Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs
© Vallender et al; licensee BioMed Central Ltd. 2010
Received: 17 September 2009
Accepted: 18 February 2010
Published: 18 February 2010
The trace amine associated receptor family is a diverse array of GPCRs that arose before the first vertebrates walked on land. Trace amine associated receptor 1 (TAAR1) is a wide spectrum aminergic receptor that acts as a modulator in brain monoaminergic systems. Other trace amine associated receptors appear to relate to environmental perception and show a birth-and-death pattern in mammals similar to olfactory receptors.
Across mammals, avians, and amphibians, the TAAR1 gene is intact and appears to be under strong purifying selection based on rates of amino acid fixation compared to neutral mutations. We have found that in dogs it has become a pseudogene. Our analyses using a comparative genetics approach revealed that the pseudogenization event predated the emergence of the Canini tribe rather than being coincident with canine domestication. By assessing the effects of the TAAR1 agonist β-phenylethylamine on [3H]dopamine uptake in canine striatal synaptosomes and comparing the degree and pattern of uptake inhibition to that seen in other mammals, including TAAR1 knockout mice, wild type mice and rhesus monkey, we found that the TAAR1 pseudogenization event resulted in an uncompensated loss of function.
The gene family has seen expansions among certain mammals, notably rodents, and reductions in others, including primates. By placing the trace amine associated receptors in an evolutionary context we can better understand their function and their potential associations with behavior and neurological disease.
Trace amine associated receptors (TAARs) are a family of G-protein coupled receptors that originated prior to the emergence of jawed vertebrates . The most widely studied of these receptors is TAAR1, which has been shown to bind a wide spectrum of biogenic amines and psychoactive compounds [2, 3] (Additional file 1) and is a known modulator of monoaminergic activity . Trace amines themselves have proven elusive to understand; the only receptors that they have been found to bind to are TAAR1 [2, 3, 5, 6] and TAAR4 (in rat only) , though they also appear to be substrates at various monoamine transporters and catabolic enzymes [5–7].
TAAR1 expression in brain is observed in a variety of species including human , rhesus macaque , mouse , and rat  with its distribution widespread. Notably, expression overlaps with regions important in brain monoaminergic function and co-expression of TAAR1 and the dopamine transporter (DAT) has been observed in dopaminergic neurons . Studies replacing TAAR1 with LacZ in knock-out mice support these findings with the staining of brain sections in these mice displaying LacZ expression throughout dopaminergic and serotonergic regions . These findings, coupled with a dysregulation of trace amines in psychiatric disease , have made understanding the function of this gene particularly relevant.
Though the TAAR gene family is present in some form in all jawed vertebrates, the number of genes observed in any given species varies considerably . While the placental mammalian ancestor is thought to have harbored nine distinct TAAR genes, even within this relatively recent clade there has been significant gene gain and loss; mouse, rat, and cow have added to the repertoire, while primates and dog have seen losses [1, 11, 12]. Functional work on members of the TAAR gene family other than TAAR1 is sparse, but what has been done suggests that other TAAR gene products do not bind the traditional TAAR1 ligands (e.g., β-phenylethylamine (β-PEA)) [2, 13], but instead show a distinct ligand set related to environmental perception . Expression studies fail to find broad expression of these other TAAR family members in the brain but rather observe localization predominantly in the olfactory apparatus [1, 14].
This functional dichotomy between TAAR1 and its cousins is significant. While other members of the TAAR gene family have seen recurrent pseudogenization and duplication, TAAR1 has been evolutionarily stable. TAAR1 was the first to arise and remains the only TAAR gene present in every species studied with the possible exception of the neotelost fish who nevertheless harbors another, evolutionarily similar, TAAR1 cousin [1, 15]. Yet, despite this conservation, TAAR1 shows sequence divergence across species and species-specific pharmacological profiles with drug potency (EC50) differences of 10-fold or more common [1, 11, 16, 17]. While it remains unclear what practical effect these differences have in vivo, it is noteworthy that this variation exists, despite the conservation of the gene itself and its unique evolutionary history.
In order to better understand the evolutionary context in which this receptor family evolved and to better grasp its likely functional significance we have explored the relationship of various TAAR homologs in twenty mammals, a marsupial, a monotreme and an outgroup avian. We have characterized the gain and loss of TAAR homologs among mammals and performed evolutionary analyses to better understand the selective constraints under which they operate. We have also more fully investigated the evolutionary history of TAAR1 with a focus on the carnivores and the functional implications of a loss of TAAR1 in dogs.
Results and Discussion
Initially we gathered all reported TAAR homologs from eleven species for whom annotated genomic sequence was available (Mus musculus, mouse; Rattus norvegicus, rat; Pan troglodytes; chimpanzee; Homo sapiens, human; Bos taurus, cow; Sus scrofa, pig; Equus caballus, horse;Canis familiaris, dog; Monodelphis domesticus, opossum; Ornithorhynchus anatinus, platypus; and Gallus gallus, chicken. We also amplified and sequenced TAAR homologs from three new world monkey species (Callithrix jacchus, common marmoset; Saguinus oedipus, cotton-top tamarin; and Saimiri sciureus, squirrel monkey), sequence confirmed the genomic data available for rhesus macaque, Macaca mulatta, and identified putative orangutan (Pongo pygmaeus) homologs through trans-alignments with the as yet unannotated genomic sequence.
