Pharmacological characterization of membrane-expressed human trace amine-associated receptor 1 (TAAR1) by a bioluminescence resonance energy transfer cAMP biosensor.
Trace amines are neurotransmitters whose role in regulating invertebrate physiology has been appreciated for many decades. Recent studies indicate that trace amines may also play a role in mammalian physiology by binding to a novel family of G protein-coupled receptors (GPCRs) that are found throughout the central nervous system. A major obstacle impeding the careful pharmacological characterization of trace amine associated receptors (TAARs) is their extremely poor membrane expression in model cell systems, and a molecular basis for this phenomenon has not been determined. In the present study, we show that the addition of an asparagine-linked glycosylation site to the N terminus of the human trace amine associated receptor 1 (TAAR1) is sufficient to enable its plasma membrane expression, and thus its pharmacological characterization with a novel cAMP EPAC (exchange protein directly activated by cAMP) protein based bioluminescence resonance energy transfer (BRET) biosensor. We applied this novel cAMP BRET biosensor to evaluate the activity of putative TAAR1 ligands. This study represents the first comprehensive investigation of the membrane-expressed human TAAR1 pharmacology. Our strategy to express TAARs and to identify their ligands using a cAMP BRET assay could provide a foundation for characterizing the functional role of trace amines in vivo and suggests a strategy to apply to groups of poorly expressing GPCRs that have remained difficult to investigate in model systems.
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Larry S
LS
Barak
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Ali
A
Salahpour
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Xiaodong
X
Zhang
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Bernard
B
Masri
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Tatyana D
TD
Sotnikova
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Amy J
AJ
Ramsey
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Jonathan D
JD
Violin
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Robert J
RJ
Lefkowitz
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Marc G
MG
Caron
Departments of Cell Biology (L.S.B., A.S., X.Z., B.M., T.D.S., A.J.R., M.G.C., R.R.G.) and Biochemistry (J.D.V., R.J.L.) and the Howard Hughes Medical Institute (J.D.V., R.J.L.), Duke University, Durham, North Carolina.
Raul R
RR
Gainetdinov
D015374:Biosensing Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism*; D003913:Dextroamphetamine / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D010627:Phenethylamines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D000095383:Trace Amine-Associated Receptors
D000242:Cyclic AMP; D003913:Dextroamphetamine; D020662:Guanine Nucleotide Exchange Factors; D010627:Phenethylamines; D043562:Receptors, G-Protein-Coupled; D000095383:Trace Amine-Associated Receptors; C116122:RAPGEF3 protein, human; C029261:phenethylamine