The molecular basis of species-specific ligand activation of trace amine-associated receptor 1 (TAAR(1)).
ABSTRACT: The trace amine-associated receptor 1 (TAAR(1)) is an aminergic G protein-coupled receptor (GPCR) potently activated by 3-iodothyronamine (1), an endogenous derivative of thyroid hormone. Structure-activity relationship studies on 1 and related agonists showed that the rat and mouse species of TAAR(1) accommodated structural modifications and functional groups on the ethylamine portion and the biaryl ether moiety of the molecule. However, the two receptors clearly exhibited distinct, species-specific ligand preferences despite being remarkably similar with 93% sequence similarity. In this study, we generated single and double mutants of rat and mouse TAAR(1) to probe the molecular recognition of agonists and the underlying basis for the ligand selectivity of rat and mouse TAAR(1). Key, nonconserved specificity determinant residues in transmembranes helices 4 and 7 within the ligand binding site appear to be the primary source of a number of the observed ligand preferences. Residue 7.39 in transmembrane 7 dictated the preference for a beta-phenyl ring, while residue 4.56 in transmembrane 4 was partially responsible for the lower potency of 1 and tyramine for the mouse receptor. Additionally, 1 and tyramine were found to have the same binding mode in rat TAAR(1) despite structure-activity relationship data suggesting the possibility of each molecule having different binding orientations. These findings provide valuable insights into the critical binding site residues involved in the ligand-receptor interaction that can influence compound selectivity and functional activity of aminergic GPCRs.
Project description:Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous ?-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular ?-adrenergic receptor subtypes.
Project description:AIMS:The trace amine-associated receptor (Taar) family displays high species- and subtype-specific pharmacology. Several trace amines such as ?-phenylethylamine (?-PEA), p-tyramine and tryptamine are agonists at TA(1) but poorly activate rat and mouse Taar4. PRINCIPAL RESULTS:Using rat TA(1) and Taar4 chimera, we identified determinants in transmembrane helices 3 and 6, which, when replaced by the corresponding portion of rat TA(1) , can rescue cell surface expression of rat Taar4. When expressed at the cell surface, rat Taar4 pharmacology was very similar to that of TA(1) and coupled to the G?(s) -protein/AC pathway. Our data suggest that binding pockets of Taar for surrogate agonists overlap between paralogs. CONCLUSIONS:This implicates that the repertoire of Taar ensures functional redundancy, tissue- and cell-specific expression and/or different downstream signalling rather than different agonist specificity.
Project description:Classical biogenic amines (adrenaline, noradrenaline, dopamine, serotonin and histamine) interact with specific families of G protein-coupled receptors (GPCRs). The term 'trace amines' is used when referring to p-tyramine, beta-phenylethylamine, tryptamine and octopamine, compounds that are present in mammalian tissues at very low (nanomolar) concentrations. The pharmacological effects of trace amines are usually attributed to their interference with the aminergic pathways, but in 2001 a new gene was identified, that codes for a GPCR responding to p-tyramine and beta-phenylethylamine but not to classical biogenic amines. Several closely related genes were subsequently identified and designated as the trace amine-associated receptors (TAARs). Pharmacological investigations in vitro show that many TAAR subtypes may not respond to p-tyramine, beta-phenylethylamine, tryptamine or octopamine, suggesting the existence of additional endogenous ligands. A novel endogenous thyroid hormone derivative, 3-iodothyronamine, has been found to interact with TAAR1 and possibly other TAAR subtypes. In vivo, micromolar concentrations of 3-iodothyronamine determine functional effects which are opposite to those produced on a longer time scale by thyroid hormones, including reduction in body temperature and decrease in cardiac contractility. Expression of all TAAR subtypes except TAAR1 has been reported in mouse olfactory epithelium, and several volatile amines were shown to interact with specific TAAR subtypes. In addition, there is evidence that TAAR1 is targeted by amphetamines and other psychotropic agents, while genetic linkage studies show a significant association between the TAAR gene family locus and susceptibility to schizophrenia or bipolar affective disorder.