We also considered the relative rate of amino acid fixation (dN/dS) among the lineages. Without exception, all lineages showed dN/dS values less than one indicative of negative, purifying, selection (see Additional file 3). Because of the observed differences between ligands and expression patterns associated with TAAR1 as compared to other family members, we performed a branches test to identify if there was a significant difference between the dN/dS values among TAAR1 genes compared to other family members. Interestingly there was a significant difference (p < 0.001) but with TAAR1 showing an elevated, though still purifying, dN/dS of 0.256 compared to the remainder of the family at a dN/dS of 0.164. We compared this to the TAAR5 clade which also is robustly conserved without pseudogenization. TAAR5 is evolutionarily statistically indistinguishable from other TAAR family members.
While TAAR1 is inactivated in dogs, it is not certain that its function has been lost. While analysis of the dog genome failed to identify a specific recent duplication event of TAAR1, it did identify two other TAAR family members with intact coding regions and so it is conceivable, if unlikely, that a compensatory functional change may have appeared. It is also notable that the dog genome, in its current state, remains incomplete. While the TAAR1 genomic region maps to a chromosome with flanking genes (VNN1 upstream and STX7 downstream) identical to the genes that flank the TAAR regions in other species, TAAR2, TAAR4, and TAAR5 map to a contig that has not been assembled onto a chromosome. TAAR3, TAAR6, TAAR7, TAAR8, and TAAR9 homologs are not readily apparent in the dog genome though several large gaps exist in the region downstream of the TAAR2/4/5 cluster where TAAR6/7/8/9 would be expected. In all the dog genome assemblies, the TAAR locus is far from finished quality. Because of this we sought to determine whether TAAR1 functionality, if not TAAR1 itself, could be observed in dogs.
Two groups have separately created TAAR1 knockout mice and not only are these mice fully viable, but the mice show no gross physical or behavioral abnormalities and identification of any non-pharmacologically induced phenotype has not been forthcoming [9, 22]. Yet the conservation of the gene for the last 450 million years across numerous species belies an importance. Concurrent with a role as a monoaminergic modulator, knock-out mice show changes in their dopaminergic system compared to wild types including increased high-affinity dopamine receptor D2 and higher neuronal firing rates in the ventral tegmental area [9, 22], which is important in brain reward circuitry . It may be noteworthy then that while the D2 receptor antagonist pimozide does not change the self-administration of β-PEA in dogs , it does block β-PEA induced locomotion in mice . Further, while the self-administration of β-PEA in dogs is unaffected by the D2 antagonist, it increases self-administration of amphetamine both in dogs  and rats .
Our understanding of neuroaminergic signaling has recently become more complicated with the emergence of the TAAR gene family. These genes represent a heretofore unexplored mechanism of neuromodulation. They have gained a further importance with findings suggesting an association with schizophrenia [27, 28]. In these studies we catalog the TAAR family members across mammals, demonstrating a pattern of pseudogenization and duplication among most of the family members. In primates particularly, only two members, TAAR1 and TAAR5, are completely conserved, suggesting perhaps a greater importance for these genes. Yet despite this evolutionary flux there seems to be a strong purifying selection across all intact genes.
Both because of its conservation as well as the fact that only it binds neurotransmitters, TAAR1 has proven particularly interesting. Indeed TAAR1 knock-out mouse may show some of the same endophenotypes as schizophrenic patients  and have been touted as a model system. Yet here we demonstrate that there exist naturally occurring TAAR1 knockouts: dogs and their close relatives. Among these species there does not appear to be an intact open reading frame and ex vivo studies show effects similar to those seen in TAAR1 knockout mice. Yet it remains unclear what, if any effects, this loss of function has had. While it is tantalizing to speculate that many behavioral characteristics of dogs, including their relative ease of training, may involve reward encoding and perception, and suggest that this is related to TAAR1 effects on the dopamine system, it remains unproven. Indeed, any association between dog behavior and TAAR1 functional loss must also account for the other Canini species.
That dogs and their brethren can well tolerate the loss of TAAR1 despite its evolutionary conservation must provoke questions as to the relevance of the gene to schizophrenia or other mental health diseases. While it has been suggested that perhaps another member of the trace amine associated receptor family is contributory to the schizophrenic phenotype , the widespread loss of TAAR genes suggests an evolutionary lability that seems contraindicative. Rather the ligand binding studies suggest a dichotomy between TAAR1 and the remainder of the family that is broadly supported by the observed differences in gene phylogenies. The suggestion from rodents that TAAR family members other than TAAR1 are related to chemosensory perception  is bolstered by these findings as well. The patterns of species-specific duplications and pseudogenization events are reminiscent of that seen for olfactory genes .
