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

Abstract

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...

The Role of ERp44 in Maturation of Serotonin Transporter Protein

“…The JAR cell line in the transient transfection system offers an in situ environment in processing of the newly synthesized SERT. As reported previously (10,31), transient transfection produced an at least 50-fold higher level of protein than the endogenous system. Therefore, using JAR cells only mimics the endogenous condition for the transiently transfected plasmids.…”

“…In other words, 5-HT levels improved the rate of transport not simply by saturating the transporter but by altering its functional efficiency and/or concentration. Based on our previous studies (31,36,37), we favor the biphasic relationship between the extracellular 5-HT levels with the number of SERT molecules on the plasma membrane. Specifically, the number of SERT molecules on the plasma membrane and the 5-HT uptake rates of cells initially rise as extracellular 5-HT levels are increased but then fall below normal as the 5-HT level continues to rise.…”

Loss of Serotonin Transporter Function Alters ADP-mediated Glycoprotein αIIbβ3 Activation through Dysregulation of the 5-HT2A Receptor

“…This process, known as serotonylation, involves the covalent attachment of 5-HT molecules to proteins mediated by the enzyme transglutaminase, resulting in transamination of small GTPases important for platelet ␣-granule secretion (20,25). In our experiments, we demonstrated no difference in P-selectin antibody as a measure for ␣-granule exocytosis in SERT Ϫ/Ϫ platelet following thrombin activation (Fig.…”

The C-Terminal Region of Serotonin Transporter Is Important for Its Trafficking and Glycosylation

“…Thereafter, it is trafficked to the Golgi apparatus and undergoes mature-glycosylation there, followed by trafficking to the plasma membrane (17 -19). Many SERT-binding proteins, including syntaxin 1A (20), MacMARCKS (21), , SCAMP2 (23), nNOS (24), Rab4 (25), M6B (26), α-synuclein (27), and integrin α IIb β 3 (28), are thought to regulate SERT function via membrane trafficking and the glycosylation process. The different regulation of membrane trafficking has been pointed out in some of the missense mutants of SERT identified in families susceptible to autism (13,29).…”