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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
Received for publication May 15, 2008.
Accepted for publication June 3, 2008.
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Abstract |
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; Premont et al., 2001; Branchek and Blackburn, 2003; Berry, 2004; Grandy, 2007). TA dysregulation has been linked to various psychiatric and neurological disorders, including schizophrenia and Parkinson's disease. As such, the TAs offer tempting targets for ameliorating the symptoms of central nervous system diseases when used in combination with standard therapies or for reducing doses and hence the side effects that accompany the long-term use of some currently prescribed medications. This is particularly relevant for Parkinson's disease, where long-term L-DOPA therapy often produces drug-related dyskinesias (Hornykiewicz, 2002; Fahn, 2003; Mercuri and Bernardi, 2005).
Trace amines are structurally related to several psychotropic molecules that naturally occur in plants, including amphetamine-like compounds. Amphetamines are recognized inhibitors of inward monoamine transport (Jones et al., 1998), and it was generally proposed that TAs also affect the monoamine system indirectly via interaction with plasma membrane transporters, such as the dopamine transporter (Sotnikova et al., 2004). In this view, the TAs function simply as neuromodulators of conventional mammalian brain amine transmitters, and at physiological levels, TAs generally should have only minor effects on neuronal excitability in the absence of conventional monoamines. However, binding sites for tryptamine, tyramine and β-PEA have been reported in rat brain and a family of trace amine G-protein-coupled receptors (GPCRs) has been recently identified in rodents and humans (Borowsky et al., 2001; Bunzow et al., 2001; Lindemann and Hoener, 2005; Grandy, 2007). Thus solid support exists for a potential direct behavioral mechanism in which trace amines influence neuronal activity.
In humans, six trace amine-associated receptors (TAARs) and three pseudogenes have been identified (Lindemann and Hoener, 2005). Similar to their mouse counterparts on chromosome 10, human TAARs are coded for by intronless genes clustered tightly on chromosome 6. TAAR1 is the best characterized of them (Borowsky et al., 2001; Bunzow et al., 2001; Grandy, 2007). TAAR1 couples to Gs, and when assessed using cAMP, is activated by TAs such as β-PEA in addition to metabolites of catecholamines, amphetamines (including MDMA), and other compounds known to affect monoaminergic transmission (Borowsky et al., 2001; Bunzow et al., 2001; Kim and von Zastrow, 2001; Scanlan et al., 2004; Lindemann and Hoener, 2005; Miller et al., 2005; Grandy, 2007; Reese et al., 2007; Wainscott et al., 2007; Wolinsky et al., 2007; Xie and Miller, 2007; Xie et al., 2007).
In the brain, the mRNA for TAAR1 is distributed throughout the limbic system and in regions containing catecholaminergic cell bodies and their projections (Borowsky et al., 2001; Bunzow et al., 2001; Lindemann and Hoener, 2005; Xie et al., 2007). Thus, TAAR1 is well positioned to modulate locomotor, emotional, and motivated behaviors that are traditionally associated with monoaminergic activity. Recent evidence indicates that at least some TAARs can also serve as a new class of chemosensory receptors in mammals (Liberles and Buck, 2006). Because an imbalance in the function of TAs might have important implications in the pathogenesis of several disorders, finding selective small-molecule agonists and antagonists for these receptors could provide novel approaches to disease management. However, a major obstacle in studying and screening TA receptors is their extremely poor plasma membrane expression in heterologous cell systems (Borowsky et al., 2001; Bunzow et al., 2001; Lindemann and Hoener, 2005; Miller et al., 2005; Grandy, 2007; Wainscott et al., 2007; Wolinsky et al., 2007), a scenario remarkably similar to that of the large GPCR subfamily of chemosensory odorant receptors (Saito et al., 2004; Zhuang and Matsunami, 2007).
