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J. Biol. Chem., Vol. 283, Issue 14, 9388-9398, April 4, 2008

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

Billow A. Ahmed, Brandon C. Jeffus, Syed I. A. Bukhari, Justin T. Harney, Resat Unal, Vladimir V. Lupashin, Peter van der Sluijs^¶, and Fusun Kilic¹

From the Departments of Biochemistry and Molecular Biology and Physiology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 and the ^¶Department of Cell Biology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands

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.

Received for publication, August 2, 2007 , and in revised form, January 23, 2008.

^* This work was supported by National Science foundation Grants MCB-0234822 and MCB-0645163 (to V. L.), by the Netherlands Organization for Scientific Research (to P. v. d. S.), and by the National Alliance for Research on Schizophrenia and Depression, American Heart Association (to F. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 301 West Markham St., Mail Slot 516, Little Rock, AR 72205. Tel.: 501-526-6488; Fax: 501-686-8169; E-mail: kilicfusun{at}uams.edu.