Down-regulation of the serotonin transporter in hyperreactive platelets counteracts the pro-thrombotic effect of serotonin.

An elevated plasma concentration of serotonin ([5-HT]) is a common feature of cardiovascular disease often associated with enhanced platelet activation and thrombosis. Whether elevated in vivo plasma 5-HT per se represents an independent risk factor for platelet hyperreactivity or only is an epiphenomenon of cardiovascular disease is poorly understood. We examined in vitro and in vivo platelet function following a 24h elevation of plasma [5-HT] in mice. In vivo administration of 5-HT using osmotic minipumps increased plasma [5-HT] in treated mice compared to control mice instrumented with saline loaded pumps. 5-HT infusion did not increase systolic blood pressure, but markers of platelet activation including P-selectin and (PE)Jon/A staining were increased and these findings coincided with the enhanced aggregation of isolated platelets in response to type I fibrillar collagen. Tail bleeding times and the time to occlusion following chemical damage to the carotid artery were shortened in 5-HT-infused mice. 5-HT-infused mice were treated with paroxetine (Prx) to block 5-HT uptake via the serotonin transporter (SERT). Prx lowered platelet [5-HT] and attenuated platelet activation and aggregation. These results and our biochemical indices of enhanced 5-HT intracellular signaling in the platelets of 5-HT-infused mice reveal a mechanistic link between elevated plasma [5-HT], abnormal intracellular 5-HT signaling and accentuated platelet aggregation. Although a down-regulation of the serotonin transporter (SERT) on the platelet surface may counteract the pro-thrombotic influence of elevated plasma [5HT], this compensatory mechanism may fail to prevent the increased thrombotic risk caused by elevated plasma [5-HT].

The cellular distribution of serotonin transporter is impeded on serotonin-altered vimentin network.

BACKGROUND: The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments. METHODOLOGY/PRINCIPAL FINDINGS: We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Delta14 and Delta20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S(611), T(613), and T(616) arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter. CONCLUSIONS/SIGNIFICANCE: Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network.

Serotonin transamidates Rab4 and facilitates its binding to the C terminus of serotonin transporter.

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.

Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter. A specific role for sialic acid residues.

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec4 cells) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions.