Trace amine associated receptor 1 signaling in activated lymphocytes

Abstract

Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.

Figure 1. EPPTB blocks TAAR1 activation by methamphetamine in TAAR1-transfected HEK293 cells

A dual luciferase assay was performed to observe the response of mouse TAAR1 to EPPTB in vitro. HEK293-derived cells that stably express mouse TAAR1 (top) or HEK293 cells (bottom) were transiently transfected with a reporter vector CRE-Luc (cAMP sensitive) and a control vector pGL4.73 for 24 h and then treated with the indicated drugs for 20 h. (Top left) TAAR1 response to methamphetamine and EPPTB. (Top right) Blockade effect of EPPTB (10 μM) on the response of TAAR1 to methamphetamine. (Bottom left) Effect of methamphetamine on cells in the absence of TAAR1. (Bottom right) Effect of EPPTB on cells in the absence of TAAR1.

Figure 2. Selective upregulation of NFAT and CREB by methamphetamine in TAAR1-transfected HEK29 cells

Comparison of 45 different signal transduction pathways following vehicle or methamphetamine (1 μM) treatment in untransfected and rhesus monkey TAAR1-transfected HEK293 cells. Signal transduction pathways that are active in untransfected HEK293 cells treated with vehicle (a) were compared with those detected in untransfected HEK293 cells treated with methamphetamine (b) or rhesus monkey TAAR1-transfected HEK293 cells treated with methamphetamine (c). Black bars highlight TAAR1-dependent activation of the cAMP/PKA and PKC/Ca⁺⁺ pathways in response to methamphetamine.

Figure 3. Methamphetamine-induced Lenti-CRE or Lenti-NFAT reporter expression is TAAR1-dependent

HEK293 cells stably transfected with rhesus monkey TAAR1 were transformed with a Lenti-CRE Reporter or with a Lenti-NFAT Reporter. Transformed cell lines were treated with vehicle, EPPTB (10 μM), methamphetamine (1 μM), or methamphetamine in combination with EPPTB. Levels of Firefly luciferase expressed by the reporter constructs were determined in cell lysates, and relative light units (RLU) are displayed as a percent of the baseline RLU values. **: p < 0.0001 for indicated comparisons.

Figure 4. TAAR1 signaling promotes phosphorylation of both PKA and PKC

HEK293 cells and HEK293 cells stably expressing rhesus monkey TAAR1 were left untreated (1) or were treated with vehicle (2), 1 μM methamphetamine (3), 1 μM methamphetamine and 10 μM H89 (4), 1 μM methamphetamine and 10 μM R032-0432 (5), 100 μM 8-Br-cAMP (6), or 1 μM μ-PMA and were then collected and lysed to detect phosphorylated PKA and phosphorylated PKC by Western blot. Methamphetamine significantly increased phosphorylation of both PKA and PKC in TAAR1 cells but not in HEK293 cells. The PKA inhibitor H89 blocked methamphetamine-induced PKA phosphorylation selectively, whereas the PKC inhibitor R032-0432 blocked methamphetamine-induced PKC phosphorylation selectively in TAAR1 cells, but had no significant effect in HEK293 cells. The cyclic AMP analog 8-Br-cAMP evoked PKA phosphorylation and the phorbol ester β-PMA evoked PKC phosphorylation comparably in TAAR1 cells as well as HEK293 cells. ** (1) p < 0.01 by one-way ANNOVA/Turkey post-hoc test, compared to the vehicle treatment; ** (2) p < 0.01 by one-way ANNOVA/Turkey post-hoc test, compared to the methamphetamine treatment.

Figure 5. Phosphorylation of PKA and PKC by methamphetamine is TAAR1-dependent in rhesus monkey immortalized B lymphocytes

(a) TAAR1 expression in four immortalized rhesus monkey B cell lines compared to TAAR1 expression in rhesus monkey TAAR1-transfected HEK293 cells assessed by Western blot using an anti-TAAR1 antibody. (b) Phosphorylation status of PKA and PKC in untreated B cells and following vehicle, EPPTB (10 μM), methamphetamine (1 μM), and EPPTB plus methamphetamine treatments were assessed by Western blot using anti-phospho-PKA and anti-phospho-PKC antibodies. Data shown are representative of two independent experiments. (c) Quantification and comparison of PKA and PKC phosphorylation was done by densitometry using IMAGEJ software. Data are averaged from two different immortalized B cell lines assessed independently. **: p < 0.0001 for indicated comparisons.

Figure 6. TAAR1 expression upregulation following immune activation and TAAR1-dependent phosphorylation of PKA and PKC by methamphetamine in rhesus monkey PBMC

(a) TAAR1 expression was upregulated in PBMC by stimulation with PHA (1 μg/ml for 48 h) and is compared to TAAR1 expression in rhesus monkey TAAR1-transfected HEK293 cells, as assessed by Western blot in cells obtained from two different rhesus monkeys (top and bottom). (b) Phosphorylation status of PKA and PKC in untreated PBMC and following vehicle, EPPTB (10 μM), methamphetamine (1 μM), and EPPTB plus methamphetamine treatments were assessed by Western blot using anti-phospho-PKA and anti-phospho-PKC antibodies. Data shown are derived from one rhesus monkey and is representative of two independent experiments using PBMC generated from two different rhesus monkeys. (c) Quantification and comparison of PKA and PKC phosphorylation in PHA-treated PBMC was done by densitometry using IMAGEJ software. Data are averaged from the two independent experiments.**: p < 0.0001 for indicated comparisons.