cyclic-gmp and Choriocarcinoma

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec4 cells) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions.

Topics: Animals; Carrier Proteins; CHO Cells; Choriocarcinoma; Cricetinae; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Female; Gene Expression; Glycosylation; Macromolecular Substances; Membrane Glycoproteins; Membrane Transport Proteins; Mutagenesis, Site-Directed; N-Acetylneuraminic Acid; Nerve Tissue Proteins; Nonmuscle Myosin Type IIA; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Phosphorylation; Pregnancy; Protein Conformation; Rats; Serotonin; Serotonin Plasma Membrane Transport Proteins; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured; Uterine Neoplasms

The serotonin transporter expressed in brain, platelets, and placenta is a primary target for antidepressants, cocaine, and amphetamines. Here we report that interleukin-1 beta is a potent regulator of the expression of this transporter. The activity of the serotonin transporter in human JAR choriocarcinoma cells is stimulated by this cytokine and the stimulation is accompanied by an increase in the steady state levels of the transporter mRNAs and in the transporter density. The increase in mRNA levels is effectively blocked by actinomycin D. The stimulatory effect of interleukin-1 beta is not associated with any change in the cellular levels of cAMP, indicating involvement of a cAMP-independent pathway. Even though cholera toxin, which is known to increase cAMP levels, also upregulates the serotonin transporter in JAR cells, the effects of interleukin-1 beta and cholera toxin on the transporter activity are additive. This constitutes the first report on the identification of a physiologically occurring hormone/cytokine which upregulates the expression of the human serotonin transporter gene.

Topics: Biological Transport; Carrier Proteins; Cell Line; Cell Membrane; Choriocarcinoma; Cocaine; Cyclic AMP; Cyclic GMP; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Imipramine; Interleukin-1; Iodine Radioisotopes; Kinetics; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Pregnancy; RNA, Messenger; Serotonin; Serotonin Plasma Membrane Transport Proteins; Time Factors; Tumor Cells, Cultured; Uterine Neoplasms

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with staurosporine caused a marked stimulation of serotonin transport activity in these cells. The stimulatory effect was noticeable at nanomolar concentrations of staurosporine, and a treatment time of > 4 h was required for staurosporine to elicit the effect. At 40 nM and with a treatment time of 16 h, the stimulation of the transport activity was 3.5-6.0-fold. None of the several other protein kinase inhibitors tested had similar effect except KT 5720, a protein kinase A inhibitor, which showed a small but significant (approximately 1.4-fold) stimulatory effect at a concentration of 5 microM. Blockade of RNA synthesis and protein synthesis in the cells prevented completely the stimulation of the transport activity induced by staurosporine. The stimulation was observed not only in intact cells but also in plasma membrane vesicles prepared from staurosporine-treated cells. The stimulation was accompanied by a 5-7-fold increase in the steady state levels of the transporter-specific mRNAs, by a 7-fold increase in the maximal velocity of the transport process, and by a 6-fold increase in the transporter density in the plasma membrane. Even though both staurosporine and cholera toxin had similar effects on the serotonin transport activity in these cells, the effect was not additive when the cells were treated with both reagents together. While treatment of the cells with cholera toxin markedly elevated intracellular levels of cAMP, staurosporine did not have any effect on the cellular levels of this cyclic nucleotide. It is concluded that staurosporine up-regulates the serotonin transport activity in JAR cells by increasing the steady state levels of the serotonin transporter mRNA and by the consequent increase in the transporter density in the plasma membrane and that the process involves a cAMP-independent signaling pathway.

Topics: Alkaloids; Carrier Proteins; Cholera Toxin; Choriocarcinoma; Cocaine; Cyclic AMP; Cyclic GMP; Cycloheximide; Dactinomycin; Female; Humans; Isoquinolines; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Pregnancy; RNA, Messenger; Serotonin; Serotonin Plasma Membrane Transport Proteins; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation