Introduction

Top Abstract Introduction Experimental Procedures Results and Discussion References

In vertebrates, the trace amines -phenethylamine (-PEA), para-tyramine, tryptamine, and octopamine are found in peripheral tissues as well as the central nervous system (Tallman et al., 1976; Paterson et al., 1990). In vivo, -PEA and p-tyramine can be synthesized from phenylalanine or tyrosine by the enzyme amino acid decarboxylase. (Boulton and Dyck, 1974; Tallman et al., 1976). The trace amines are found in low amounts (accounting for less than 1% of the biogenic amines in most brain regions) and have been thought of as metabolic byproducts of catecholamine biosynthesis. Investigations into the effects of trace amines on smooth muscle and glandular preparations early in the twentieth century clearly demonstrated that amines produced by putrefaction and lacking the catechol nucleus were capable of producing robust sympathomimetic effects (Barger and Dale, 1910). Currently it is thought that p-tyramine and -PEA manifest their peripheral effects by promoting the efflux of catecholamines from sympathetic neurons and adrenals (Schonfeld and Trendelenburg, 1989; Mundorf et al., 1999) which results in the indirect stimulation of adrenergic receptors (Black et al., 1980). The abilities of p-tyramine and -PEA to deplete neurotransmitter from storage vesicles, compete with neurotransmitters for uptake, and stimulate outward neurotransmitter flux through the plasma membrane carriers are similar to the actions of the -PEA analog, -methyl--phenethylamine, better known as amphetamine (Seiden et al., 1993; Amara and Sonders, 1998).

Amphetamines were originally marketed as stimulants and appetite suppressants, but their clinical use is now mostly limited to treating attention deficit hyperactivity disorder (Seiden et al., 1993). Although listed as controlled substances, amphetamines are widely consumed because of their ability to produce wakefulness and intense euphoria. Some substituted amphetamines, such as MDMA ("ecstasy") and DOI, are taken for their "empathogenic" and hallucinogenic effects. (Shulgin and Shulgin, 1991; Eisner, 1994). Numerous liabilities are associated with the use of amphetamines, including hyperthermia (Byard et al., 1998), neurotoxicity (Ricaurte and McCann, 1992), psychosis (Seiden et al., 1993), and psychological dependence (Murray, 1998). In addition to the actions of amphetamines at biogenic amine transporters, it is also clear that a subset of amphetamine analogs, especially those with hallucinogenic properties, can act directly on 5-HT receptors because they have much higher affinities for these sites than for the transporters (Marek and Aghajanian, 1998).

We report herein the discovery and functional expression of a rat G protein-coupled receptor with homology to members of the catecholamine receptor family. This receptor stimulates cAMP production when exposed to the trace amines p-tyramine and -PEA. A pharmacological survey revealed that this rat trace amine receptor (rTAR1) is directly activated by a wide variety of clinically and socially important drugs, which include amphetamines, ergot derivatives, and adrenergic agents. Surprisingly, rTAR1 is more potently activated by the presumably "inactive" catecholamine metabolites 3-methoxytyramine (3-MT), normetanephrine, and metanephrine than by the neurotransmitters dopamine, norepinephrine, and epinephrine themselves.

In addition to enabling studies on its pharmacology and distribution, knowledge of rTAR1's DNA sequence led to the isolation of the rat and human genes. The human gene is located on chromosome 6 at q23, which falls within a region that has been identified by several schizophrenia linkage studies (Cao et al., 1997; Martinez et al., 1999; Levinson et al., 2000; Mowry and Nancarrow, 2001). Recently, Borowsky et al. (2001) reported the cloning of a family of GPCRs that includes the rTAR1¹ and the human ortholog that we describe here. Considering the broad spectrum of endogenous and exogenous molecules that activate rTAR1 and the multiplicity of related receptor genes, this family of trace amine receptors seems likely to mediate a variety of physiological functions that have yet to be fully understood.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results and Discussion References

Materials. Oligonucleotide primers were synthesized by Invitrogen (Carlsbad, CA). S(+)- and R()-4-OH-amphetamine were kindly provided by the National Institute on Drug Abuse Drug Supply System (Bethesda, MD). 5-Carboxamidotryptamine and sumatriptan were gifts from Dr. John T. Williams (Vollum Institute for Advanced Biomedical Research, Oregon Health and Science University). All other drugs were purchased from Sigma Chemical Co. (St. Louis, MO), Aldrich (Milwaukee, WI), Tocris Cookson Inc. (Ballwin, MO), or Alltech Associates (State College, PA).

