Glycosyl Modification Facilitates Homo- and Hetero-oligomerization of the Serotonin Transporter

Ozaslan, Deniz; Wang, Xin Wei; Ahmed, Billow A.; Kocabas, Arif; McCastlain, John C.; Bene, Anca; Kilic, Fusun

doi:10.1074/jbc.m306360200

Cited by 50 publications

(62 citation statements)

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“…Cell Surface Biotinylation-Cell surface expression of SERT was detected after biotinylation with membrane-impermeant NHS-SS-biotin as described previously (42)(43)(44)(45). Briefly, upon the biotinylation reaction, the cells were treated with 100 mM glycine to quench unreacted NHS-SS-biotin and lysed in Trisbuffered saline containing 1% SDS, 1% Triton X-100, and PIM/ phenylmethylsulfonyl fluoride.…”

Section: Methodsmentioning

confidence: 99%

“…The SERT Ab gave one major band at 90 kDa in CHO or HeLa cells expressing SERT, which was absent in non-transfected cells. Signals of the 90-kDa band were quantitated with a VersaDoc 1000 analysis system (42). Using a standard curve of SERT prepared form the lysates of CHO cells expressing SERT, the integrated density value for each band was converted to an equivalent amount of SERT for Ab (44).…”

Section: Methodsmentioning

confidence: 99%

“…5HT Uptake Assay in Heterologous Expression System-CHO cells in 24-well culture plates were transfected with SERT and assayed 24 h post-transfection (42)(43)(44)(45). Briefly, cells were washed with phosphate-buffered saline (PBS) containing 0.1 mM CaCl 2 and 1 mM MgCl 2 and incubated for 10 min with this buffer and then assayed for their 5HT uptake by incubating with 20.5 nM [1,2-3 H]5HT (3400 cpm/pmol) as described previously (32)(33)(34).…”

Section: Methodsmentioning

confidence: 99%

“…For all studies, cells were transiently transfected with either SERT and/or Rab4 constructs using a 1:2.5 ratio of Lipofectamine 2000 reagent to DNA in Opti-MEM. SERT expression was determined with quantitative WB on a VersaDoc 1000 analysis system (42)(43)(44)(45).…”

Section: Methodsmentioning

confidence: 99%

“…The biotinylated proteins (500 l) were recovered with an excess of streptavidin-agarose beads (400 l) during overnight incubation. Biotinylated proteins were eluted in 100 l of sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose, and were detected with the SERT Ab as described (42)(43)(44)(45).…”

Section: Methodsmentioning

confidence: 99%

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Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

Ahmed

Jeffus

Bukhari

et al. 2008

Journal of Biological Chemistry

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The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT] ex ) is elevated. It is well reported that stimulation of cells with high [5HT] ex induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT] ex , Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616 -624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT] ex "paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation.The serotonin transporter (SERT) 2 is a member of the Cl Ϫ -and Na ϩ -dependent monoamine transporter family, which also includes the dopamine transporter (DAT) and the norepinephrine transporter. SERT is a 630-amino acid plasma membrane-bound glycoprotein. Hydropathy analysis predicts that SERT contains 12 transmembrane domains and that both the N and C termini are exposed to the cytoplasm. The primary function of SERT in the central nervous system involves the regulation of serotonergic signaling via transport of serotonin (5-hydroxytryptamine (5HT)) molecules from the synaptic cleft into the pre-synaptic terminal for re-utilization. SERT is also expressed in non-neuronal cells, including platelets, placental, intestinal and adrenal cell lines, but the exact function of SERT in these cell lines is still under investigation (1-5).The C-and N-terminal regions of monoamine transporter proteins have just recently garnered increased attention for their importance in transport function and localization. Significant work has been accomplished in identifying the importance of the C-terminal region of DAT and norepinephrine transporter in transporter function, expression, and localization (6 -9).The proteins interacting with the N terminus of SERT are syntaxin 1A (10, 11) and secretory carrier membrane protein 2 (12). SERT also complexes with Hic-5 (13) and ␣-synuclein (14), but the functional significance of these interactions are not known. PICK1 (15), MacMARCKS (16), the actin cytoskeleton (49), neuronal nitric-oxide synthase, and Se...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

Ahmed

Jeffus

Bukhari

et al. 2008

Journal of Biological Chemistry

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Neurotransmitter Transporters

Leitzell

Wang

et al. 2007

Handbook of Contemporary Neuropharmacology

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Neurotransmitter transporters belong to the superfamily of solute carrier. Transporters regulate transmitter levels in the extracellular space and in transmitter‐containing vesiclesby controlling the movement of transmitter between various intracellular andextracellular compartments in brain. This function is achieved in part by coupling the movement of transmitter against its concentration gradient to the movement of associated ions down their electrochemical gradients. Disruption of the normal transport process isassociated with a variety of brain disorders including Alzheimer's disease, depression, epilepsy, Parkinson's disease, stroke, and substance abuse. Moreover, some transportersin this gene family are primary targets of potent psychoactive drugs such as cocaine, amphetamine, and antidepressants. Neurons and glia have multiple mechanisms by which they control transporter expression and function on a variety of time scales. This regulation is not only important for understanding the role of transporters in physiological and pathological processes, but may provide mechanistic targets for therapeutics aimed at disorders related to abnormal transmitter levels.

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Nitric oxide stimulates γ‐aminobutyric acid release and inhibits glycine release in retina

Eldred

2005

J of Comparative Neurology

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Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of γ-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways. Keywords turtle; neurotransmitter; release, uptake; modulation Nitric oxide (NO) plays diverse roles in the regulation of neurotransmission mediated by conventional neurotransmitters. In the brain or retina of different species, NO has been found to affect the release of acetylcholine

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Glycosyl Modification Facilitates Homo- and Hetero-oligomerization of the Serotonin Transporter

Cited by 50 publications

References 56 publications

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

Neurotransmitter Transporters

Nitric oxide stimulates γ‐aminobutyric acid release and inhibits glycine release in retina

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