Convergent olfactory trace amine-associated receptors detect biogenic polyamines with distinct motifs via a conserved binding site

Biogenic amines activate G-protein-coupled receptors (GPCRs) in the central nervous system in vertebrate animals. Several biogenic amines, when excreted, stimulate trace amine-associated receptors (TAARs), a group of GPCRs in the main olfactory epithelium, and elicit innate behaviors. How TAARs recognize amines with varying numbers of amino groups is largely unknown. We reasoned that a comparison between lamprey and mammalian olfactory TAARs, which are thought to have evolved independently but show convergent responses to polyamines, may reveal structural determinants of amine recognition. Here, we demonstrate that sea lamprey TAAR365 (sTAAR365) responds strongly to biogenic polyamines cadaverine, putrescine, and spermine, and shares a similar response profile as a mammalian TAAR (mTAAR9). Docking and site-directed mutagenesis analyses show that both sTAAR365 and mTAAR9 recognize the two amino groups of cadaverine with the conserved Asp3.32 and Tyr6.51 residues. sTAAR365, which has remarkable sensitivity for cadaverine (EC50 = 4 nM), uses an extra residue, Thr7.42, to stabilize ligand binding. These cadaverine recognition sites also interact with amines with four and three amino groups (spermine and spermidine, respectively). Glu7.36 of sTAAR365 cooperates with Asp3.32 and Thr7.42 to recognize spermine, whereas mTAAR9 recognizes spermidine through an additional aromatic residue, Tyr7.43. These results suggest a conserved mechanism whereby independently evolved TAAR receptors recognize amines with two, three, or four amino groups using the same recognition sites, at which sTAAR365 and mTAAR9 evolved distinct motifs. These motifs interact directly with the amino groups of the polyamines, a class of potent and ecologically important odorants, mediating olfactory signaling. Biogenic amines activate G-protein-coupled receptors (GPCRs) in the central nervous system in vertebrate animals. Several biogenic amines, when excreted, stimulate trace amine-associated receptors (TAARs), a group of GPCRs in the main olfactory epithelium, and elicit innate behaviors. How TAARs recognize amines with varying numbers of amino groups is largely unknown. We reasoned that a comparison between lamprey and mammalian olfactory TAARs, which are thought to have evolved independently but show convergent responses to polyamines, may reveal structural determinants of amine recognition. Here, we demonstrate that sea lamprey TAAR365 (sTAAR365) responds strongly to biogenic polyamines cadaverine, putrescine, and spermine, and shares a similar response profile as a mammalian TAAR (mTAAR9). Docking and site-directed mutagenesis analyses show that both sTAAR365 and mTAAR9 recognize the two amino groups of cadaverine with the conserved Asp3.32 and Tyr6.51 residues. sTAAR365, which has remarkable sensitivity for cadaverine (EC50 = 4 nM), uses an extra residue, Thr7.42, to stabilize ligand binding. These cadaverine recognition sites also interact with amines with four and three amino groups (spermine and spermidine, respectively). Glu7.36 of sTAAR365 cooperates with Asp3.32 and Thr7.42 to recognize spermine, whereas mTAAR9 recognizes spermidine through an additional aromatic residue, Tyr7.43. These results suggest a conserved mechanism whereby independently evolved TAAR receptors recognize amines with two, three, or four amino groups using the same recognition sites, at which sTAAR365 and mTAAR9 evolved distinct motifs. These motifs interact directly with the amino groups of the polyamines, a class of potent and ecologically important odorants, mediating olfactory signaling. Biogenic amines are a group of signaling molecules that activate G-protein-coupled receptors (GPCRs) and regulate a wide variety of neurophysiologic and behavioral functions. Recognition of amine neurotransmitters, which are often monoamines that activate the aminergic family of GPCRs in vertebrate central nervous systems, has been examined extensively (1Kandel E.R. Schwartz J.H. Jessell T.M. Siegelbaum S.A. Hudspeth A.J. Principles of Neural Science. McGraw-Hill Medical, New York, NY2000Google Scholar, 2Liu Y. Zhao J. Guo W. Emotional roles of mono-aminergic neurotransmitters in major depressive disorder and anxiety disorders.Front. Psychol. 