0026-895X/01/6006-1181–1188$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 6 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 1304/951292 Mol Pharmacol 60:1181–1188, 2001 Printed in U.S.A. ACCELERATED COMMUNICATION Amphetamine, 3,4-Methylenedioxymethamphetamine, Lysergic Acid Diethylamide, and Metabolites of the Catecholamine Neurotransmitters Are Agonists of a Rat Trace Amine Receptor JAMES R. BUNZOW, MARK S. SONDERS, SEKSIRI ARTTAMANGKUL, LAURA M. HARRISON, GE ZHANG, DENISE I. QUIGLEY, TRISTAN DARLAND, KATHERINE L. SUCHLAND, SHAILAJA PASUMAMULA, JAMES L. KENNEDY, SUSAN B. OLSON, R. ELLEN MAGENIS, SUSAN G. AMARA, and DAVID K. GRANDY Departments of Physiology & Pharmacology (J.R.B., S.A., L.M.H., G.Z., D.I.Q., T.D., K.L.S., S.P., S.B.O., R.E.M., D.K.G.) and Molecular and Medical Genetics (S.B.O., R.E.M), School of Medicine, the Vollum Institute (M.S.S., S.G.A.), and the Howard Hughes Medical Institute (S.G.A.), Oregon Health & Science University, Portland, Oregon; and Centre for Addiction and Mental Health, University of Toronto, Canada (J.L.K.) Received August 21, 2001; accepted September 28, 2001 This paper is available online at http://molpharm.aspetjournals.org ABSTRACT The trace amine para-tyramine is structurally and functionally re- goline derivatives, adrenergic ligands, and 3-methylated metabo- lated to the amphetamines and the biogenic amine neurotrans- lites of the catecholamine neurotransmitters are also good ago- mitters. It is currently thought that the biological activities elicited nists at the rat trace amine receptor 1 (rTAR1). These results by trace amines such as p-tyramine and the psychostimulant suggest that the trace amines and catecholamine metabolites amphetamines are manifestations of their ability to inhibit the may serve as the endogenous ligands of a novel intercellular clearance of extracellular transmitter and/or stimulate the efflux of signaling system found widely throughout the vertebrate brain and transmitter from intracellular stores. Here we report the discovery periphery. Furthermore, the discovery that amphetamines, includ- and pharmacological characterization of a rat G protein-coupled ing 3,4-methylenedioxymethamphetamine (MDMA; “ecstasy”), receptor that stimulates the production of cAMP when exposed to are potent rTAR1 agonists suggests that the effects of these the trace amines p-tyramine, ␤-phenethylamine, tryptamine, and widely used drugs may be mediated in part by this receptor as well octopamine. An extensive pharmacological survey revealed that as their previously characterized targets, the neurotransmitter psychostimulant and hallucinogenic amphetamines, numerous er- transporter proteins. In vertebrates, the trace amines ␤-phenethylamine (Boulton and Dyck, 1974; Tallman et al., 1976). The trace (␤-PEA), para-tyramine, tryptamine, and octopamine are amines are found in low amounts (accounting for less than found in peripheral tissues as well as the central nervous 1% of the biogenic amines in most brain regions) and have system (Tallman et al., 1976; Paterson et al., 1990). In vivo, been thought of as metabolic byproducts of catecholamine ␤-PEA and p-tyramine can be synthesized from phenylala- biosynthesis. Investigations into the effects of trace amines nine or tyrosine by the enzyme amino acid decarboxylase. on smooth muscle and glandular preparations early in the twentieth century clearly demonstrated that amines pro- This work was supported by National Institute on Drug Abuse (NIDA) duced by putrefaction and lacking the catechol nucleus were Grants DA10703 and DA12408, NIDA/National Institute on Alcohol Abuse capable of producing robust sympathomimetic effects (Barger and Alcoholism Training Grant DA07262-09, and the Howard Hughes Medical Institute. and Dale, 1910). Currently it is thought that p-tyramine and 1 Because the predicted amino acid sequences of the rat and human trace ␤-PEA manifest their peripheral effects by promoting the amine receptors described here are identical to the rTAR1 and hTAR1 se- quences reported by Borowsky et al. (2001), we employ the same nomencla- efflux of catecholamines from sympathetic neurons and adre- ture. nals (Schonfeld and Trendelenburg, 1989; Mundorf et al., ABBREVIATIONS: ␤-PEA, ␤–phenethylamine; MDMA, 3,4-methylenedioxymethamphetamine; GPCR, G protein-coupled receptor; 5-HT, 5-hy- droxytryptamine (serotonin); 3-MT, 3-methoxytyramine (3-methyldopamine); TAR1, trace amine receptor 1; PCR, polymerase chain reaction; RT, reverse transcription; HEK, human embryonic kidney; KRH, Krebs-Ringer-HEPES; PBS, phosphate-buffered saline; BAC, bacterial artificial chromosome; DA, dopamine; NE, norepinephrine; Epi, epinephrine; COMT, catechol-O-methyltransferase; DOI, 2-amino,1-(2,5-dimethoxy-4- iodophenyl)propane. 