Amphetamines signal through intracellular TAAR1 receptors coupled to Gα13 and GαS in discrete subcellular domains

Abstract

The extensive use of amphetamines to treat attention deficit hyperactivity disorders in children provides a compelling rationale for understanding the mechanisms of action of amphetamines and amphetamine-related drugs. We have previously shown that acute amphetamine (AMPH) regulates the trafficking of both dopamine and glutamate transporters in dopamine neurons by increasing activation of the small GTPase RhoA and of protein kinase A. Here we demonstrate that these downstream signaling events depend upon the direct activation of a trace amine-associated receptor, TAAR1, an intracellular G-protein coupled receptor (GPCR) that can be activated by amphetamines, trace amines, and biogenic amine metabolites. Using cell lines and mouse lines in which TAAR1 expression has been disrupted, we demonstrate that TAAR1 mediates the effects of AMPH on both RhoA and cAMP signaling. Inhibition of different Gα signaling pathways in cell lines and in vivo using small cell-permeable peptides confirms that the endogenous intracellular TAAR1 couples to G₁₃ and to G_S α-subunits to increase RhoA and PKA activity, respectively. Results from experiments with RhoA- and PKA-FRET sensors targeted to different subcellular compartments indicate that AMPH-elicited PKA activation occurs throughout the cell, whereas G₁₃-mediated RhoA activation is concentrated near the endoplasmic reticulum. These observations define TAAR1 as an obligate intracellular target for amphetamines in dopamine neurons and support a model in which distinct pools of TAAR1 mediate the activation of signaling pathways in different compartments to regulate excitatory and dopaminergic neurotransmission.

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

Fig. 2

AMPH does not activate RhoA or PKA in cells that lack the TAAR1 gene. HEK293 cells in which the TAAR1 gene is intact or knocked out using CRISPR-Cas9 technology (TAAR1 KO Cells) were transiently transfected with the DAT and AKAR4- or Rho-FRET sensors to measure and the activation of PKA or RhoA, respectively (a and b). AMPH did not stimulate PKA or RhoA in the TAAR1 KO line (blue lines), while the responses in their wild-type counterparts (black lines) are similar to those observed in Fig. 1. To assess the effect of the TAAR1 gene deletion on RhoA-mediated downregulation of DAT and EAAT3, we transiently expressed DAT (c) or DAT and EAAT3 in wild-type and TAAR1 KO cells and (d) measured AMPH-induced trafficking of the transporters with ³H-neurotransmitter uptake assays. AMPH-induced loss of the activity of both transporters was ameliorated in the knockout cell lines. This effect was rescued by co-transfection with a Cas9 nuclease-resistant TAAR1 construct (TAAR1-R). The time course of RhoA activation was assessed using an affinity-based pulldown assay followed by western blotting (see Materials and methods) in TAAR1 KO HEK293 cells (blue) and wild-type HEK293 cells (black, e). AMPH-induced RhoA activation peaked at 10 min and returned to baseline by 30 min. PKA-mediated phosphorylation of RhoA at S188 (f) peaked at the 30-min time point (**p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons test; n = 9 unless otherwise noted in figure)

Fig. 3

TAAR1 signaling through PKA and RhoA occur in different intracellular sites. SK-N-SH neuroblastoma cell were transfected with DAT and either AKAR4 (a) or the Rho-FRET (b) sensors to which tags were added to the carboxy or amino termini to dictate localization to various subcellular domains (see Materials and methods). Cellular nuclei were stained with DAPI (blue). PKA activation by AMPH was detected in all compartments (c) but favored non-raft membranes. RhoA activation (d, f) was most robust near the ER (red). (*p < 0.05 and **p < 0.01 by one-way ANOVA with Dunnett’s multiple comparisons test compared with the highest responding compartment; n≥10 cells.)

