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Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT).

Abstract：

:Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.

journal_name

Neurochem Int

journal_title

Neurochemistry international

authors

Mochizuki H,Amano T,Seki T,Matsubayashi H,Mitsuhata C,Morita K,Kitayama S,Dohi T,Mishima HK,Sakai N

doi

10.1016/j.neuint.2004.08.008

subject

Has Abstract

pub_date

2005-01-01 00:00:00

pages

93-105

issue

2

eissn

0197-0186

issn

1872-9754

pii

S0197-0186(04)00178-0

journal_volume

46

pub_type

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