Fig. 1
Neurotransmitters, trace amines, and AMPHs activate the small GTPase RhoA in HEK293 cells and midbrain neurons. In HEK293 cells transiently transfected with the DAT and either the FRET sensor for RhoA (a) or PKA (b) activation, the application of 10 μM AMPH, indicated by the vertical dashed line at 2 min, stimulates activation of these enzymes. However, in cells expressing only the FRET sensor and not the DAT, AMPH does not lead to RhoA or PKA activation (black lines). Activated RhoA was isolated with a GST-isolation assay in HEK293 cells transiently transfected with DAT (c). RhoA activation was detected in response to a number of DAT transported TAAR1 agonists. Octopamine did not stimulate RhoA until the cells were permeablized with streptolysin O before octopamine application. The non-transported DAT inhibitors cocaine (100 μM) and methylphenidate (100 μM) did not stimulate RhoA activation on their own and were sufficient to block AMPH-induced RhoA activation (d). Biotinylation of cell-surface proteins in acute midbrain slices of wildtype or TAAR1 knockout animals was performed after vehicle or AMPH treatment. AMPH-induced DAT and EAAT3 internalization was abolished in TAAR1 knockout animals (e). RhoA activation (f, g) observed after 15 min of AMPH exposure in acute slices from wild-type mice was absent in the homozygous littermate knockout animals. Heterozygous animals displayed an intermediate level of RhoA activation. Asterisk indicates *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test for c, e, f and two-way ANOVA with Sidak’s multiple comparisons test for d; n ≥ 3