2018; 9: 2201Crossref PubMed Scopus (57) Google Scholar, 3Liu Y. Zhao J. Fan X. Guo W. Dysfunction in serotonergic and noradrenergic systems and somatic symptoms in psychiatric disorders.Front. Psychiatry. 2019; 10: 286Crossref PubMed Scopus (20) Google Scholar). Besides, some excreted biogenic amines function as odorants and are detected by another family of GPCRs, the olfactory trace amine-associated receptors (TAARs) (4Andersen G. Marcinek P. Sulzinger N. Schieberle P. Krautwurst D. Food sources and biomolecular targets of tyramine.Nutr. Rev. 2019; 77: 107-115Crossref PubMed Scopus (16) Google Scholar, 5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar, 6Liberles S.D. Buck L.B. A second class of chemosensory receptors in the olfactory epithelium.Nature. 2006; 442: 645-650Crossref PubMed Scopus (541) Google Scholar, 7Ferrero D.M. Lemon J.K. Fluegge D. Pashkovski S.L. Korzan W.J. Datta S.R. Spehr M. Fendt M. Liberles S.D. Detection and avoidance of a carnivore odor by prey.Proc. Natl. Acad. Sci. U. S. 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To date, the structural basis of a TAAR receptor recognizes amines with one or two amino groups having been examined (11Sharma K. Ahuja G. Hussain A. Balfanz S. Baumann A. Korsching S.I. Elimination of a ligand gating site generates a supersensitive olfactory receptor.Sci. Rep. 2016; 6: 28359Crossref PubMed Scopus (9) Google Scholar, 12Ferrero D.M. Wacker D. Roque M.A. Baldwin M.W. Stevens R.C. Liberles S.D. Agonists for 13 trace amine-associated receptors provide insight into the molecular basis of odor selectivity.ACS Chem. Biol. 2012; 7: 1184-1189Crossref PubMed Scopus (66) Google Scholar, 13Li Q. Tachie-Baffour Y. Liu Z. Baldwin M.W. Kruse A.C. Liberles S.D. Non-classical amine recognition evolved in a large clade of olfactory receptors.Elife. 2015; 4e10441Crossref PubMed Scopus (32) Google Scholar). However, how TAAR receptors recognize polyamines with three or four amino groups has not been determined. Thus, exploring the mechanism whereby TAARs respond to polyamines with two, three, or four amino groups will complete the story on how biogenic amines with one through four amino groups are recognized by GPCRs. Odorous polyamines are found in natural excretions (urine, feces, and semen), decomposed tissues, and food sources, and can elicit significant physiological changes and behavioral responses in various species examined (4Andersen G. Marcinek P. Sulzinger N. Schieberle P. Krautwurst D. Food sources and biomolecular targets of tyramine.Nutr. Rev. 2019; 77: 107-115Crossref PubMed Scopus (16) Google Scholar, 5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar, 6Liberles S.D. Buck L.B. A second class of chemosensory receptors in the olfactory epithelium.Nature. 2006; 442: 645-650Crossref PubMed Scopus (541) Google Scholar, 7Ferrero D.M. Lemon J.K. Fluegge D. Pashkovski S.L. Korzan W.J. Datta S.R. Spehr M. Fendt M. Liberles S.D. Detection and avoidance of a carnivore odor by prey.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 11235-11240Crossref PubMed Scopus (246) Google Scholar, 8Ferrero D.M. Liberles S.D. The secret codes of mammalian scents.Wiley Interdiscip. Rev. Syst. Biol. Med. 2010; 2: 23-33Crossref PubMed Scopus (39) Google Scholar, 9Hussain A. Saraiva L.R. Ferrero D.M. Ahuja G. Krishna V.S. Liberles S.D. Korsching S.I. High-affinity olfactory receptor for the death-associated odor cadaverine.