

G Protein ◽

Protein Complex ◽

Adaptor Protein ◽

Receptor Internalization ◽

Wild Type ◽

Endocytic Machinery ◽

Protease Activated Receptor 1 ◽

Clathrin Adaptor ◽

G Protein Coupled ◽

Type Receptor

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

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Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

10.1101/2020.04.30.070011 ◽

2020 ◽

Cited By ~ 1

Author(s):

Martina Sundqvist ◽

André Holdfeldt ◽

Shane C. Wright ◽

Thor C. Møller ◽

Esther Siaw ◽

...

Keyword(s):

Nadph Oxidase ◽

Neutrophil Migration ◽

Adaptor Protein ◽

Formyl Peptide Receptor ◽

Human Neutrophils ◽

Ros Production ◽

Receptor Endocytosis ◽

Formyl Peptide ◽

Clathrin Adaptor ◽

Nadph Oxidase Activation

AbstractFormyl peptide receptor 2 (FPR2), a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent β- arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis reveled that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated neutrophil ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from human neutrophils.

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Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Molecular Biology of the Cell ◽

10.1091/mbc.10.3.677 ◽

1999 ◽

Vol 10 (3) ◽

pp. 677-691 ◽

Cited By ~ 110

Author(s):

Carol Pitcher ◽

Stefan Höning ◽

Anja Fingerhut ◽

Katherine Bowers ◽

Mark Marsh

Keyword(s):

Phorbol Esters ◽

Protein Complexes ◽

Adaptor Protein ◽

Cytoplasmic Domain ◽

Serine Phosphorylation ◽

Differentiation Antigen ◽

Coated Pits ◽

Early Endosomes ◽

Clathrin Adaptor ◽

Cluster Of Differentiation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

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Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

10.1101/162024 ◽

2017 ◽

Author(s):

Delia Bucher ◽

Felix Frey ◽

Kem A. Sochacki ◽

Susann Kummer ◽

Jan-Philip Bergeest ◽

...

Keyword(s):

Mammalian Cells ◽

Adaptor Protein ◽

Membrane Tension ◽

Coated Pits ◽

Lattice Curvature ◽

Cellular Processes ◽

Molecular Events ◽

Electron And Light Microscopy ◽

Mechanical Factors ◽

Clathrin Adaptor

AbstractAlthough essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

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μ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate

Journal of Cell Science ◽

10.1242/jcs.114.24.4469 ◽

2001 ◽

Vol 114 (24) ◽

pp. 4469-4476 ◽

Cited By ~ 1

Author(s):

Christoph Meyer ◽

Eeva-Liisa Eskelinen ◽

Medigeshi Ramarao Guruprasad ◽

Kurt von Figura ◽

Peter Schu

Keyword(s):

Plasma Membrane ◽

Fold Increase ◽

Retrograde Transport ◽

Receptor Internalization ◽

Recycling Rate ◽

Coated Pits ◽

Trans Golgi Network ◽

Internalization Rate ◽

Clathrin Adaptor ◽

Golgi Network

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in μ1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In μ1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.

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α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015319 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18076-18090

Author(s):

Meir Schechter ◽

Merav Atias ◽

Suaad Abd Elhadi ◽

Dana Davidi ◽

Daniel Gitler ◽

...

Keyword(s):

Membrane Trafficking ◽

Active Form ◽

Adaptor Protein ◽

Intrinsically Disordered Protein ◽

Coated Pits ◽

Intrinsically Disordered ◽

Repeat Domain ◽

Steady State Levels ◽

Acidic Phospholipids ◽

Clathrin Adaptor

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P2 levels on the plasma membrane. In accord with their effects on PI(4,5)P2 levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P2 levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.

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Phosphorylation of AMPA receptor subunit GluA1 regulates clathrin-mediated receptor internalization

Journal of Cell Science ◽

10.1242/jcs.257972 ◽

2021 ◽

Author(s):

Matheus F. Sathler ◽

Latika Khatri ◽

Jessica P. Roberts ◽

Isabella G. Schmidt ◽

Anastasiya Zaytseva ◽

...

Keyword(s):

Synaptic Plasticity ◽

Ampa Receptors ◽

Long Term Potentiation ◽

Surface Expression ◽

Synaptic Strength ◽

Receptor Internalization ◽

Synaptic Membrane ◽

Coated Pits ◽

Ampa Receptor Subunit ◽

Clathrin Adaptor

Synaptic strength is altered during synaptic plasticity by controlling the number of AMPA receptors (AMPARs) at excitatory synapses. During long-term potentiation and synaptic up-scaling, AMPARs are accumulated at synapses to increase synaptic strength. Neuronal activity leads to phosphorylation of AMPAR subunit GluA1 and subsequent elevation of GluA1 surface expression, either by an increase in receptor forward trafficking to the synaptic membrane or a decrease in receptor internalization. However, the molecular pathways underlying GluA1 phosphorylation-induced elevation of surface AMPAR expression are not completely understood. Here, we employ fluorescence recovery after photobleaching (FRAP) to reveal that phosphorylation of GluA1 Serine 845 (S845) predominantly plays a role in receptor internalization than forward trafficking during synaptic plasticity. Notably, internalization of AMPARs depends upon the clathrin adaptor, AP2, which recruits cargo proteins into endocytic clathrin coated pits. In fact, we further reveal that an increase in GluA1 S845 phosphorylation by two distinct forms of synaptic plasticity diminishes the binding of the AP2 adaptor, reducing internalization, and resulting in elevation of GluA1 surface expression. We thus demonstrate a mechanism of GluA1 phosphorylation-regulated clathrin-mediated internalization of AMPARs.

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