Fig. 4

AMPH-induced RhoA activation is mediated by G₁₃. HEK293 cells in which various endogenous α-subunits of GPCRs were knocked out were co-transfected with DAT or DAT and EAAT3 (a). G_S and G_q knockout cell lines exhibited similar sensitivity to AMPH as wild-type HEK293 cells with a decreased capacity to transport ³H-neurotransmitters following a 30-min pretreatment with AMPH (10 μM). Cells in which G_12/13 were knocked  out, however, lost sensitivity to AMPH pretreatment in regards to both DAT and EAAT3 trafficking. Wild-type HEK293 cells were co-transfected with DAT or DAT and EAAT3 along with mini-genes that interfere with the function of various α-subunits of GPCRs (b; Gilchrist et al. 1999 and 2001). The effect of AMPH on DAT and EAAT3 function were unaltered by the mini-genes that interfere with G_S, G₁₁ or G₁₂. However, the effects of AMPH on DAT and EAAT3 were inhibited by co-expression of the G₁₃ interfering minigene. We made cell-permeable versions of the alpha-interfering mini-genes by creating peptides of the interfering sequences with the addition of the TAT domain (YGRKKRRQRRR). HEK293 cells that were transiently transfected with DAT were treated with the TAT peptides for 30 min and then with AMPH (10 μM) for 30 min. Similar to the co-expression results, only the G₁₃ interfering peptide selectively disrupted AMPH-mediated DAT internalization (c). Selectivity of the G₁₃ and G_S peptides was determined by recording responses of the Rho-FRET (d) or the AKAR4-FRET sensor (e) in HEK293 cells expressing DAT. AMPH-induced RhoA activation was prevented by pretreatment with the TAT-interfering peptide directed at G₁₃ (red), while the G_S interfering peptide (green) did not alter RhoA activation (d). AMPH-induced PKA activation detected by AKAR4 was blocked by the G_S interfering peptide (green), but the G₁₃ (red) response was similar to scrambled control (black, e). G₁₃ expression (red and top panel) was detected in TH(+) cultured neurons (f, green and bottom panel). ³H-DA uptake in primary midbrain cultures was measured to reflect cell-surface localization of the DAT (g). With TAT-scrambled peptides, AMPH pretreatment (30 min, 10 μM) lead to a loss of DAT-mediated ³H-dopamine transport capacity but those treated with the TAT-13 interfering peptide, had no sensitivity to the AMPH pretreatment. (Asterisk indicates *p < 0.05, **p < 0.01, *** p < 0.001, and **** p < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons test compared with vehicle control; n≥10 wells per condition)

Fig. 5

G₁₃ mediates AMPH-induced internalization of DAT and EAAT3, as well as AMPH-induced potentiation of glutamatergic responses. In acute brain slices, cell-surface expression of DAT and EAAT3 were assessed by biotinylation assays (a and b). Pretreatment with the G₁₃ interfering peptide blocks AMPH-mediated internalization of both of the neurotransmitter transporters. The G₁₃ inhibiting peptide also blocks AMPH-mediated potentiation of NMDA synaptic currents in mouse midbrain dopamine neurons (C and D). Example traces of evoked NMDA synaptic currents in Mg²⁺-free extracellular solution. Left, in the presence of an intracellular scrambled version of the peptide (10 µM) AMPH (red) potentiates the baseline synaptic current (blue), similar to previous observations. Right, AMPH (red) has no effect on the baseline current (blue) in the presence of the G₁₃ inhibitor peptide applied intracellularly (10 µM). Compiled data showing a lack of significant potentiation by AMPH in the cells recorded with the G₁₃ inhibitor peptide (d). Cartoon of AMPH-mediated signaling through two populations of TAAR1 (t₍₁₃₎ = 5.16, p = 0.0002; p < 0.001 by paired t-test; *p < 0.05 **p < 0.01 by two-way ANOVA with Sidak’s multiple comparisons test for panels A and E; n≥3)

Fig. 6

AMPH enters the cells through the DAT found at the plasma membrane. Once inside the cell, AMPH binds to G13-coupled TAAR1 receptors that stimulate RhoA activation near the ER. RhoA mediates endocytosis of the neurotransporters DAT and EAAT3. AMPH also stimulates GS-coupled TAAR1 receptors that propagate PKA signaling throughout the neuron. Downstream PKA activation leads to phosphorylation of the RhoA and stops transporter internalization. (Created using SMART server medical art.)