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 19579-19584Crossref PubMed Scopus (128) Google Scholar, 10Muñoz-Esparza N.C. Latorre-Moratalla M.L. Comas-Basté O. Toro-Funes N. Veciana-Nogués M.T. Vidal-Carou M.C. Polyamines in food.Front. Nutr. 2019; 6: 108Crossref PubMed Scopus (76) Google Scholar, 11Sharma K. Ahuja G. Hussain A. Balfanz S. Baumann A. Korsching S.I. Elimination of a ligand gating site generates a supersensitive olfactory receptor.Sci. Rep. 2016; 6: 28359Crossref PubMed Scopus (9) Google Scholar, 12Ferrero D.M. Wacker D. Roque M.A. Baldwin M.W. Stevens R.C. Liberles S.D. Agonists for 13 trace amine-associated receptors provide insight into the molecular basis of odor selectivity.ACS Chem. Biol. 2012; 7: 1184-1189Crossref PubMed Scopus (66) Google Scholar, 13Li Q. Tachie-Baffour Y. Liu Z. Baldwin M.W. Kruse A.C. Liberles S.D. Non-classical amine recognition evolved in a large clade of olfactory receptors.Elife. 2015; 4e10441Crossref PubMed Scopus (32) Google Scholar, 14Li Q. Liberles S.D. Aversion and attraction through olfaction.Curr. Biol. 2015; 25: R120-R129Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 15Li Q. Korzan W.J. Ferrero D.M. Chang R.B. Roy D.S. Buchi M. Lemon J.K. Kaur A.W. Stowers L. Fendt M. Liberles S.D. Synchronous evolution of an odor biosynthesis pathway and behavioral response.Curr. Biol. 2013; 23: 11-20Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Cadaverine and putrescine, the foul-smelling diamines produced by microbial metabolism of putrefied animal tissue, repel zebrafish by activating an olfactory TAAR receptor (zTAAR13c) (9Hussain A. Saraiva L.R. Ferrero D.M. Ahuja G. Krishna V.S. Liberles S.D. Korsching S.I. High-affinity olfactory receptor for the death-associated odor cadaverine.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 19579-19584Crossref PubMed Scopus (128) Google Scholar). Similarly, cadaverine activates an olfactory TAAR receptor in mouse (mTAAR9) and elicits either neutral or aversive behavioral responses, depending on the particular behavioral paradigm (16Saraiva L.R. Kondoh K. Ye X. Yoon K.H. Hernandez M. Buck L.B. Combinatorial effects of odorants on mouse behavior.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E3300-E3306Crossref PubMed Scopus (76) Google Scholar, 17Dewan A. Pacifico R. Zhan R. Rinberg D. Bozza T. Non-redundant coding of aversive odours in the main olfactory pathway.Nature. 2013; 497: 486-489Crossref PubMed Scopus (134) Google Scholar). In contrast, putrescine is attractive to mice, although the cognate receptor or receptors have not been identified (16Saraiva L.R. Kondoh K. Ye X. Yoon K.H. Hernandez M. Buck L.B. Combinatorial effects of odorants on mouse behavior.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E3300-E3306Crossref PubMed Scopus (76) Google Scholar). Also, both cadaverine and putrescine can elicit feeding behaviors in rat and goldfish (18Heale V.R. Petersen K. Vanderwolf C.H. Effect of colchicine-induced cell loss in the dentate gyrus and Ammon's horn on the olfactory control of feeding in rats.Brain Res. 1996; 712: 213-220Crossref PubMed Scopus (17) Google Scholar, 19Rolen S.H. Sorensen P.W. Mattson D. Caprio J. Polyamines as olfactory stimuli in the goldfish Carassius auratus.J. Exp. Biol. 2003; 206: 1683-1696Crossref PubMed Scopus (84) Google Scholar). In addition, spermine, an abundant polyamine in the semen of male sea lamprey, acts as a male sex pheromone that specifically attracts ovulated females (5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar). A sea lamprey TAAR receptor, sTAAR348, is proposed to play a key role in mediating the pheromone function of spermine (5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar). Likewise, spermine and spermidine (a biosynthetic precursor of spermine) activate mTAAR9 and elicit neutral or attractive behavioral preferences, respectively (16Saraiva L.R. Kondoh K. Ye X. Yoon K.H. Hernandez M. Buck L.B. Combinatorial effects of odorants on mouse behavior.