This work demonstrates the strengths of applying evolutionary studies to functional analyses. From the phylogenies of the TAAR genes, TAAR1 appears qualitatively different in its pattern of gene gain and loss. This coupled with our knowledge of TAAR ligand binding suggests a dichotomy of function among the receptors. At the same time the tolerance of loss of TAAR1 in dogs offers a warning for association studies with disease; the subtleties of effect must be considered. Understanding the paradox of evolutionarily ancient conservation and an accommodation of recent loss will shed significant light on neuroaminergic biology and the genetic basis underlying brain and behavior.
TAAR1 was amplified from genomic DNA by trial-and-error using primers designed against the dog genome as well as conserved regions between the dog and cat genomes. Following verification of product size and specificity on a 1.5% agarose gel, the PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's protocol. Sequencing was performed using the CEQ8000 Genetic Analysis System and CEQ DTSC Quick Start Kit (Beckman Coulter). For all species under study, both strands were sequenced and analyzed using Vector NTI (Invitrogen).
TAAR1 knockout mice and wild type mice were derived from six pairs of heterozygous mice given to us as a gift by Lundbeck Research USA, Inc. All procedures were conducted in accordance with the Animal Experimentation Protocol approved by the Harvard Medical Area Standing Committee on Animals. Fresh post-mortem brain tissue from mongrel dog sacrificed for unrelated purposes was obtained with the help of M. Contreras, N. McDannold, N. Vykhodtseva and Y. Zhang at the Harvard Medical School. TAAR1 knockout and wild-type mouse brain tissue was collected from our mouse colony at the New England Primate Research Center (Southborough, MA). Dog striatum offered enough tissue to allow preparation from a single animal while mouse tissues were pooled. All procedures were conducted in accordance with the Animal Experimentation Protocol approved by the Harvard Medical Area Standing Committee on Animals. Fresh tissues were homogenized in 1.5-ml Eppendorf centrifuge tubes with 10× volume of ice-cold unbuffered 0.32 M sucrose solution (pH 7.0), using a motor-driven pellet pestle. The homogenate was centrifuged (1000 g, 10 min at 4°C) to yield a crude nuclear pellet and low-speed supernatant. The low-speed supernatant fraction was carefully transferred into another fresh tube and centrifuged at 10,000 g and 4°C for 20 min to yield a synaptosome-containing pellet. The resulting pellet was resuspended in an appropriate volume of ice-cold uptake buffer (a modified Krebs buffer: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1 μM pargyline, 2 mg/ml glucose, and 0.2 mg/ml ascorbic acid, pH 7.5) for further assays.
50 uL of the synaptosome preparation was added into 1.5-ml Eppendorf centrifuge tubes and exposed to [3H]dopamine (10 nM; 60 Ci/mmol; PerkinElmer Life and Analytical Sciences) only or combined with 100 nM β-PEA at 25°C in uptake buffer for various times as indicated. The uptake by the synaptosomes at 30 min in [3H]dopamine only was taken as maximal uptake (100%). Non-specific uptake was defined in the presence of 10 μM methylphenidate. Uptake reactions were terminated by addition of 1 ml of ice-cold uptake buffer into the tubes and immediate centrifugation at 1,000 g (for cells) or 10,000 g (for synaptosomes) at 4°C for 3 min. The resulting pellets were rinsed twice with 1 ml of ice-cold uptake buffer and then incubated in 1× PLB buffer for 30 min on a shaking platform at 200 rpm, prior to being transferred into scintillation vials containing 4 ml of Beckman ReadySafe scintillation cocktail and counted on a Beckman LS6000IC scintillation spectrophotometer for 1 min/sample.
For studies of intact genes, nucleotide sequences were aligned in-frame using ClustalW, while alignments including pseudogenes were made directly from nucleotide sequences. Trees were generated with RAxML [19, 31, 32] using a maximum likelihood inference and validation with 1000 bootstrap analyses. CAFE was used to attempt to model the birth and death processes in primates with a maximum likelihood value of λ, probability of gene birth or death per million years, calculated as 0.0141. However with existing data the distribution of pseudogenes was not significantly different than expected. dN/dS (KA/KS) values for protein-coding sequences were calculated using PAML (using model 1, free ratio, in codeml) as well as the Li et al.  and Yang and Neilsen  methods with no significant differences observed.
PAML was used to test for branch-specific evolution in codeml. The parameters are shown below in Additional file 4. Specifically we tested the hypotheses that either the TAAR1 clade or TAAR5 clade of Figure 1 had a significantly different dN/dS ratio than the remainder of the tree. Significance values were obtained by comparing the likelihood ratio statistics (2 Δℓ) to a with degrees of freedom equal to the difference between number of parameters in the model. PAML was also used to test site-specific evolution. Parameters for site-specific evolution are shown in Additional file 5. The hypothesis tested whether there were any sites under positive selection in TAAR1. Significance was calculated as above.
The authors would like to thank R.K. Wayne, O.A. Ryder, M.A. Contreras, and N. McDannold for tissue and/or genomic DNA. This work was supported by NIH grants MH082507 (to E.J.V.), MH077995 (to G.M.M.), DA022323 (to G.M.M.), DA016606 (to G.M.M.), AA016194 (to G.M.M.), DA025697 (to G.M.M.), DA025802 (to G.M.M.) and RR00168.
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