Cloning and Expression of Nucleic Acids. First strand cDNA was synthesized from the rat pancreatic tumor cell line AR42J (American Type Culture Collection, Manassas, VA) and was used as a template for the original polymerase chain reactions (PCRs) employing a pair of degenerate oligonucleotide primers based on a derived consensus sequence of the third and sixth transmembrane domains of known members of the catecholamine receptor family: primer TM III, 5'-GAGTCGACCTGTG(C/T)G(C/T)(C/G)AT(C/T)(A/G)CIIT(G/T)GAC(C/A)G(C/G)TAC-3'; primer TM VI, 5'-CAGAATTCAG(T/A)AGGGCAICCAGCAGAI(G/C)(G/A)(T/C)GAA-3' (where I = inosine).

Tissue Distribution of Receptor mRNA by RT-PCR. Total RNA was extracted from freshly dissected rat (Sprague-Dawley) brain regions and peripheral tissues using the Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA). In the course of these studies, all animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Tissue fragments were mechanically homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, NY). The RNA was treated with DNase to remove all genomic DNA. For the synthesis of first strand cDNA, equal amounts of total RNA for each reaction (1.0 µg) were mixed with ProSTAR RT-PCR reagents (Stratagene, La, Jolla, CA). A PCR using oligonucleotide primers complementary to the rat receptor was then performed. All reactions for the samples were carried out in parallel using the same reaction mixtures for the cDNA synthesis and the PCR so that a semiquantitative measure of RNA quantity could be evaluated by ethidium bromide staining of the PCR products on an agarose gel.

cAMP Assays. HEK293 cells were harvested in Krebs-Ringer-HEPES buffer (KRH) and preincubated in KRH with 200 µM 3-isobutyl-1-methylxanthine. For drug treatments, cells were incubated in KRH with 100 µM 3-isobutyl-1-methylxanthine with the test compound (or 10 µM forskolin) for 1 h at 37°C. The cells were then boiled for 20 min after adding an equal volume of 0.5 mM sodium acetate buffer, centrifuged to remove cell debris, and the resulting extract was analyzed for cAMP content using competitive binding of [³H]cAMP to a cAMP binding protein (Diagnostic Products Corp., Los Angeles, CA). Data were normalized according to protein content as determined using the Bradford reagent (Bio-Rad). Concentration-response curves were plotted and EC₅₀ values calculated with Prism software (GraphPad, San Diego, CA).

Immunofluorescence Microscopy. HEK293 cells stably expressing the rTAR1sequence or the hD1R were maintained in Dulbecco's minimal essential medium containing 10% fetal calf serum and 700 µg/ml G418 (Invitrogen). Confluent cells were detached with a PBS solution containing 0.05% trypsin and 0.53 mM EDTA, harvested, and after diluting 1:10, plated on glass microscope coverslips coated with poly(D-lysine) and left to grow in the incubator for 48 h. Cells were washed twice with PBS, fixed with 2.5% paraformaldehyde in PBS for another 20 min, then incubated with anti-FLAG monoclonal antibody (1:500; Sigma Chemical Co., St. Louis MO) in blocking solution (3% dry milk, 1 mM CaCl₂, 50 mM Tris HCl, pH 7.5) with or with out 0.1% Triton X-100, for 30 min. After 3 washes with Tris-BS containing 1 mM CaCl₂, cells were incubated in blocking solution containing goat anti-mouse IgG conjugated to Cy5 (1:200; Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 30 min. Cells were then washed three times before being mounted onto microscope slides with Mowiol (Sigma/Aldrich, Milwaukee, WI). Confocal microscopy was performed using MRC-1000 laser scanning confocal imaging system (Bio-Rad) equipped with an Optiphot II Nikon microscope (Nikon, Tokyo, Japan) and a Plan Apo 60 × 1.4 oil immersion objective.

Human Chromosomal Localization. The complete coding region of the rTAR1 was used to screen a human genomic DNA library (CLONTECH). This attempt yielded a 600-bp fragment of the receptor's amino terminal sequence. A full-length genomic clone was eventually identified in a human bacterial artificial chromosome (BAC) after PCR screening of a BAC library (Research Genetics Inc., Huntsville, AL) using primers based on the original partial human sequence. DNA from the hTAR-containing BAC was nick-translated using digoxigenin-11-UTP and used to probe spreads of metaphase chromosomes, as described previously (Grandy et al., 1990). Localization of a human trace amine receptor (hTAR) gene was determined by fluorescent in situ hybridization. Chromosome identification was accomplished by sequential G-banding, as described previously (Grandy et al., 1990).

	Results and Discussion

Top Abstract Introduction Experimental Procedures Results and Discussion References

Cloning of Rat and Human Trace Amine Receptors. In an effort to identify novel GPCRs that are activated by dopamine and other related biogenic amines, RT-PCRs were performed on RNA isolated from cell lines whose tissue of origin was known to receive sympathetic innervation. These RT-PCRs employed degenerate oligonucleotide primers that incorporated conserved sequences present in the putative transmembrane domains III and VI of the G protein-coupled catecholamine receptor gene family. One of the cell lines examined in this way was the rat pancreatic tumor cell line ARJ42. DNA sequence analysis of the RT-PCR products produced from the ARJ42 cell line revealed the presence of a cDNA fragment, r2-3, that predicted a novel amino acid sequence related to several known biogenic amine-recognizing GPCRs.

View larger version (97K): [in this window] [in a new window]   Fig. 1.   Deduced amino acid sequences for the rat and human trace amine receptors aligned with other homologous G protein-coupled receptors. Putative transmembrane domains are numbered with Roman numerals. Amino acids conserved in four or more sequences are outlined in black. Abbreviations used: rTAR, rat trace amine receptor1; hTAR, human trace amine receptor1; NT Receptor, orphan neurotransmitter receptor (Zeng et al., 1998); orphan receptors GPCR57 and GPCR58 (Lee et al., 2000); hD1R, human dopamine D1 receptor; h2R, human 2 adrenergic receptor; r5ht4R, rat serotonin 5HT4 receptor. Arrowheads indicate positions designated as 5.42 and 5.43.

Pharmacological Characterization of the Rat Trace Amine Receptor. Although the distribution of r2-3 mRNA did not immediately suggest a potential agonist for the putative receptor, the striking similarity in amino acid sequence between r2-3 and biogenic amine GPCRs (Fig. 1) indicated that catecholamines or related compounds might activate the receptor. When tested in functional assays of cAMP production using HEK293 cells stably expressing r2-3, DA did stimulate synthesis of the second messenger. However, p-tyramine and -PEA were considerably more potent. Nanomolar concentrations of the trace amines generated responses comparable with that of 10 µM forskolin in the cells expressing r2-3 (Fig. 2B) but not in HEK293 cells that were stably transfected with the empty expression vector. Subsequently, other closely related trace amines, including tryptamine, octopamine, and synephrine, were tested and found to stimulate cAMP production (Fig. 2A). These molecules had EC₅₀ values (Table 1) in the following rank order (lowest to highest): p-tyramine < -PEA < tryptamine < synephrine < octopamine < meta-tyramine  dopamine < 5-HT NE, Epi. Based on this preference for the trace amines over the catecholamines and 5HT, the rat orphan receptor coded for by r2-3 was renamed the rTAR1.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Agonist-induced cAMP production in HEK293 cells expressing rTAR1. A, concentration-response for naturally occurring compounds. B, concentration-response for synthetic compounds. The curves depicted are from a single representative experiment performed in duplicate. Concentrations units are log (M). Results from all concentration-response experiments are summarized in Table 1.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Pharmacological survey of drugs eliciting cAMP production through the rTAR1 stably expressed in HEK293 cells. Bars indicate the activity of test compounds administered alone (1 µM concentrations except where specified) as a fraction of the cAMP produced in the same assay by 1 µM p-tyramine. Forskolin (10 µM) was tested in all experiments and elicited comparable stimulation. Error bars indicate standard error of n = 2 to 8 different experiments. Open bars designate known antagonists of biogenic amine receptors or nontransported inhibitors of DA transporter protein. 3-Ph-propylamine, 3-phenyl-1-propylamine; Norepi, norepinephrine; NMN, -4-dihydroxy-3-methoxy--phenethylamine (normetanephrine); MN, -4-dihydroxy-3-methoxy-N-methyl--phenethylamine (metanephrine); 4-MT, 4-methoxytyramine; 3,4-DiMeO-PEA, 3,4-dimethoxy--phenethylamine; HVA, 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid); DOI, 2-amino,1-[2,5-dimethoxy-4-iodophenyl]propane; DHE, dihydroergotamine; d-LSD, d-lysergic acid diethylamide; N,N-DMT, N,N-dimethyltryptamine; 5-MeO-DMT, 5-methoxy-N,N-dimethyltryptamine; 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, 8-hydroxy-N,N-dipropylamino-2-amino-1,2,3,4-tetrahydronapthalene; mCPP, 1-(3-chlorophenyl)piperazine; -CIT, ()-2--carbomethoxy-3--(4-fluorophenyl)tropane; MPP+, 1-methyl-4-phenyltetrahydropyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. * P < 0.01, two-tailed t test, n  3 experiments. §, Drugs tested in the presence of 0.3 µM propranolol to eliminate a modest component of cAMP production attributable to endogenous -adrenergic receptors in the HEK293 cells that could be activated by Epi, NE, and the -agonists. Responses to DA, 3-MT, MN, NMN, p-tyramine, -PEA, and amphetamines were unaffected by inclusion of propranolol or pindolol and were not observed in cells transfected with an empty vector or sequence encoding the hD1 receptor (with the exception of DA).

Cellular Localization of the Rat Trace Amine Receptor. When considering the physiological role of rTAR1 activation the question arises as to its localization in the cell. HEK293 cells stably expressing the epitope-tagged rTAR1 (see Experimental Procedures) were examined in parallel with HEK293 cells stably expressing another G_s-coupled receptor, the human dopamine D1 receptor (hD1R), similarly tagged at its N terminus. The results of a confocal immunofluorescence study were somewhat surprising in that the distribution of rTAR1 in the HEK293 cells (Fig. 4, A and B) appeared as intracellular puncta, in marked contrast to the localization of hD1R at the plasma membrane (Fig. 4C). Whether this localization is representative of the protein's localization in vivo remains to be seen, but it does raise the interesting possibilities that 1) rTAR1 requires an accessory protein for proper trafficking to the plasma membrane that is lacking in HEK293 cells or 2) rTAR1 may reside primarily intracellularly, perhaps even in vesicular membranes. The notion that rTAR1 might function in an intracellular environment is supported in part by knowledge that such receptors would have access to several agonists: rTAR1 agonists could be synthesized in the cytoplasm (e.g., -PEA, p-tyramine, DA) of biogenic amine-producing cells or could be imported into the cytoplasm and/or vesicular lumen because they serve as substrates of plasma membrane and vesicular transporters (e.g., amphetamines, DA).

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Cellular distribution of the M1 flag-tagged rTAR1 stably expressed in HEK293 cells. Cells were treated with anti-FLAG monoclonal antibody in the absence (A) or presence (B) of 0.1% Triton X-100, followed by Cy5 goat anti-mouse IgG, and were visualized using a confocal microscope. According to this pattern of immunoreactivity, the rTAR1 seem to be almost exclusively retained within the cytoplasm. C, for comparison, HEK293 cells stably expressing hD1R carrying the M1 epitope tag were stained in parallel in the presence of Triton X-100. As expected, the hD1R was primarily localized around the surface of the cell.

Chromosomal Localization of a Human Trace Amine Receptor Gene. With the identification of GPCRs for trace amines and the evidence suggesting that these compounds are involved in monoamine release (Schonfeld and Trendelenburg, 1989; Mundorf et al., 1999), drug abuse (Shannon and Degregorio, 1982), hypertension (Brown et al., 1989), anxiety (Lapin, 1990), schizophrenia (Boulton, 1982), Parkinson's disease (Da Prada et al., 1984), and diabetes (Mosnaim et al., 1982), it was of interest to determine the chromosomal localization of the human TAR gene (hTAR1) to establish whether or not it lies within a region of interest based on linkage or association studies. For the chromosomal mapping of hTAR1, fluorescence in situ hybridization was performed on human metaphase chromosomes. Hybridization signals were visualized over the long arms of both chromosomes 6 (data not shown) in a position consistent with a chromosomal localization of 6q23.2, where at least two other orphan GPCRs related to hTAR1 reside (NT receptor, Zeng et al., 1998; GPCR57, GPCR58, Lee et al., 2000). This chromosomal localization is particularly noteworthy because it is one of the few regions that has been reproducibly associated with schizophrenia in linkage studies (Cao et al., 1997; Martinez et al., 1999; Levinson et al., 2000; Mowry and Nancarrow, 2001), suggesting the possibility that hTAR1 may be involved in the mechanism of psychosis. The relevance of this receptor to the etiology of psychosis is enhanced by the evidence that 3-MT is a potent and efficacious agonist. 3-MT is the major metabolite of dopamine produced by the enzyme COMT, a variant of which was recently found to be transmitted with greater frequency to schizophrenic offspring in a family-based association study (Egan et al., 2001).