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1.
Elife ; 82019 05 08.
Article in English | MEDLINE | ID: mdl-31066673

ABSTRACT

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.


Subject(s)
Clathrin/metabolism , Endocytosis , ErbB Receptors/metabolism , Recombinant Proteins/metabolism , ErbB Receptors/genetics , Gene Editing , Hydrogen-Ion Concentration , Protein Engineering , Recombinant Proteins/genetics , Spectrum Analysis
2.
Mol Biol Cell ; 30(1): 4-16, 2019 01 01.
Article in English | MEDLINE | ID: mdl-30403549

ABSTRACT

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Mutant Proteins/biosynthesis , Mutant Proteins/metabolism , Proteolysis , Sequence Homology, Amino Acid , Small Ubiquitin-Related Modifier Proteins/metabolism , Bronchi/pathology , Cell Line , Cell Membrane/metabolism , Cystic Fibrosis/pathology , Endoplasmic Reticulum/metabolism , Epithelial Cells/metabolism , Gene Knockdown Techniques , Humans , Nuclear Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Inhibitors of Activated STAT/metabolism , Protein Stability , Sumoylation , Transcription Factors/metabolism , Ubiquitination
3.
J Biol Chem ; 293(35): 13682-13695, 2018 08 31.
Article in English | MEDLINE | ID: mdl-29986884

ABSTRACT

The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants. BAG3 silencing was the most effective, leading to improved F508del-CFTR stability, trafficking, and restoration of cell-surface function, both alone and in combination with the FDA-approved CFTR corrector, VX-809. We also found that the BAG3 silencing-mediated correction of F508del-CFTR restores the autophagy pathway, which is defective in F508del-CFTR-expressing cells, likely because of the maladaptive stress response in CF pathophysiology. These results highlight the potential therapeutic benefits of targeting the cellular chaperone system to improve the functional folding of CFTR variants contributing to CF and possibly other protein-misfolding-associated diseases.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Autophagy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , HSP70 Heat-Shock Proteins/metabolism , Mutation , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Cell Line , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Protein Stability , Protein Transport , RNA Interference , RNA, Small Interfering/genetics , Up-Regulation
4.
Elife ; 72018 04 09.
Article in English | MEDLINE | ID: mdl-29630493

ABSTRACT

Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.


Subject(s)
Clathrin/metabolism , Dopamine Plasma Membrane Transport Proteins/chemistry , Dopamine Plasma Membrane Transport Proteins/metabolism , Endocytosis , Protein Multimerization , Small Molecule Libraries/pharmacology , Actin Cytoskeleton/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Dopaminergic Neurons/cytology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Dynamins/metabolism , Humans , Membrane Microdomains/drug effects , Membrane Microdomains/metabolism , Mice , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Neuroblastoma/pathology , Protein Conformation , Protein Transport
5.
Biochemistry ; 57(5): 861-871, 2018 02 06.
Article in English | MEDLINE | ID: mdl-29283245

ABSTRACT

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged ß2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.


Subject(s)
Carbocyanines/analysis , Coloring Agents/analysis , Endocytosis/physiology , Exocytosis/physiology , Fluorescent Dyes/analysis , Protein Transport/physiology , Rosaniline Dyes/analysis , Single-Chain Antibodies/analysis , Endosomes/metabolism , Endosomes/ultrastructure , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Indicators and Reagents/analysis , Microscopy, Confocal , Receptors, Adrenergic, beta-2/metabolism
6.
Methods ; 96: 40-45, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26361332

ABSTRACT

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.