The human variant of the TAAR1 has remained even more difficult to express in model systems than its poorly expressed rodent analog (Lindemann and Hoener, 2005; Grandy, 2007), thus impeding measurements of biochemical and pharmacological properties of this receptor. To improve our ability to study hTAAR1, we investigated potential reasons for the poor hTAAR1 expression. In this report, we demonstrate that an N-glycosylated variant of the human TAAR1 expresses at the plasma membrane of HEK-293 cells well enough to be studied using antibody labeling. We have determined in this system that hTAAR1 can interact with β-arrestin2. Moreover, we have used an N-glycosylated variant of the human TAAR1 for identifying and studying TAAR1 ligands by constructing a novel cell-based screening assay that employs a bioluminescence resonance energy transfer (BRET) exchange protein activated by cAMP (EPAC) cAMP biosensor.
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Materials and Methods |
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Construction of Expression Vectors. A cDNA leader sequence containing the triple-HA epitope was constructed using the polymerase chain reaction for insertion into the plasmid pcDNA3. Full-length human TAAR1 cDNA without a stop codon was amplified by PCR with 5' and 3' in-frame restriction enzyme sites of EcoRI and KpnI, respectively. A cDNA encoding GFP in the absence of its initiation start codon was amplified by PCR with 5' (in-frame) and 3' (downstream of stop codon) restriction enzyme sites of KpnI and XbaI, respectively. Enzyme-digested TAAR1 and GFP PCR products were ligated into EcoRI and XbaI restriction enzyme sites of pcDNA3 vector with N-terminal triple HA tag to generate the triple HA-β2N9-TAAR1-GFP construct. The cDNA corresponding to the first nine amino acids of the β2-adrenergic receptor was inserted in-frame between the triple HA and TAAR1.
For the GFP-negative construct, the GFP moiety was removed from the above construct by PCR amplification using 5' T7 promoter sequence primer upstream of triple-HA sequence and 3' primer containing a stop codon followed by the restriction enzyme site of XbaI. The PCR product was then digested with HindIII (upstream of triple HA sequence) and XbaI, and subcloned into the pcDNA3 vector. Two complementary sequences encoding the EcoRV/EcoRI-digested β2N9 DNA fragment were allowed to anneal in buffer containing 10 mM Tris-EDTA, pH 8.0, and 50 mM NaCl and were ligated into EcoRV/EcoRI restriction enzyme sites. The cDNA illustrative of these modifications is shown below; upper case characters correspond to the three triple-HA inserts, and the last segment corresponds to the β2-adrenergic receptor. The corresponding cDNA sequence is: [tccaccatggctagctacccaTACGATGTTCCTGACTATGCGggcTATCCCTATGACGTCCCGGACTATGCAggatccTATCCATATGACGTTCCAGATTACGCCagatctgatatcATGGGGCAACCCGGGAACGGCAGCGCCgaattcATGATGCCCTTTTGCC]. The translated sequence is: (stmas YPYDVPDY ag YPYD VPDY ags YPYDVPDY arsdi MGQPGNGSA ef M MPFC).
The BRET-based cyclic AMP biosensor (Ponsioen et al., 2004; Holz et al., 2006; Jiang et al., 2007) was constructed by the modification of ICUE2, an existing FRET-based intramolecular biosensor in which enhanced cyan fluorescent protein and citrine flank residues 149 to 881 of Epac1 (exchange proteins directly activated by cAMP) (DiPilato et al., 2004; Violin et al., 2008). The enhanced cyan fluorescent protein moiety of ICUE2 was removed by BamHI and KpnI restriction and replaced with a humanized Renilla reniformis luciferase gene that was PCR-amplified from phRluc-C1 (PerkinElmer Life and Analytical Sciences) and was engineered with BamHI and KpnI restriction sites that preserve the frame of translation. Primers for the PCR amplification of Rluc were 5'-GGATCCATGACCAGCAAGGTGTACGACC and 5'-GGTACCCCCCCTGCTCGTTCTTCAGCAC.
