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Graduate
Physical and Life Sciences
PhD Pharmacology
Abstract ID# 1081
Is TAAR1 a Potential Therapeutic Target for Immune Dysregulation in Drug Abuse?
Fleischer, Lisa M; Tamashunas, Nina and Miller, Gregory M
Addiction Sciences Laboratory, Northeastern University, Boston MA 02115
Abstract
Discovered in 2001, Trace Amine Associated Receptor 1 (TAAR1) is a direct target of
amphetamine, methamphetamine and MDMA. It is expressed in the brain reward circuity and
modulates dopamine transporter function and dopamine neuron firing rates. Newly-developed
compounds that specifically target TAAR1 have recently been investigated in animal models
as candidate therapeutics for methamphetamine, cocaine and alcohol abuse. These studies
involving classic behavioral measures of drug response, as well as drug self-administration,
strongly implicate TAAR1 as a potential therapeutic target for the treatment of addiction. In
addition to its central actions, we demonstrated that TAAR1 is upregulated in peripheral blood
mononuclear cells (PBMC) and B cells following immune activation, and that subsequent
activation of TAAR1 by methamphetamine stimulates cAMP, similar to the function of
adenosine A2 receptors which are also present in immune cells and play a critical role in the
immune response. Here, we are investigating the relationship between TAAR1 and the
adenosine A2 receptor at the level of cellular signaling and receptor dimerization. We
hypothesize that both receptors synergistically elevate cAMP through their Gs coupling, and
that specific TAAR1 drugs may have immunomodulatory effects through this shared signaling
mechanism. This hypothesis is supported by the observation that methamphetamine is a
potent TAAR1 agonist that has profound effects on immune system function. Accordingly,
deciphering the role of TAAR1 in methamphetamine action and immune regulation may lead
to the development of novel addiction therapeutics that combat both central addictive
mechanisms, as well as immunological aberrations that occur in drug abuse.
Introduction
• Trace-Amine Associated Receptor 1 (TAAR1) is a Gs-linked GPCR first cloned in 2001.
• TAAR1 is a target for trace amines, common biogenic amines, thyronamines,
amphetamine, methamphetamine (METH), and MDMA.
• TAAR1 is expressed in some dopamine neurons, and has been shown to modulate
DAT function, D2 function, and DA neuronal firing rates.
• Newly-developed specific TAAR1-targeted agonists are being tested as therapeutics for
psychiatric and addiction disorders.
• In addition to brain, TAAR1 is expressed in immune cells.
Data and Results
In addition to brain, TAAR1 is expressed in immune cells
METH promotes PKA and PKC Phosphorylation through TAAR1
•
Rhesus and Human
Cells Lines
•
•
•
•
TAAR1 has a wide agonist spectrum
METH is a potent TAAR1 agonist
Antibodies:
Phospho-PKA (Abcam ab5815);
Phospho-PKC (Abcam
ab23513);
TAAR1 (Imgenex IMG-71855)
We treated HEK/TAAR1 cells and HEK293
cells with vehicle or METH, with and without
activators and inhibitors of PKA and PKC.
We performed Western blotting experiments to
measure levels of phospho-PKA and phosphoPKC.
We found that specific activators of PKA and
PKC promote upregulation of phosphor-PKA
and phosphor-PKC, respectively, in both
HEK/TAAR1 cells and HEK293 cells.
However, METH promoted upregulation of
phospho-PKA and phospho-PKC only in
HEK/TAAR1 cells.
Antibodies: Phospho-PKA (Abcam ab5815);
Phospho-PKC (Abcam ab23513); TAAR1
(Imgenex IMG-71855)
Drugs:
Methamphetamine (1mM); PKA Inhibitor: H89 (10 mM);
PKC Inhibitor: Ro32-0432 (10 mM); PKA activator:
8-Br-cAMP (100 mM) PKC activator: b-PMA (1 mM)
TAAR1 is upregulated in activated immune cells, and
METH promotes PKA and PKC phosphorylation only in the
presence of TAAR1
Aims and Methods
We hypothesize that TAAR1 is an immunomodulator. We speculate that TAAR1 signaling
may be synergistic with Adenosine A2A receptor signaling. A2A is also Gs-linked and plays
a prominent role in orchestrating the immune response. Further, we speculate that TAAR1
mediates some of the immunological actions of methamphetamine, and may serve as a
therapeutic target.
The preliminary data shown here suggests that immunological actions of METH may be
mediated through TAAR1. Moving forward, we will explore whether TAAR1 signaling is
interactive with A2A signaling, and whether TAAR1 and A2A form heteromers.
• We used a CRE-Luciferase assay to perform a dose-response study using
common biogenic amines, trace amines and amphetamine-like
psychostimulants. Data is generated in TAAR1-transfected HEK293 cells.
The TAAR1 agonist METH selectively activates NFAT (Ca++/PKC)
and CREB (cAMP/PKA) pathways
1) Characterize human cell lines for TAAR1 and A2A co-expression by developing
sensitive and specific PCR probes for TAAR1 and for A2A detection in:
-PM1 cells (human CD4+ T-lymphoid cells)
-SH-S5Y5 cells (human neuroblastoma, bone marrow cells)
Vehicle in HEK293 cells
2) Assess cellular signaling pathways utilized by TAAR1 and A2A using Cignal45
assays and Lenti-reporter cell lines to assess TAAR1 and A2A signaling.
METH in HEK293 cells
3) Determine whether TAAR1 and A2A form heteromers in vitro using YFP- and Luctagged TAAR1 and A2A constructs and assessing BRET in transfected cells that natively
express TAAR1 and A2.
METH in HEK/TAAR1 cells
Conclusions
•
•
•
•
Preliminary findings indicate that:
TAAR1 is expressed in a variety of immune cell types.
METH is an agonist at TAAR1.
TAAR1 activates both the cAMP/PKA and the Ca++/PKC pathways.
TAAR1 is upregulated in activated immune cells, and METH stimulation of both the
cAMP/PKA and the Ca++/PKC pathways is TAAR1-dependent.
Our future studies will explore whether TAAR1 signaling is interactive with A2A signaling
and whether TAAR1 and A2A form heteromers.
• We used a Cignal45 array assay (SABiosciences) to determine whether additional
signaling pathways are stimulated by TAAR1 activation.
• Consistently, we found that NFAT (Ca++/PKC) and CREB (cAMP/PKA) pathways
were activated by METH in a TAAR1-dependent manner.
TAAR1 expression upregulation following immune activation and TAAR1dependent phosphorylation of PKA and PKC by METH 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/HEK293 cells.
b) Phosphorylation status of PKA and PKC in untreated PBMC and following
vehicle, EPPTB (10 μM), methamphetamine (1 μM), and EPPTB plus METH
treatments assessed by Western blot using anti-phospho-PKA and antiphospho-PKC antibodies.
c) Quantification and comparison of PKA and PKC phosphorylation in PHAtreated PBMC was done by densitometry using IMAGEJ software. **: p<0.0001
for indicated comparisons.
Support and References
NIDA 025697 (GM), 022323 (GM), DA030177 (GM).
Some data shown is published in:
Panas et al, J Neuroimmune Pharmacol (2012) 7:866–876.