Project description:The trace amine-associated receptor 1 (TAAR(1)) is a biogenic amine G protein-coupled receptor (GPCR) that is potently activated by 3-iodothyronamine (1, T(1)AM) in vitro. Compound 1 is an endogenous derivative of the thyroid hormone thyroxine which rapidly induces hypothermia, anergia, and bradycardia when administered to mice. To explore the role of TAAR(1) in mediating the effects of 1, we rationally designed and synthesized rat TAAR(1) superagonists and lead antagonists using the rotamer toggle switch model of aminergic GPCR activation. The functional activity of a ligand is proposed to be correlated to its probable interactions with the rotamer switch residues; agonists allow the rotamer switch residues to toggle to their active conformation, whereas antagonists interfere with this conformational transition. These agonist and antagonist design principles provide a conceptual model for understanding the relationship between the molecular structure of a drug and its pharmacological properties.
Project description:Trace amine-associated receptors (TAARs) in mammals recently have been shown to function as olfactory receptors. We have delineated the taar gene family in jawless, cartilaginous, and bony fish (zero, 2, and >100 genes, respectively). We conclude that taar genes are evolutionary much younger than the related OR and ORA/V1R olfactory receptor families, which are present already in lamprey, a jawless vertebrate. The 2 cartilaginous fish genes appear to be ancestral for 2 taar classes, each with mammalian and bony fish (teleost) representatives. Unexpectedly, a whole new clade, class III, of taar genes originated even later, within the teleost lineage. Taar genes from all 3 classes are expressed in subsets of zebrafish olfactory receptor neurons, supporting their function as olfactory receptors. The highly conserved TAAR1 (shark, mammalian, and teleost orthologs) is not expressed in the olfactory epithelium and may constitute the sole remnant of a primordial, nonolfactory function of this family. Class III comprises three-fourths of all teleost taar genes and is characterized by the complete loss of the aminergic ligand-binding motif, stringently conserved in the other 2 classes. Two independent intron gains in class III taar genes represent extraordinary evolutionary dynamics, considering the virtual absence of intron gains during vertebrate evolution. The d(N)/d(S) analysis suggests both minimal global negative selection and an unparalleled degree of local positive selection as another hallmark of class III genes. The accelerated evolution of class III teleost taar genes conceivably might mark the birth of another olfactory receptor gene family.
Project description:Trace amine-associated receptors (TAARs) are a member of the G-protein-coupled receptor superfamily and are known to be expressed in olfactory sensory neurons. A limited number of molecular evolutionary studies have been done for TAARs so far. To elucidate how lineage-specific evolution contributed to their functional divergence, we examined 30 metazoan genomes. In total, 493 TAAR gene candidates (including 84 pseudogenes) were identified from 26 vertebrate genomes. TAARs were not identified from non-vertebrate genomes. An ancestral-type TAAR-like gene appeared to have emerged in lamprey. We found four therian-specific TAAR subfamilies (one eutherian-specific and three metatherian-specific) in addition to previously known nine subfamilies. Many species-specific TAAR gene duplications and losses contributed to a large variation of TAAR gene numbers among mammals, ranging from 0 in dolphin to 26 in flying fox. TAARs are classified into two groups based on binding preferences for primary or tertiary amines as well as their sequence similarities. Primary amine-detecting TAARs (TAAR1-4) have emerged earlier, generally have single-copy orthologs (very few duplication or loss), and have evolved under strong functional constraints. In contrast, tertiary amine-detecting TAARs (TAAR5-9) have emerged more recently and the majority of them experienced higher rates of gene duplications. Protein members that belong to the tertiary amine-detecting TAAR group also showed the patterns of positive selection especially in the area surrounding the ligand-binding pocket, which could have affected ligand-binding activities and specificities. Expansions of the tertiary amine-detecting TAAR gene family may have played important roles in terrestrial adaptations of therian mammals. Molecular evolution of the TAAR gene family appears to be governed by a complex, species-specific, interplay between environmental and evolutionary factors.