Cell Culture and Transfection of Cell Lines. Cells were cultured in MEM supplemented with 10% fetal bovine serum and 1:100 penicillin/streptomycin in a humidified 5% CO2 atmosphere. Transfections were carried out by the calcium phosphate precipitation method. HEK-293T cells or U2OS cells were transfected with aliquots containing 1 to 2 µg of β-arrestin2 cDNA and/or 5 µg of receptor cDNA per milliliter. Cells were plated for immunofluorescence studies into the wells of 35-mm glass-bottomed Matek dishes containing 2 ml of MEM/10% fetal bovine serum. Transfection solution (125 µl) was added for a period lasting between 6 h and overnight, after which the transfection solution was replaced with fresh serum containing media. For BRET studies, the HEK-293T cells were transfected with 5 µg of cDNA in 1 ml of transfection solution in 100-mm plastic dishes overnight and selected to permanently express the EPAC sensor using phleomycin (Zeocin; 200-400 µg/ml). These cells were transfected as mentioned above with TAAR1 for evaluation of compounds in the screening assay.
Fluorescence Microscopy. Cells were plated at a density of 2 to 4 x104/well in 35-mm Matek glass cover-slip-bottomed dishes. Anti-HA antibody was diluted 1:400. Alexa Fluor 568 goat anti-mouse antibody was used at a 1:1500 dilution. Fixed cells were first washed twice in PBS followed by incubation with 4% paraformaldehyde for 15 min at room temperature, rewashed, and then incubated for 40-min intervals in PBS with 2% bovine serum albumin with either anti-HA or, subsequently, secondary antibody using an intervening three washes with PBS between antibody treatments. Live cells were incubated with the same antibody dilutions in MEM at room temperature or 37°C. β-Arrestin translocation was assessed as described previously (Barak et al., 1997). Cells were examined with a Zeiss LSM-510 confocal microscope equipped with a 100x/numerical aperture 1.4 oil objective using excitation at 488 nm for the GFP tag and excitation at 568 nm for Alexa Fluor 568 antibody, and using the accompanying fluorescein and rhodamine emission filter sets, respectively.
Immunoblotting. HEK-293T cells were transfected with the various HA-tagged receptor subtypes and lysed the following day in a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100. Cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-HA antibody at a dilution of 1:5000.
BRET Screening Assays. HEK-293T cells permanently transfected with the EPAC sensor and transiently with the TAAR1 were split into 96-well plates at 15 to 20 x 104 cells per well. On the following day, they were washed twice with PBS, and 80 µl of PBS containing calcium and magnesium was added to each well followed by addition of 10 µl of a 50 µM coelenterazine solution (final concentration, 5 µM). After 10-min incubation, either 10 µl of vehicle or 10x concentrated solution of drug in PBS was added, and the plate was then placed into a Mithras LB940 instrument (Berthold Technologies, Bad Wildbad, Germany) that allowed the sequential integration of the luminescent signals detected in the 465 to 505 nm and 505 to 555 nm windows using filters with the appropriate band pass and by using MicroWin 2000 software (Berthold Technologies). The BRET signal is determined by calculating the ratio of the light emitted at 505 to 555 nm to the light emitted at 465 to 505 nm.
