The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine N.

doi:10.1074/jbc.m113.495754

Cited by 28 publications

(33 citation statements)

References 47 publications

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“…To further substantiate the distinct postendocytic fate of NET, we investigated the colocalization of internalized JHC 1-64-labeled NET with the lysosomal marker LysoTracker that has previously been shown to colocalize with constitutively internalized DAT and SERT (22,24,49). Consistent with sorting to a recycling pathway and not to degradation, we observed by live confocal imaging limited colocalization of internalized JHC 1-64-labeled NET with LysoTracker (Fig.…”

Section: Internalized Net Colocalizes With Rab11-positive Recycling Esupporting

confidence: 64%

“…Altogether, our results suggest that to a major extent, constitutively internalized NET is sorted to the Rab11 recycling compartment in SCG neurons and that impairment of the Rab11 pathway results in decreased NET function. NET and DAT Have Distinct Trafficking Properties-We have recently characterized in detail the postendocytic fate of constitutively internalized DAT and SERT, which both appeared to be sorted largely to late endosomes and lysosomal degradation (21,22). Thus, we did not expect that constitutively internalized NET would be sorted primarily to the Rab11 recycling compartment in primary neurons.…”

Section: Internalized Net Colocalizes With Rab11-positive Recycling Ementioning

confidence: 99%

“…Their fate upon internalization, however, has not been defined. Whereas some studies suggest that the transporters are recycled back to the plasma membrane (18,23), other studies support the notion that DAT and SERT primarily are sorted to Rab7-positive late endosomes and subsequently to lysosomal degradation upon both constitutive and stimulated internalization (22,24,25).…”

Section: Reuptake Of Synaptically Released Norepinephrine (Ne)mentioning

confidence: 99%

“…JHC 1-64 contains a rhodamine moiety, extending from the tropane nitrogen of the parent compound 2␤-carbomethoxy-3␤-(3,4-dichlorophenyl)tropane with an ethylamino linker (35). We have previously used JHC 1-64 to visualize cell surface expression and internalization of both DAT and SERT (20,22,24,35). Importantly, JHC 1-64 also displays high affinity for NET, as reflected by the potency by which JHC 1-64 inhibits […”

Section: Jhc 1-64 Enables Specific Labeling Of Endogenous Net Inmentioning

confidence: 99%

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Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

Vuorenpää

Jørgensen

Newman

et al. 2016

Journal of Biological Chemistry

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The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the "long loop" recycling marker Rab11, whereas less overlap was seen with the "short loop" recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by nonconserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function. Reuptake of synaptically released norepinephrine (NE)2 is the primary mechanism to control duration and strength of noradrenergic neurotransmission. The reuptake is mediated by the NE transporter (NET), which is present in noradrenergic neurons both in the brain and in the peripheral sympathetic nerve system (1-3). NET belongs to the SLC6 (solute carrier 6) gene family (also referred to as neurotransmitter-sodium symporters) that also includes transporters for the neurotransmitters dopamine, serotonin, glycine, and ␥-aminobutyric acid (GABA) (4 -6). NET is targeted by many clinically important antidepressant drugs and by several widely abused drugs, such as cocaine and amphetamine. In addition, impaired NET function has been implicated in cardiovascular diseases and in disorders of mood and cognition (7-10).It is assumed that the amount of NET present in the presynaptic plasma membrane is tightly controlled to sustain NE homeostasis, but the underlying molecular and cellular mechanisms are poorly understood. Several mechanisms have been suggested to regulate NET distribution, including protein kinase C (PKC), whose activation (e.g. by G q -coupled muscarinic acetylcholine receptors) can increase NET internal...

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Section: Internalized Net Colocalizes With Rab11-positive Recycling Esupporting

confidence: 64%

Section: Internalized Net Colocalizes With Rab11-positive Recycling Ementioning

confidence: 99%

Section: Reuptake Of Synaptically Released Norepinephrine (Ne)mentioning

confidence: 99%

Section: Jhc 1-64 Enables Specific Labeling Of Endogenous Net Inmentioning

confidence: 99%

See 2 more Smart Citations

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

Vuorenpää

Jørgensen

Newman

et al. 2016

Journal of Biological Chemistry

Self Cite

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“…In some cases, extracellular epitope antibodies have been developed, either based on native transporter sequence or engineered epitope tags (Sorkina et al, 2009;Navaroli and Melikian, 2010;Rahbek-Clemmensen et al, 2014), that can be used in nonpermeabilized cells to assess transporter surface localization. These approaches have the advantage of being able to track the subcellular location of transporters in living cells (e.g., endocytosis), although here the caveat is that antibody binding may perturb transporter structure, affecting rates or extent of trafficking events.…”

Section: B Assessment Of Transporter Surface Expression-steady-statementioning

confidence: 99%

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

Bermingham

Blakely

2016

Pharmacol Rev

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Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays

et al. 2016

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In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

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The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

Cited by 28 publications

References 47 publications

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays

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