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At least 865 records · Page 48Linked to original sources
PubMed2000

Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization.

Internalization of postsynaptic AMPA receptors depresses excitatory transmission, but the underlying dynamics and mechanisms of this process are unclear. Using immunofluorescence and surface biotinylation, we characterized and quantified basal and regulated AMPA receptor endocytosis in cultured hippocampal neurons, in response to synaptic activity, AMPA and insulin. AMPA-induced AMPA receptor internalization is mediated in part by secondary activation of voltage-dependent calcium channels, and in part by ligand binding independent of receptor activation. Although both require dynamin, insulin- and AMPA-induced AMPA receptor internalization are differentially dependent on protein phosphatases and sequence determinants within the cytoplasmic tails of GluR1 and GluR2 subunits. AMPA receptors internalized in response to AMPA stimulation enter a recycling endosome system, whereas those internalized in response to insulin diverge into a distinct compartment. Thus, the molecular mechanisms and intracellular sorting of AMPA receptors are diverse, and depend on the internalizing stimulus.

Animals↗
PubMed2002

Motoring around the Golgi.

The Golgi apparatus is a dynamic organelle through which nascent secretory and transmembrane proteins are transported, post-translationally modified and finally packaged into carrier vesicles for transport along the cytoskeleton to a variety of destinations. In the past decade, studies have shown that a number of 'molecular motors' are involved in maintaining the proper structure and function of the Golgi apparatus. Here, we review just some of the many functions performed by these mechanochemical enzymes - dyneins, kinesins, myosins and dynamin - in relation to the Golgi apparatus.

Animals↗
PubMed2005

Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization.

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.

Animals↗
PubMed2005

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.

Animals↗
PubMed2004

Entry of newly synthesized GLUT4 into the insulin-responsive storage compartment is GGA dependent.

Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2-3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6-9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process.

3T3-L1 Cells↗
PubMed2004

Regulation of Bin1 SH3 domain binding by phosphoinositides.

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity.

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

Antigen Presentation↗
PubMed2003

Expression patterns and subcellular localization of the Down syndrome candidate protein MNB/DYRK1A suggest a role in late neuronal differentiation.

The Minibrain (Mnb) gene belongs to a new protein kinase family, which is evolutionarily conserved, and probably plays several roles during brain development and in adulthood. In Drosophila, mnb is involved in postembryonic neurogenesis and in learning/memory. In humans, MNB has been mapped within the Down syndrome critical region of chromosome 21 and is overexpressed in the Down syndrome embryonic brain. It has been widely proposed that MNB is involved in the neurobiological alterations associated with Down syndrome. Nevertheless, little is known about the functional role that MNB plays in vertebrate brain development. We have recently shown [Hämmerle et al. (2002) Dev. Biol., 246, 259-273] that in early vertebrate embryos, Mnb is transiently expressed in neural progenitor cells during the transition from proliferating to neurogenic divisions. Here we have studied in detail a second wave of Mnb expression, which takes place in the brain of intermediate and late vertebrate embryos. In these stages, MNB seems to be restricted to certain populations of neurons, as no consistent expression was detected in astroglial or oligodendroglial cells. Interestingly, MNB expression takes place at the time of dendritic tree differentiation and is initiated by a transient translocation from the cytoplasm to the nucleus. Afterwards, MNB protein is transported to the growing dendritic tree, where it colocalizes with Dynamin 1, a putative substrate of MNB kinases. We propose that MNB kinase is involved in the signalling mechanisms that regulate dendrite differentiation. This functional role helps to build a new hypothesis for the implication of MNB/DYRK1A in the developmental aetiology of Down syndrome neuropathologies.

Aging↗
PubMed2004

DNM1DN: a new class of paralogous genomic segments (duplicons) with highly conserved copies on chromosomes Y and 15.

Screening a testis cDNA selection library for Y-linked genes yielded 79 cDNAs. Of these, 9 matched the 3' region of the dynamin 1 gene (DNM1) on chromosome 9q34 with >90% identity. Fluoresence in situ hybridisation and PCR amplification were used to localise a large number of DNM1-like sequences to human chromosomes 15 and Y. PCR amplification of overlapping Y-linked YACs allowed a more accurate mapping of the Y-linked DNM1-like cDNAs to a euchromatic locus in close proximity to heterochromatin at Yq11.23. A search of the genome database identified 64 highly homologous copies of the DNM1 fragment. Most of these copies were localised to chromosomes 15 and Y, but others mapped to chromosomes 5, 8, 10, 12, 19 and 22. These sequences exhibit all the major features of a duplicon and have been designated DNM1DN (DNM1 duplicon). Evolutionary studies using fluorescence in situ hybridisation indicate that transposition of the DNM1DN sequence to chromosome 15 took place earlier in primate evolution than the transposition to the Y chromosome. The translocation to the Y took place at a time following the divergence of a common ancestor from gorilla, approximately 4-7 million years ago.

Animals↗
PubMed2006

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

Animals↗
PubMed2002

Conditional disruption of synaptic transmission induces male-male courtship behavior in Drosophila.

It is reported here that male-male courtship behavior is evoked instantaneously in the fruit fly Drosophila by conditional disruption of synaptic transmission. A temperature-sensitive allele of the Drosophila dynamin gene shibire (shi(ts1)) was expressed by using the GAL4/UAS system to disrupt synaptic transmission from GAL4-positive neurons in a temperature-dependent manner. An enhancer-trap GAL4 line C309 directing shi(ts1) expression in central and peripheral neurons (C309/UAS-shi(ts1)) initiated stereotypical precopulatory behavior toward other mature males immediately after a temperature shift from the permissive to restrictive temperature. At the restrictive temperature, C309/UAS-shi(ts1) males formed "courtship chains" and exhibited abnormally high levels of head-to-head interactions. The temperature-induced male-male courtship is attributable not to an increase in sexual attractiveness but to an increase in sexual activity of C309/UAS-shi(ts1) males. Interestingly, the temperature-induced increase in sexual activity is specific toward male partners, because C309/UAS-shi(ts1) males courted receptive virgin females less vigorously and copulated less efficiently after shifted to the restrictive temperature. Among the GAL4-positive neurons in C309, conditional disruption of certain cholinergic neurons but not the mushroom body intrinsic neurons plays a critical role in the induction of male-male courtship. These neurons may be involved in inhibitory systems that normally suppress aberrant male-male courtship. The presented strategy that can induce behavioral abnormalities by disrupting synaptic transmission in an acute and noninvasive manner will allow further exploration as to how distinct neuronal groups control sexual orientation and other aspects of reproductive behavior in Drosophila.

Alleles↗
PubMed2000

Receptor-mediated endocytosis of soluble and membrane-tethered Sonic hedgehog by Patched-1.

Patched (Ptc) is the ligand-binding component of the Hedgehog (Hh) receptor complex. In the Drosophila embryo, Ptc and Hh colocalize in vesicular punctate structures. However, receptor-mediated endocytosis of Hh proteins has not been demonstrated. By using chick neural plate explants, we show that Sonic hedgehog (Shh)-responsive neural precursor cells internalize recombinant and endogenous Shh and provide direct evidence for a gradient of endogenous Shh in the ventral neural tube. Shh internalization is blocked by a monoclonal antibody whose epitope overlaps the Ptc-binding site of Shh. These findings suggest that Shh internalization is mediated by Ptc-1 and may be linked to signaling. Concordantly, transfection of mammalian cell lines with a Ptc-1 cDNA confers the ability to internalize multiple forms of Shh, including transmembrane-anchored Shh, by a dynamin-dependent process.

Animals↗
PubMed1998

Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase.

The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and beta-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of beta-arrestin or dynamin, beta2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and beta-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade.

Arrestins↗
PubMed1998

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Insulin stimulates sequestration of beta-adrenergic receptors and enhanced association of beta-adrenergic receptors with Grb2 via tyrosine 350.

G-protein-linked receptors, such as the beta2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305-25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of beta2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing beta2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of beta-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with beta-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of beta-adrenergic receptors. Insulin administration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the beta-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with beta-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to beta2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the beta-adrenergic receptor are shown to be enhanced by insulin, beta-agonist, or both. The Tyr-350 --> Phe mutant form of the beta2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of beta2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the beta-adrenergic receptor, via a new mechanism, i.e. internalization.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization.

Thromboxane A2 (TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2 receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rbeta, but not the TXA2Ralpha, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rbeta is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the alpha and beta isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rbeta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rbeta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1999

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.

1-Methyl-3-isobutylxanthine↗
PubMed1999

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

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

Adaptor Protein Complex 2↗