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Results |
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; Grandy, 2007; Zhuang and Matsunami, 2007). Our initial attempts at expression were also disappointing, but in the process of evaluating TAAR1 plasma membrane expression, we noted a marked absence of glycosylated bands that are common to other rhodopsin-family GPCRs. Moreover, in silico prediction suggested that the N terminus of TAAR1 should be poorly glycosylated. Therefore, in an attempt to augment TAAR1 glycosylation and the subsequent expression of the receptor at the plasma membrane (Fig. 1A), the first nine amino acids of the human β2-adrenergic receptor (β2-AR) were inserted between the HA tag and the N terminus of TAAR1, creating the HA-βTAAR1 (note that an initial attempt to augment expression using the N-terminal rhodopsin signal sequence proved unsuccessful). This nine-residue segment contains an asparagine glycosylation site at position 6 (Rands et al., 1990). Moreover, for the β2-AR, this glycosylation enhances receptor expression at the plasma membrane (Rands et al., 1990). Without the added insert, we observed for a TAAR1-GFP chimera a homogeneous intracellular GFP distribution consistent with degraded TAAR1 (Fig. 1B, top left) or receptor trapped in the endoplasmic reticulum (Fig. 1B, top right). With addition of the first nine amino acids of the human β2-adrenergic receptor, we observed a substantial accumulation, at 24 h, of what appears to be plasma membrane-localized receptor at the edge of the cells, with loss of the majority of internal staining (Fig. 1B, bottom). Immunoblotting of cell lysates for HA antibody (Fig. 1C) demonstrates extended bands for both GFP and non-GFP receptor variants characteristic of glycosylated receptor that runs above sharper-well defined bands typically characteristic of the immature receptor isoforms (Sadeghi et al., 1997; Barak et al., 2001). In addition, paraformaldehyde fixation without a permeabilization step of the HA-tagged βTAAR1 transfected cells indicates that the HA tag on the receptor is located at the external face of the plasma membrane (Fig. 1D).
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). Figure 2B shows that in nonpermeabilized live antibody-labeled cells, the C-terminal GFP distribution coincides with the HA epitope distribution, indicating that a fully formed full-length receptor is being expressed at the plasma membrane. Our ability to express βTAAR1 at the plasma membrane is not simply confined to HEK cells, in that transient labeling of the cell edge by βTAAR1 can also be demonstrated in a U2OS cell line (Fig. 2C). To demonstrate receptor functionality, we evaluated whether the βTAAR1 could undergo desensitization to known agonists using a β-arrestin2-GFP translocation assay. However, because of the relatively smaller expression levels of the βTAAR1, we first aggregated the receptors using mouse monoclonal anti-HA and Alexa Fluor 568 goat anti-mouse antibodies. As seen in Fig. 2D, the aggregated βTAAR1 in the presence of p-tyramine results in the translocation of a small amount of β-arrestin2-GFP, indicating that βTAAR1 can recruit β-arrestin2 similar to other GPCRs. However, this modest translocation resembles that of the dopamine D3 receptor, which is known to desensitize relatively poorly in a β-arrestin-dependent manner (Kim et al., 2001, 2005). In addition, the reduced translocation may also be indicative of the lesser expression of βTAAR1 relative to the β2-AR. Although the β-arrestin translocation assay can be used to measure receptor activation, evaluate both agonists, and antagonists at many receptors, in this case, the weak response precludes using the direct visualization assay for this receptor variant. Therefore, we investigated a novel cAMP signaling sensor that could function well at these smaller but still greatly improved TAAR1 expression levels.