Subject(s)
Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/agonists , High-Throughput Screening Assays , Recombinant Fusion Proteins/genetics , Small Molecule Libraries/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Fluorescent Dyes/chemistry , Gene Expression , HEK293 Cells , Humans , Molecular Imaging , Mutation , Plasmids/chemistry , Plasmids/metabolism , Protein Engineering , Recombinant Fusion Proteins/metabolism , Rosaniline Dyes/chemistry , Single-Chain Antibodies/chemistry , Transfection
7.
J Neurosci ; 31(17): 6605-15, 2011 Apr 27.
Article in English | MEDLINE | ID: mdl-21525301

ABSTRACT

The serotonin transporter (SERT) is the principal mechanism for terminating serotonin (5-HT) signals in the nervous system and is a site of action for a variety of psychoactive drugs including antidepressants, amphetamines, and cocaine. Here we show that human SERTs (hSERTs) and rat SERTs are capable of robust dopamine (DA) uptake through a process that differs mechanistically from 5-HT transport in several unanticipated ways. DA transport by hSERT has a higher maximum velocity than 5-HT transport, requires significantly higher Na(+) and Cl(-) concentrations to sustain transport, is inhibited noncompetitively by 5-HT, and is more sensitive to SERT inhibitors, including selective serotonin reuptake inhibitors. We use a thiol-reactive methane thiosulfonate (MTS) reagent to modify a conformationally sensitive cysteine residue to demonstrate that hSERT spends more time in an outward facing conformation when transporting DA than when transporting 5-HT. Cotransfection of an inactive or an MTS-sensitive SERT with wild-type SERT subunits reveals an absence of cooperative interactions between subunits during DA but not 5-HT transport. To establish the physiological relevance of this mechanism for DA clearance, we show using in vivo high-speed chronoamperometry that SERT has the capacity to clear extracellularly applied DA in the hippocampal CA3 region of anesthetized rats. Together, these observations suggest the possibility that SERT serves as a DA transporter in vivo and highlight the idea that there can be distinct modes of transport of alternative physiological substrates by SERT.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/metabolism , Dopamine/metabolism , Serotonin Plasma Membrane Transport Proteins/physiology , Analysis of Variance , Animals , CA3 Region, Hippocampal/cytology , COS Cells , Cell Line, Transformed , Chlorocebus aethiops , Citalopram/pharmacology , Cocaine/analogs & derivatives , Cocaine/pharmacokinetics , Dopamine/pharmacology , Dopamine Plasma Membrane Transport Proteins/genetics , Dose-Response Relationship, Drug , Electrochemical Techniques , Humans , Male , Mutagenesis, Site-Directed/methods , Radioligand Assay/methods , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Sprague-Dawley , Serotonin/metabolism , Serotonin/pharmacology , Serotonin Plasma Membrane Transport Proteins/genetics , Selective Serotonin Reuptake Inhibitors/pharmacology , Sodium/metabolism , Transfection/methods , Tritium/metabolism
8.
Mol Biol Cell ; 19(7): 2818-29, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18434601

ABSTRACT

The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/metabolism , Dopamine/metabolism , Dual Specificity Phosphatase 6/genetics , Gene Expression Regulation , Animals , Biological Transport , Cell Line, Tumor , Dual Specificity Phosphatase 6/physiology , Dynamins/metabolism , Endocytosis , Genetic Complementation Test , Humans , MAP Kinase Signaling System , Models, Biological , Xenopus laevis , p38 Mitogen-Activated Protein Kinases/metabolism
9.
Biochemistry ; 45(4): 1331-7, 2006 Jan 31.
Article in English | MEDLINE | ID: mdl-16430230

ABSTRACT

The plasma membrane serotonin transporter (SERT) has an important role in terminating serotonergic neurotransmission by re-uptake of 5-HT from the synaptic cleft. The expression of SERT on the cell surface is therefore a critical factor. In this study, we examined the role of the carboxyl terminus of SERT in trafficking to the plasma membrane. 5-HT uptake activity was used to measure the effects of systematic deletions or alanine substitutions in the C-terminus. We found that deletion of 16 amino acids in the distal C-terminus had no effect on uptake activity, whereas further deletion was detrimental for the function of SERT. Cell surface biotinylation was used to determine the role of the C-terminus in localization and trafficking. We showed that the C-terminus is crucial for the delivery of SERT to the plasma membrane and that the deletion of this part of the transporter results in a lack of mature glycosylation and impaired trafficking to the plasma membrane. Furthermore, the C-terminally truncated mutants were shown to have a dominant negative effect on wild-type SERT uptake activity.