Project description:BACKGROUND AND PURPOSE: Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. EXPERIMENTAL APPROACH: In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. KEY RESULTS: Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. CONCLUSIONS AND IMPLICATIONS: We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.
Project description:Trace amine-associated receptors (TAARs) are vertebrate olfactory receptors. However, ligand recognition properties of TAARs remain poorly understood, as most are "orphan receptors" without known agonists. Here, we identify the first ligands for many rodent TAARs and classify these receptors into two subfamilies based on the phylogeny and binding preference for primary or tertiary amines. Some mouse and rat orthologs have similar response profiles, although independent Taar7 gene expansions led to highly related receptors with altered ligand specificities. Using chimeric TAAR7 receptors, we identified an odor contact site in transmembrane helix III that functions as a selectivity filter. Homology models based on the ?(2) adrenergic receptor structure indicate spatial proximity of this site to the ligand. Gain-of-function mutations at this site created olfactory receptors with radically altered odor recognition properties. These studies provide new TAAR ligands, valuable tools for studying receptor function, and general insights into the molecular pharmacology of G protein-coupled receptors.
Project description:Biogenic amines regulate a variety of behaviors. Their functions are predominantly mediated through G-protein-coupled 7-transmembrane domain receptors (GPCR), 16 of which are predicted to exist in the genome sequence of the nematode Caenorhabditis elegans. We describe here the expression pattern of several of these aminergic receptors, including two serotonin receptors (ser-1 and ser-4), one tyramine receptor (ser-2), and two dopamine receptors (dop-1 and dop-2). Moreover, we describe distinct but partially overlapping expression patterns of different splice forms of the ser-2 tyramine receptor locus. We find that each of the aminergic receptor genes is expressed in restricted regions of the nervous system and that many of them reveal significant overlap with the expression of regulatory factors of the LIM homeobox (Lhx) gene family. We demonstrate that the expression of several of the biogenic amine receptors is abrogated in specific cell types in Lhx gene mutants, thus establishing a role for these Lhx genes in regulating aspects of neurotransmission. We extend these findings with other cell fate markers and show that the lim-4 Lhx gene is required for several but not all aspects of RID motor neuron differentiation and that the lim-6 Lhx gene is required for specific aspects of RIS interneuron differentiation. We also use aminergic receptor gfp reporter fusions as tools to visualize the anatomy of specific neurons in Lhx mutant backgrounds and find that the development of the elaborate dendritic branching pattern of the PVD harsh touch sensory neuron requires the mec-3 Lhx gene. Lastly, we analyze a mutant allele of the ser-2 tyramine receptor, a target of the ttx-3 Lhx gene in the AIY interneuron class. ser-2 mutants display none of the defects previously shown to be associated with loss of AIY function.
Project description:There is strong evidence that olfaction plays a key role in the homing of salmonids to their natal spawning grounds, particularly in the freshwater phase. However, the physiological and genetic mechanisms behind this biological phenomenon are largely unknown. It has been shown that Pacific salmon respond to dissolved free amino acids from their natal streams. This indicates that amino acids comprise part of the olfcatory cues for imprinting and homing in salmonids. As trace amine-associated receptors (TAARs), a class of olfactory receptors that are close relatives of the G protein-coupled aminergic neurotransmitter receptors, recognize amino acid metabolites, we hypothesize that TAARs play an important role in salmon homing by recognizing olfactory cues. Therefore, to better understand homing in Atlantic salmon, we set out to characterize the TAAR genes in this species. We searched the first assembly of the Atlantic salmon genome for sequences resembling TAARs previously characterized in other teleosts. We identified 27 putatively functional TAAR genes and 25 putative TAAR pseudogenes, which cluster primarily on chromosome 21 (Ssa21). Phylogenetic analysis of TAAR amino acid sequences from 15 vertebrate species revealed the TAAR gene family arose after the divergence of jawed and jawless vertebrates. The TAARs group into three classes with salmon possessing class I and class III TAARs. Within each class, evolution is characterized by species-specific gene expansions, which is in contrast to what is observed in other olfactory receptor families (e.g., OlfCs and oras).