Figure 2. 3-MT induces activation of human TAAR1 in cAMP assay and causes CREB and Erk2 phosphorylation in HEK-293 cells.

(A) cAMP response to tyramine and 3-MT in cells expressing hTAAR1 receptor. Dowex and Alumina column chromatography was used to measure [3H]-cAMP accumulation in HEK-293 cells transfected with the hTAAR1 receptor and treated with the concentrations of compounds shown in the Figure for 15 minutes at room temperature. Results are the mean ± SEM of two (tyramine) or three (3-MT) independent experiments performed in duplicate. EC50 for tyramine was estimated as 320±100 nM and for 3-MT as 700±180 nM. No effects of tyramine and 3-MT were observed in corresponding Mock cells expressing endogenous receptors only (data not shown). The inserted images obtained with a Zeiss LSM510 confocal microscope show the fluorescence from the immunostaining of HA epitope tagged hTAAR1 receptors expressed at the plasma membrane compartment of non permeabilized HEK-293 cells (left image), and an overlay of the fluorescence on a phase image of the same cells (right image) [21]. (B) and (C) Time-course of effect of 3-MT (10 µM) on Erk2 (B) and CREB (C) phosphorylation in HEK-293 cells expressing hTAAR1. hTAAR1 was expressed in cells as described previously [21] and treated with vehicle or 3-MT (10 µM). The cells were lysed and then analyzed by Western blot for Erk2 and CREB phosphorylation. 3-MT produced time dependent phosphorylation of Erk2 and CREB in cells expressing hTAAR1 while no effects were observed in vehicle-treated controls. A significant effect in comparison to untreated cells (time point 0) was observed at 2, 5, 10 and 20 min time points for ERK2 phosphorylation and at 10 and 20 min periods for CREB phosphorylation (n = 4 independent experiments per group, p<0.05, one-way ANOVA followed by Dunnet's multiple comparison test). No effect of 3-MT was observed in corresponding Mock cells without hTAAR1 expression (data not shown).