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BRET Sensor for Measuring TAAR1 in Real Time in Live Cells. cAMP production provides sufficient amplification to measure the functional activity of TAAR1 and so far has been the only practical way to approach the pharmacological study of this very poorly expressed receptor. A significant limitation of current methods to measure cAMP in cells for compound library screening is the extended activation period necessary to accumulate enough of the second messenger product to measure in antibody mediated assays. Here we evaluated a biosensor for real time screening of cAMP production in TAAR1-transfected cells. The cAMP BRET biosensor was generated by modification of the original ICUE2 cAMP FRET biosensor (Violin et al., 2008). The sensor consists of an N-terminal truncated variant of the EPAC tagged with the R. reniformis luciferase and a yellow fluorescent protein variant (citrine) attached at the N and C termini, respectively (Fig. 3A). Upon binding cAMP, the signal of the biosensor decreases because of a conformational change that presumably increases the distance between the R. reniformis luciferase donor and the yellow fluorescent protein acceptor. The utility of the biosensor for measuring βTAAR1 cAMP signaling in real time is demonstrated in Fig. 3B, where application of 1 µM β-PEA, a TAAR1 agonist, produces a sustained reduction in the BRET signal for βTARR1 (wild-type TAAR1 did not produce changes in the BRET signal; data not shown). Moreover, the addition of IBMX to the incubation media resulted in a significant increase of the BRET signal to β-PEA (Fig. 3B) and isoproterenol (not shown), without changing the EC50 values of responses to either ligands (data not shown). The enhancement of the BRET signal with IBMX is consistent with its role as a phosphodiesterase inhibitor that prevents cAMP degradation. Figure 3C shows that, by increasing the β-PEA concentration, a concomitant increase in the βTAAR1 BRET signal occurs and that the resulting signal amplitude remains stable for at least 20 min after induction of cAMP. In contrast, in these HEK-293 cells isoproterenol stimulation of the endogenous β2-AR produces a transient change in the BRET signal that is maximal at 400 s and then returns slowly toward baseline (Fig. 3D), an indication of a desensitization of the signaling apparatus and a reduction in cAMP concentration (Violin et al., 2008).
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Taken together, the β-arrestin/βTAAR1 translocation and the cAMP BRET data indicate that the βTAAR1 has poorer affinity for β-arrestin2 than does the β2-AR. To confirm a βTAAR1/β-arrestin desensitization response by BRET, we transiently overexpressed both proteins in the same cell (Fig. 3E). In the presence of overexpressed β-arrestin2, a return to baseline (i.e., desensitization) of the β-PEA-mediated BRET signal, as well as a reduction in the magnitude of the BRET signal, is apparent after 5 min.
To demonstrate the general utility of the EPAC BRET sensor for determining cAMP concentrations, we cotransfected the sensor with the Gi-coupled D2 dopamine (DA) receptor, expecting that prestimulation of the cells by DA would dampen their cAMP responsiveness. Figure 3F demonstrates that increasing concentrations of DA diminish the magnitude of an isoproterenol-induced BRET signal toward control levels without significantly changing the time it takes the signal to desensitize.
Specificity of BRET Signaling of βTAAR1 in HEK-293 Cells. The sensitivity of the BRET EPAC assay enables measurement of the signaling of endogenous receptors (Fig. 3D). To verify the absence of TAAR1 signaling in HEK/EPAC cells not containing transfected βTAAR1 cDNA, we compared the isoproterenol and β-PEA response of non-receptor-transfected (mock) cells to cells transfected with βTAAR1 cDNA. Isoproterenol signaling was the same in both cell populations (Fig. 4A), whereas only the βTAAR1-transfected cells produced an EPAC signal significantly above baseline after β-PEA stimulation. The very small rise in the β-PEA signal in mock cells at high agonist concentrations may be due to activation of endogenous adrenergic receptors.
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; Bunzow et al., 2001; Lindemann and Hoener, 2005; Miller et al., 2005; Grandy, 2007; Wainscott et al., 2007; Wolinsky et al., 2007). The effects of amphetamine on behavior have historically been attributed to the release of internal stores of dopamine (Jones et al., 1998). However, recent observations indicated that amphetamine derivatives can also directly activate TAAR1 (Bunzow et al., 2001; Miller et al., 2005; Grandy, 2007; Reese et al., 2007; Wainscott et al., 2007; Wolinsky et al., 2007; Xie and Miller, 2007; Xie et al., 2007). Therefore, by using the EPAC cell line, the ability of these two compounds to induce cAMP production through the βTAAR1 was tested (Fig. 4B). Moreover, to confirm the results, we also measured the cAMP response in HEK cells using the standard cAMP column assay developed by Salomon et al. (1974) (Fig. 4C). The results of the column and BRET measurements are shown in Table 1. They are qualitatively similar, with the BRET assay yielding EC50 values for cAMP activation that are shifted to the left by 3- to 4-fold.
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BRET Measurement of TAAR1 for Screening. The above results indicated that the TAAR1 BRET assay should be sensitive and specific enough for general screening of TAAR1 ligands using a higher throughput format. Therefore, we measured the dose response of approximately 40 more compounds that could be potential ligands of the TAAR1 in antagonist and agonist assays. Some examples of these studies are plotted in Fig. 4D. Although among these compounds we detected no antagonist [based on their ability to counteract a β-PEA-induced BRET signal (data not shown)], we found several compounds that induced relatively potent agonist activity (Table 2) supporting previous reports on activity of these compounds at TAAR1 receptors (Bunzow et al., 2001; Miller et al., 2005; Reese et al., 2007; Wainscott et al., 2007; Wolinsky et al., 2007; Xie and Miller, 2007; Xie et al., 2007). A list of compounds that exerted no activity at βTAAR1 in the BRET assay is presented in Table 3.
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Discussion |
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; Bunzow et al., 2001; Scanlan et al., 2004; Lindemann and Hoener, 2005; Miller et al., 2005; Grandy, 2007; Reese et al., 2007; Wainscott et al., 2007; Wolinsky et al., 2007; Xie and Miller, 2007; Xie et al., 2007). Our data with HA-tagged and GFP-modified human TAAR1 provides evidence that glycosylation-stabilized expression of the receptor can occur in cells at levels sufficient for characterizing receptor biology and developing reliable in vitro cellular assays without modifying intracellular loops, developing human-rat chimeras (Lindemann and Hoener, 2005; Grandy, 2007), or coexpressing human TAAR1 with rat G
s (Wainscott et al., 2007). Furthermore, we devised and validated a novel BRET cAMP biosensor cellular assay that permits high-throughput screening for TAAR1 agonists and antagonists. Although βTAAR1 expression was 5- to 10-fold lower than what can be achieved with the β2-adrenergic receptor, we observed that an N-β2-AR peptide modified TAAR1 enables observable plasma membrane expression for which limited β-arrestin translocation occurs.
The ability of the human TAAR1 to activate adenylyl cyclase with previously identified agonists was assessed by standard methods using column chromatography and by a novel BRET method employing an EPAC biosensor. The two distinct techniques were in agreement that human TAAR1 in HEK cells responds to β-PEA and d-amphetamine, similar to what has been reported for rodent receptors or rodent modified human variants (Borowsky et al., 2001; Bunzow et al., 2001; Lindemann and Hoener, 2005; Miller et al., 2005; Reese et al., 2007; Wainscott et al., 2007; Wolinsky et al., 2007). Although the measurement of cAMP using chromatography is not well suited to high-throughput screening, we observed that the BRET method described here can be applied to large-scale screening for GPCR ligands. In fact, in our initial BRET cAMP screening, we confirmed agonistic activity at human TAAR1 of several other compounds, including the trace amines octopamine and tryptamine, the amphetamine derivatives l-amphetamine, d-methamphetamine, (+)-MDMA, and phentermine, and the catecholamine metabolites 3-MT and 4-MT (Bunzow et al., 2001; Lindemann and Hoener, 2005; Reese et al., 2007; Wainscott et al., 2007; Wolinsky et al., 2007; Xie and Miller, 2007; Xie et al., 2007). It has been noted previously that activity of these compounds at TAAR1 can differ significantly among different species (Lindemann and Hoener, 2005; Grandy, 2007; Wainscott et al., 2007). Accordingly, we observed that although these compounds were active at human TAAR1 with potencies generally similar to that observed in rodent, Rhesus monkey (Macaca mulatta) and chimeric human-rodent receptors (Bunzow et al., 2001; Lindemann and Hoener, 2005; Grandy, 2007; Wainscott et al., 2007; Wolinsky et al., 2007; Xie and Miller, 2007; Xie et al., 2007), several important differences were also noted. In particular, the most efficacious compound at human TAAR1 appeared to be l-amphetamine, followed by d-methamphetamine, with activities at least equal to or exceeding that of β-PEA. It is interesting also that trace amines octopamine and tryptamine showed partial agonist activity at human TAAR1 with potencies that were somewhat higher than those observed in previous studies with human-rat chimeric receptors (Lindemann and Hoener, 2005; Grandy, 2007; Wainscott et al., 2007). Finally, these studies also confirmed activity of catecholamine metabolites 3-MT and 4-MT at human TAAR1 (Bunzow et al., 2001; Grandy, 2007; Wainscott et al., 2007), raising an intriguing question on physiological roles that may be mediated by these metabolites via activation of TAAR1 receptors. By assessing the capability of compounds to reduce β-PEA-induced BRET signaling, we also sought to identify antagonists of TAAR1, but in a small list of compounds tested, no such activity was found. Tests of larger libraries of compounds for TAAR1 activity by using this assay are clearly warranted and are being performed currently in our lab.
