Ž . European Journal of Pharmacology 325 1997 85–92 Functional link between tyrosine phosphorylation and human serotonin transporter gene expression Puttur D. Prasad, Viviana Torres-Zamorano, Ramesh Kekuda, Frederick H. Leibach, Vadivel Ganapathy ) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA Received 25 November 1996; accepted 4 February 1997 Abstract Treatment of the JAR human placental choriocarcinoma cells with herbimycin A, an inhibitor of tyrosine kinases, led to an increase in the activity of the serotonin transporter. This effect was accompanied by an increase in the serotonin transporter density and in the steady-state levels of the serotonin transporter mRNA. A treatment time of )4 h was necessary for herbimycin A to elicit its effect. Actinomycin D and cycloheximide blocked the effect. There was no increase in the steady-state levels of the serotonin transporter mRNA when cells were treated with herbimycin A in the presence of actinomycin D. The herbimycin A-induced increase in the transporter activity was abolished by genistein, another inhibitor of tyrosine kinases. But the increase in the transporter mRNA levels caused by herbimycin A was not affected by genistein. Treatment of cells with herbimycin A resulted in an increase in the tyrosine phosphorylation of specific cellular proteins, suggesting that herbimycin A directly or indirectly activates specific tyrosine kinases. It is concluded that tyrosine phosphorylation is an essential component in the signaling pathways participating in the regulation of the human serotonin transporter gene expression. q 1997 Elsevier Science B.V. Ž . Keywords: 5-HT 5-hydroxytryptamine, serotonin transporter; Tyrosine kinase inhibitor; Tyrosine phosphorylation; Transcription; Choriocarcinoma cell, human 1. Introduction The Na q - and Cl y -coupled serotonin transporter is pri- marily expressed in serotonergic neurons, platelets and Ž placenta Rudnick and Clark, 1993; Ganapathy and . Leibach, 1995 . Molecular cloning studies have established that the transporter expressed in these tissues is identical, Ž encoded by the same gene Ramamoorthy et al., 1993a; . Lesch et al., 1993a,b . The serotonin transporter is the Ž . target for antidepressant drugs such as fluoxetine Prozac and also for abusable drugs such as cocaine and am- Ž . phetamines Rudnick and Clark, 1993 . Because of its clinical and pharmacological relevance, regulation of the serotonin transporter function is currently an area of in- tense research. For these regulatory studies, the JAR hu- man placental choriocarcinoma cells which constitutively Ž . express the serotonin transporter Cool et al., 1991 have proved to be an extremely useful model system. Two other ) Ž . Ž . Corresponding author. Tel.: 1-706 721-7652; Fax: 1-706 721-6608. human placental choriocarcinoma cells, BeWo and JEG, also express the transporter, but at a much lower level Ž . Jayanthi et al., 1994; unpublished results . To our knowl- edge, the choriocarcinoma cells are the only cell lines of human origin which natively express the serotonin trans- porter. Human platelets can be employed to investigate the regulation of the serotonin transporter, but there are limita- tions with this system. Platelets are nonnucleated cells and therefore it is not feasible to study the transporter regula- tion at the gene level. The choriocarcinoma cells are suitable for these studies. With the JAR choriocarcinoma cells, we have demonstrated that the expression of the Ž serotonin transporter gene is up-regulated by cAMP Cool . et al., 1991; Ramamoorthy et al., 1993b , staurosporine Ž . Ramamoorthy et al., 1995a and interleukin-1b Ž . Ramamoorthy et al., 1995b . cAMP-induced serotonin transporter gene expression is Ž . blocked by protein kinase A inhibitors Cool et al., 1991 , indicating participation of the enzyme in the regulatory process. Staurosporine induces the transporter gene expres- sion via a cAMP-independent mechanism, but the effects 0014-2999r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0014-2999 97 00100-3