1181 1182 Bunzow et al. 1999) which results in the indirect stimulation of adrenergic Biomedical Research, Oregon Health and Science University). All receptors (Black et al., 1980). The abilities of p-tyramine and other drugs were purchased from Sigma Chemical Co. (St. Louis, ␤-PEA to deplete neurotransmitter from storage vesicles, MO), Aldrich (Milwaukee, WI), Tocris Cookson Inc. (Ballwin, MO), or compete with neurotransmitters for uptake, and stimulate Alltech Associates (State College, PA). outward neurotransmitter flux through the plasma mem- Cloning and Expression of Nucleic Acids. First strand cDNA was synthesized from the rat pancreatic tumor cell line AR42J brane carriers are similar to the actions of the ␤-PEA analog, ␣ ␤ (American Type Culture Collection, Manassas, VA) and was used as -methyl- -phenethylamine, better known as amphetamine a template for the original polymerase chain reactions (PCRs) em- (Seiden et al., 1993; Amara and Sonders, 1998). ploying a pair of degenerate oligonucleotide primers based on a Amphetamines were originally marketed as stimulants derived consensus sequence of the third and sixth transmembrane and appetite suppressants, but their clinical use is now domains of known members of the catecholamine receptor family: mostly limited to treating attention deficit hyperactivity dis- primer TM III, 5Ј-GAGTCGACCTGTG(C/T)G(C/T)(C/G)AT(C/T)(A/ order (Seiden et al., 1993). Although listed as controlled G)CIIT(G/T)GAC(C/A)G(C/G)TAC-3Ј; primer TM VI, 5Ј-CAGAAT- substances, amphetamines are widely consumed because of TCAG(T/A)AGGGCAICCAGCAGAI(G/C)(G/A)(T/C)GAA-3Ј (where their ability to produce wakefulness and intense euphoria. I ϭ inosine). Some substituted amphetamines, such as MDMA (“ecstasy”) The conditions used were: 94°C for 90 s, 50°C for 90 s, and 72°C for and DOI, are taken for their “empathogenic” and hallucino- 120 s, for 35 cycles. Products from 400 to 750 base pairs were purified from a 1.0% agarose gel using Prep-A-Gene (Bio-Rad, Hercules, CA), genic effects. (Shulgin and Shulgin, 1991; Eisner, 1994). Nu- digested with EcoRI and SalI, and subcloned into the vector pBlue- merous liabilities are associated with the use of amphet- script (Stratagene, La Jolla, CA). Plasmid DNA from these clones amines, including hyperthermia (Byard et al., 1998), was purifed, and the nucleotide sequence of the insert was deter- neurotoxicity (Ricaurte and McCann, 1992), psychosis (Sei- mined by the dideoxynucleotide chain termination method. The de- den et al., 1993), and psychological dependence (Murray, duced amino acid sequence of a 0.4-kilobase PCR fragment displayed 1998). In addition to the actions of amphetamines at biogenic regions of homology to the known catecholamine receptors. This amine transporters, it is also clear that a subset of amphet- clone was subsequently labeled with 32P and used to probe a rat amine analogs, especially those with hallucinogenic proper- genomic library (CLONTECH, Palo Alto, CA) that had been trans- ties, can act directly on 5-HT receptors because they have ferred to nylon membranes (Gene Screen Plus; PerkinElmer Life much higher affinities for these sites than for the transport- Science Products, Boston, MA) resulting in the identification of sev- eral full-length clones. Full-length clones were also obtained by RT- ers (Marek and Aghajanian, 1998). PCR from the rat pancreatic tumor cell line RIN5 and rat cerebel- We report herein the discovery and functional expression lum. The nucleotide sequence of the human TAR has been assigned of a rat G protein-coupled receptor with homology to mem- GenBank accession number AF200627. The nucleotide sequence of bers of the catecholamine receptor family. This receptor stim- the rat TAR has been assigned GenBank accession number ulates cAMP production when exposed to the trace amines AF421352. p-tyramine and ␤-PEA. A pharmacological survey revealed For the purpose of expressing the putative receptor in tissue that this rat trace amine receptor (rTAR1) is directly acti- culture, a 16-amino-acid signal sequence from the influenza hemag- vated by a wide variety of clinically and socially important glutinin virus followed by the 8-amino-acid M1-“Flag” epitope and a drugs, which include amphetamines, ergot derivatives, and “MetGly” linker were added to the N terminus of the cerebellar adrenergic agents. Surprisingly, rTAR1 is more potently ac- cDNA before its insertion into the expression vector pcDNA3.1/V5/ His-TOPO (Invitrogen). The resulting construct was transiently ex- tivated by the presumably “inactive” catecholamine metabo- pressed in HEK293 and COS-7 cells after LipofectAMINE-assisted lites 3-methoxytyramine (3-MT), normetanephrine, and transfection (Invitrogen). A line of G418 resistant HEK293 cells