It is noteworthy that the BRET method has been used for high-throughput screening of chemokine receptor CCR5 antagonists using a variant of a BRET-sensitive β-arrestin2 recruitment assay (Hamdan et al., 2005). During the course of this work, a similar BRET sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), was developed independently for characterizing cAMP synthesis stimulated via a sphingosine 1-phosphate/G13 pathway (Jiang et al., 2007). Moreover, the modified EPAC cAMP sensor, described here, has shown recently great utility to monitor dynamics of β2AR desensitization in live cells by fluorescence resonance energy transfer (Violin et al., 2008). The BRET data in our study indicate that the TAAR1-induced cellular concentration of cAMP is stable over an extended period. In contrast, under similar assay conditions, β2AR cAMP production decreases relatively rapidly presumably as a result of more pronounced receptor desensitization (Jiang et al., 2007; Violin et al., 2008). The TAAR1 behaves in this regard similarly to the dopamine D3 receptor, which also shows no discernable desensitization in HEK 293 cells (Kim et al., 2001, 2005).
In conclusion, we expressed human TAAR1 receptor in HEK cells at the plasma membrane at levels enabling biologic characterization and the development of a BRET signaling cellular assay for large-scale screening of TAAR1 ligands. Identification of selective ligands of the TAAR1 would be critical for investigating the functional role of this receptor in mammalian physiology in vivo and/or managing human disorders in which abnormalities in TA physiology may occur. The use of the BRET EPAC biosensor described here may become an effective approach to identify selective agonists and antagonists of a wide range of GPCRs in general. Compared with other available cAMP assays, the described BRET assay provides a user-friendly opportunity for real-time dynamic assessment of cAMP production rather than single-endpoint analysis of total accumulated cAMP. In addition, the strategy applied to express TAAR1 in HEK cells may be applicable to other GPCRs, such as olfactory receptors that exhibit very limited expression at the plasma membrane in model systems.
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Acknowledgements |
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Footnotes |
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L.S.B. and A.S. contributed equally to this work
ABBREVIATIONS: TA, trace amine; β-PEA, β-phenylethylamine; GPCR, G-protein coupled receptor; TAAR, trace amine-associated receptor; βTAAR1, human TAAR1 with the first nine amino acids of the human β2-AR inserted at the N terminus; h, human; BRET, bioluminescence resonance energy transfer; EPAC, exchange protein directly activated by cAMP; HA, hemagglutinin; PCR, polymerase chain reaction; HEK, human embryonic kidney; PBS, phosphate-buffered saline; MEM, minimal essential medium; GFP, green fluorescent protein; β2-AR, β2-adrenergic receptor; MDMA, methylenedioxymethamphetamine; DA, dopamine; IBMX, 3-isobutyl-1-methylxanthine; MT, methoxytyramine.
Address correspondence to: Marc G. Caron, Box 3287, Duke University, Durham, NC 27710. E-mail: m.caron{at}cellbio.duke.edu
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