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E3300-E3306Crossref PubMed Scopus (76) Google Scholar). However, the mechanisms for TAARs in recognizing polyamines have not been fully determined. We argue that vertebrate TAARs have retained a conserved mechanism for polyamine recognition, even though the behavioral responses to the polyamines are species-specific and context-dependent. Olfactory TAAR gene families are present in all vertebrate species (20Eyun S.I. Moriyama H. Hoffmann F.G. Moriyama E.N. Molecular evolution and functional divergence of trace amine-associated receptors.PLoS One. 2016; 11e0151023Crossref PubMed Scopus (22) Google Scholar, 21Hussain A. Saraiva L.R. Korsching S.I. Positive Darwinian selection and the birth of an olfactory receptor clade in teleosts.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 4313-4318Crossref PubMed Scopus (109) Google Scholar). Phylogenetic analysis revealed that TAARs of sea lamprey (a jawless vertebrate) cluster into an independent clade that is distantly related to the TAAR clade of jawed animals (20Eyun S.I. Moriyama H. Hoffmann F.G. Moriyama E.N. Molecular evolution and functional divergence of trace amine-associated receptors.PLoS One. 2016; 11e0151023Crossref PubMed Scopus (22) Google Scholar, 21Hussain A. Saraiva L.R. Korsching S.I. Positive Darwinian selection and the birth of an olfactory receptor clade in teleosts.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 4313-4318Crossref PubMed Scopus (109) Google Scholar, 22Li Q. Chapter 4-odor sensing by trace amineassociated receptors.in: Zufall F. Munger S.D. Chemosensory Transduction. In Methods Mol Biol. Academic Press, Cambridge, MA2018: 21-31Google Scholar). Given that sea lamprey and mouse TAARs both detect the same group of polyamines, this provides an excellent opportunity to study the functional convergence of two independently evolved TAAR subfamilies. Many olfactory TAARs retain amine recognition motifs that are conserved in classical aminergic receptors, including an aspartate residue in transmembrane helix III (Asp3.32; Ballesteros–Weinstein indexing) and a tryptophan residue in transmembrane helix VII (Trp7.40) (22Li Q. Chapter 4-odor sensing by trace amineassociated receptors.in: Zufall F. Munger S.D. Chemosensory Transduction. In Methods Mol Biol. Academic Press, Cambridge, MA2018: 21-31Google Scholar, 23Huang E.S. Construction of a sequence motif characteristic of aminergic G protein-coupled receptors.Protein Sci. 2003; 12: 1360-1367Crossref PubMed Scopus (49) Google Scholar). Molecular docking and mutagenesis studies of mammalian TAAR1, mTAAR7e, and mTAAR7f demonstrate that the negatively charged residue Asp3.32 is critical for amine recognition and forms a salt bridge with the ligand amino group. Other highly variable residues in the transmembrane domains contribute to the selectivity for ligands and serve as scaffolds that stabilize ligand binding (12Ferrero D.M. Wacker D. Roque M.A. Baldwin M.W. Stevens R.C. Liberles S.D. Agonists for 13 trace amine-associated receptors provide insight into the molecular basis of odor selectivity.ACS Chem. Biol. 2012; 7: 1184-1189Crossref PubMed Scopus (66) Google Scholar). By contrast, a large number of teleost-TAARs lack Asp3.32, and instead, use Asp5.42 to form a salt bridge with an amino group of the biogenic amine. Several TAARs such as zTAAR13c, contain both Asp3.32 and Asp5.42 and recognize dicationic molecules including cadaverine and putrescine (13Li Q. Tachie-Baffour Y. Liu Z. Baldwin M.W. Kruse A.C. Liberles S.D. Non-classical amine recognition evolved in a large clade of olfactory receptors.Elife. 2015; 4e10441Crossref PubMed Scopus (32) Google Scholar). Notably, almost all sea lamprey and mouse TAARs have only a single negatively charged residue, Asp3.32 or Glu3.32 in transmembrane helix III that could theoretically recognize only one amino group of the polyamines. The structural basis for these TAARs to stabilize their interaction with the other amino groups in polyamines remains elusive. It is likely that vertebrate olfactory TAARs feature a salt bridge that engages a ligand amino group and have other scaffolds that contribute to the specificity of polyamine recognition. However, the structures of these predicted scaffolds and their function in recognizing amines with two or more amino groups have not been elucidated. We hypothesized that vertebrate TAARs rely on residues that form a cation–pi interaction or a hydrogen bond with the amino groups in addition to the salt bridge formed by Asp3.32 to recognize polyamines. In this study, we identified a sea lamprey olfactory TAAR receptor (sTAAR365) that shows a strikingly similar response profile to cadaverine, putrescine, and spermine as does mTAAR9. Through a systematic comparison of these two distant receptors with convergent functions, we show that sTAAR365 and mTAAR9 both possess conserved Asp3.32 and Tyr6.51 residues that interact with the two amino groups in cadaverine. In addition, sTAAR365 uses an extra Thr7.42 that stabilizes the recognition of cadaverine, serving as part of the amine-binding motif. In sTAAR365, this motif uses an additional negatively charged residue Glu7.36 that cooperates with Asp3.32 and Thr7.42 to recognize the tetraamine spermine. Likewise, mTAAR9 recognizes the triamine spermidine through an aromatic residue, Tyr7.43. Thus, sTAAR365 and mTAAR9 recognize these polyamines through a novel motif located in the transmembrane α-helices VI and VII. Taken together, our results propose a mechanism that sTAAR365 and mTAAR9 converged on their polyamine recognition through distinct motifs in a conserved binding site. We first asked whether mammalian TAAR9 orthologs are broadly tuned to triethylamine, cadaverine, spermidine, and spermine, as has been shown in mTAAR9 (16Saraiva L.R. Kondoh K. Ye X. Yoon K.H. Hernandez M. Buck L.B. Combinatorial effects of odorants on mouse behavior.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E3300-E3306Crossref PubMed Scopus (76) Google Scholar). We examined the response of TAAR9s from rat, human, hamster, and rabbit to the stimulation of 1 mM amines using a well-established cAMP response element (CRE)-driven luciferase reporter assay based on Golf-mediated cAMP signal transduction (5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar, 24Saito H. Kubota M. Roberts R.W. Chi Q. Matsunami H. RTP family members induce functional expression of mammalian odorant receptors.Cell. 2004; 119: 679-691Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). In addition to the four mTAAR9 ligands, we included putrescine, which is also a polyamine and precursor of spermine/spermidine biosynthesis. The tested TAAR9s were not activated by putrescine but exhibited varying degrees of activation to the other four amines (Fig. 1B). Rat TAAR9 showed the maximum responses to triethylamine, cadaverine, spermidine, and spermine compared with the other mammalian species. Cat TAAR9 displayed similar activation properties (potency and efficacy) to those of mTAAR9. These mTAAR9 ligands induced concentration-dependent activities in cells expressing mouse, rat, and cat TAAR9s (Fig. 1C). In contrast, human, hamster, and rabbit TAAR9s showed minimal activities for this set of ligands. We then sought to determine if other mouse olfactory TAARs are activated by the mTAAR9 ligands. Only mTAAR8c showed modest activity to 500 μM spermidine (Fig. 1D). Triethylamine induced robust responses by mTAAR7f and moderate activities for mTAAR5 and mTAAR8c (Fig. 1D). Other TAARs were not activated by cadaverine or spermidine. Based on these findings, we concluded that several mammalian TAAR9 orthologs detect polyamines. As the mouse is a model animal for olfactory studies, we focused the remainder of our studies on mouse TAAR9 to further characterize TAAR interactions with polyamines. Next, we questioned whether the sea lamprey TAAR repertoire contains members that are broadly tuned to biogenic amines and that share similar response profiles with mTAAR9. In a previous study, we reported that sTAAR348 responds to spermine when expressed in HEK293T cells but not to other structurally related biogenic amines (5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar). Sequence alignment analyses indicated that sTAAR365 shares 74% sequence identity with sTAAR348 (Fig. S1). sTAAR348 and sTAAR365 showed 34.0% and 34.5% sequence identity, respectively, with mTAAR9 (Fig. S1). We reasoned that sTAAR365 could be a candidate as a polyamine receptor. To test this hypothesis, we used an established cAMP assay to examine the amine response properties of sTAAR365 (5Scott A.M. Zhang Z. Jia L. Li K. Zhang Q. Dexheimer T. Ellsworth E. Ren J. Chung-Davidson Y.W. Zu Y. Neubig R.R. Li W. Spermine in semen of male sea lamprey acts as a sex pheromone.PLoS Biol. 2019; 17e3000332Crossref PubMed Scopus (24) Google Scholar). sTAAR365 was activated by cadaverine, putrescine, and spermine, but not by spermidine or triethylamine (Fig. 2A). Putrescine and spermine elicited a half maximal response (EC50) at concentrations of 56 μM and 28 μM, respectively, in cells expressing sTAAR365 (Fig. 2B). These are comparable to the potency of odorant receptor agonists in similar assays, ranging from 100 nM to 100 μM (25Rinaldi A. The scent of life. The exquisite complexity of the sense of smell in animals and humans.EMBO Rep. 2007; 8: 629-633Crossref PubMed Scopus (41) Google Scholar, 26Saito H. Chi Q. Zhuang H. Matsunami H. Mainland J.D. Odor coding by a Mammalian receptor repertoire.Sci. Signal. 2009; 2ra9Crossref PubMed Scopus (351) Google Scholar, 27Katada S. Hirokawa T. Oka Y. Suwa M. Touhara K. Structural basis for a broad but selective ligand spectrum of a mouse olfactory receptor: Mapping the odorant-binding site.J. Neurosci. 2005; 25: 1806-1815Crossref PubMed Scopus (235) Google Scholar). Surprisingly, sTAAR365 was exquisitely sensitive to cadaverine, with EC50 of 4 nM, and a response threshold approaching 100 pM (Fig. 2B). This level of sensitivity rivals the olfactory responses observed through in vivo recording (17Dewan A. Pacifico R. Zhan R. Rinberg D. Bozza T. Non-redundant coding of aversive odours in the main olfactory pathway.Nature. 2013; 497: 486-489Crossref PubMed Scopus (134) Google Scholar). Moreover, the maximal efficacy (Emax) of sTAAR365 response to cadaverine was comparable to that for putrescine, whereas spermine elicited a maximal response of only one-third as much, suggesting that spermine likely acts as a partial agonist for sTAAR365. We confirmed expression of sTaar365 and mTaar9 in olfactory sensory neurons (OSNs) with in situ hybridization. For adult male and female sea lamprey, the antisense sTaar365 labeled cells were sparsely distributed in lamellae along the rostral-caudal axis of the main olfactory epithelium, displaying tall cell bodies situated in the deeper epithelium and long dendrites coursing toward the epithelium surface (Fig. S2). In comparison, no labeling was observed with the sense probe. The expression pattern of sTaar365 is very similar to that of mTaar9 in the mouse olfactory epithelium (Fig. S3). These results demonstrate that sTAAR365 and mTAAR9 are both broadly tuned and sparsely distributed in olfactory epithelia. A previous study of zTAAR13c by Li et al. (13Li Q. Tachie-Baffour Y. Liu Z. Baldwin M.W. Kruse A.C. Liberles S.D. Non-classical amine recognition evolved in a large clade of olfactory receptors.Elife. 2015; 4e10441Crossref PubMed Scopus (32) Google Scholar) proposed that Asp3.32 and Asp5.42 each interact with one of the two amino groups in diamines, such as cadaverine and putrescine. However, it is not known how TAARs recognize polyamines with more than two amino groups. We sought to model the polyamine recognition sites, including residues that directly interact with additional amino groups, in sTAAR365 and mTAAR9. We speculated that TAARs use Asp3.32 to confer critical and direct interactions with one amino group, while other nearby polar and/or aromatic residues stabilize polyamine binding by forming hydrogen bond or pi–cation interactions. To test this hypothesis, we generated sTAAR365 and mTAAR9 homology models using GPCR-I-TASSER to predict the putative recognition residues for biogenic polyamines. The models were based on the crystal structures of nine homologous templates. The primary models of sTAAR365 and mTAAR9 shared a maximal identity of 31% and 40%, respectively, to their closest homologous template, the human β2-adrenergic GPCR (Protein Data Bank Entry 2rh1A). The homology model with the highest C-score was chosen as the final structure for molecular docking. Both the sTAAR365 and mTAAR9 models displayed a canonical GPCR structure with seven hydrophobic transmembrane α-helices and an eighth intracellular helix (H8) in the C-terminus. Using the homology models, we performed Induced Fit Docking (IFD) with Schrodinger Maestro 11.5 to predict the residues of sTAAR365 and mTAAR9 involved in polyamine binding. Several poses of ligand–receptor interactions were generated, and the top result was chosen according to docking scores and glide models. For sTAAR365, the highly conserved Asp3.32 contacts both amino groups of cadaverine, one is docked 2.67 Å away from the highly conserved Asp3.32, forming a salt bridge and a hydrogen bond with the carboxyl group of Asp3.32 (Fig. 3A). The second amino group of cadaverine also forms a pi–cation interaction with Tyr6.51 and a hydrogen bond with Thr7.42 (Fig. 3A). Similar to cadaverine, putrescine was predicted to interact with Asp3.32, Tyr6.51, and Thr7.42 (Fig. 3B). Notably, the distance between the carboxyl group of Asp3.32 and its salt-bridged amino group of putrescine is predicted at 3.61 Å. The difference in the salt bridge distance predicted for cadaverine and putrescine likely explains the 1000-fold difference in their potency for sTAAR365. We then docked spermine, a polyamine with four amino groups, into sTAAR365 homology model to infer how TAARs may interact with additional amino groups. As expected, the cadaverine recognition sites Asp3.32 and Thr7.42 are involved in spermine binding (Fig. 3C). Asp3.32 forms a salt bridge and a hydrogen bond with the two middle amino groups of spermine. The distance between the charged aspartate side chain and the further amino group of spermine is 4.22 Å. Meanwhile, an amino group at one end of spermine contacts the backbone of Thr7.42 with a hydrogen bond, while the amino group at the other end is anchored on the negatively charged residue Glu7.36, located in the extracellular vestibule of TM VII, through a salt bridge and a hydrogen bond. The distance between the carboxyl group of Glu7.36 and the terminal amino group of spermine is predicted at 4.33 Å. The salt bridges involved in spermine recognition are longer than the typical cutoff value for a salt bridge at 4 Å. These relatively weak ionic interactions may explain the partial activation of sTAAR365 by spermine. Docking cadaverine into mTAA9 homology model suggested that Asp3.32 and Tyr6.51 are the primary binding sites. The carboxyl group of Asp3.32 forms a salt bridge with one amino group of cadaverine at a distance of 4.41 Å (Fig. 4A) and a hydrogen bond with the second amino group of cadaverine. Likewise, Tyr6.51 is predicted to be part of the cadaverine-binding pocket, forming a hydrogen bond with the amino group of cadaverine (Fig. 4A). However, mTAAR9 differs from sTAAR365 by having Val7.42 instead of Thr7.42. The larger distance of the salt bridge and the lack of scaffold interaction with Thr7.42 may explain the much lower potency of cadaverine for mTAAR9 compared with sTAAR365. mTAAR9 exhibits robust responses to spermidine, a biosynthetic precursor of spermine. Results from docking spermidine (with three amino groups) into the mTAAR9 model suggested that an extra residue, Tyr7.43, cooperates with Asp3.32 and Tyr6.51 to form the binding pocket (Fig. 4B). Asp3.32 recognizes the middle amino group of spermidine by a salt bridge, at a distance of 2.87 Å, and a hydrogen bond. Moreover, a pi–cation interaction is also predicted between the Tyr6.51 residue and the middle amino group. For the amino