Abstract
BackgroundThe 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 FindingsWe 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 Δ14 and Δ20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S611, T613, and T616 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/SignificanceBased 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.
Highlights
The serotonin transporter (SERT) is a member of a larger family of Na+- dependent transporters in prokaryotes and animals, which is designated the SLC6 or NSS family
We analyzed these findings with biochemical techniques following the endogenous expression of vimentin and SERT in platelets with Western Blot (W/B) assays (Fig. 1A)
The plasma membrane level of SERT is altered by the rate of the translocation transporter protein to/from the plasma membrane which is controlled through its interaction with other proteins in these pathways
Summary
The serotonin transporter (SERT) is a member of a larger family of Na+- dependent transporters in prokaryotes and animals, which is designated the SLC6 or NSS family. The interaction with MacMARCKS has been shown to modulate 5HT uptake, endocytosis, and phosphorylation of SERT via activating protein kinase C (PKC) [10] in a biphasic manner [11]. Studies have shown that PKC-dependent modulation of SERT is correlated with extracellular 5HT levels [12,13]. It has been suggested that the final 20 amino acids of the C-terminal of SERT are critical for the functional expression of the transporter [14,15]. 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
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