Subject(s)
Gene Expression Regulation/genetics , Serotonin Plasma Membrane Transport Proteins/metabolism , Amino Acid Sequence , Amino Acids/genetics , Amino Acids/metabolism , Animals , Biological Transport, Active/genetics , Biological Transport, Active/physiology , COS Cells , Cell Membrane/metabolism , Cells, Cultured , Chlorocebus aethiops/metabolism , Humans , Kinetics , Molecular Sequence Data , Mutagenesis , Point Mutation/physiology , Sensitivity and Specificity , Serotonin Plasma Membrane Transport Proteins/genetics
10.
Eur Neuropsychopharmacol ; 15(2): 193-8, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15695064

ABSTRACT

The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram/hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H]-venlafaxine/SERT complex to some extent. Thus, escitalopram shows a unique interaction with the hSERT compared with other 5-HT reuptake inhibitors (SSRIs) and, in addition to its 5-HT reuptake inhibitory properties, displays a pronounced effect via an affinity-modulating allosteric site.


Subject(s)
Citalopram/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , Membrane Transport Modulators , Membrane Transport Proteins/antagonists & inhibitors , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Selective Serotonin Reuptake Inhibitors/metabolism , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , COS Cells , Chlorocebus aethiops , Citalopram/pharmacology , Dose-Response Relationship, Drug , Humans , Protein Binding/drug effects , Protein Binding/physiology , Serotonin Plasma Membrane Transport Proteins , Selective Serotonin Reuptake Inhibitors/pharmacology , Stereoisomerism
11.
J Neurochem ; 92(1): 21-8, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15606893

ABSTRACT

The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of [3H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/- 0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S-citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S-citalopram from a complex with the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.


Subject(s)
Allosteric Site/physiology , Citalopram/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins/metabolism , Serotonin/metabolism , Animals , Cattle , Cell Line , Dose-Response Relationship, Drug , Humans , Serotonin Agents/metabolism , Serotonin Plasma Membrane Transport Proteins
12.
J Biol Chem ; 279(40): 42147-56, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15271993

ABSTRACT

The serotonin transporter (SERT) belongs to a family of sodium chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. SERT represents the main pharmacological target in the treatment of several clinical conditions, including depression and anxiety. Serotonin-selective reuptake inhibitors and tricyclic antidepressants are the most predominantly prescribed drugs in the treatment of depression. In addition to antidepressants also psychostimulants, like cocaine and amphetamines, are important SERT antagonists. In the present study, we report the cloning and characterization of chicken SERT. Although the uptake kinetic was very similar to human SERT, the pharmacological profiles differed considerably for the two species. We find that chicken SERT is capable of discriminating between different serotonin-selective reuptake inhibitors; thus, the potency of S-citalopram and paroxetine is reduced more than 40-fold. A cross-species chimera strategy was undertaken and followed by species-scanning mutagenesis. Differences in pharmacological profiles were tracked to amino acid residues 169, 172, and 586 in human SERT. Structure-activity studies on structurally related compounds indicated that species divergences in drug sensitivity between human and chicken SERT were arising from differences in coordination or recognition of an important aminomethyl pharmacophoric substructure, which is shared by all high affinity antidepressants. Consequently, we suggest that Ala(169) and Ile(172) of human SERT are important residues in sensing the N-methylation state of SERT antagonists.


Subject(s)
Carrier Proteins/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Nerve Tissue Proteins/metabolism , Selective Serotonin Reuptake Inhibitors/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Chickens , Cloning, Molecular , Conserved Sequence , Membrane Glycoproteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Nerve Tissue Proteins/genetics , Protein Binding , Serotonin Plasma Membrane Transport Proteins
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