Evolutionary Origin And The Mechanism Of Ligand Recognition Of Olfactory Trace Amine-associated Receptors (TAARs)

Olfaction is a part of peripheral sensory system that plays a vital role in animal behaviors such as feeding,avoiding enemies,courtship and maternal and child interactions.Olfactory receptors are considered to be the molecular basis for olfactory system to perceive and discriminate olfactory compounds.Trace amine-associated receptor(TAAR)family is the second major category of olfactory receptors in the main olfactory epithelium(MOE).TAARs are a kind of G protein-coupled receptors(GPCRs)which are conserved in vertebrates.TAARs are evolutionarily distant from odorant receptors(ORs),while share a higher degree of homology with biological amine receptors such as dopamine receptors and adrenergic receptors.The third transmembrane domains of most mammalian TAARs contain a highly conserved aspartic acid residue(D3.32)as the site for bioamines to bind through intermolecular forces such as salt bridge.Some teleost TAARs lost D3.32 during evolution,but retain aspartic acid residue(D5.42)in the fifth transmembrane domain to identify amines.Ligands of some TAARs of species such as human,mouse,rat and zebrafish have been identified,such as isoamylamine,trimethylamine and phenethylamine and so on.Some of these ligands found in natural animal products such as urine,can induce instinctive behaviors.Therefore,TAARs,as a kind of olfactory receptors,play an important role in social communication in animals.However,most TAARs are "orphan" receptors,whose ligands are still unknown.Besides,the molecular mechanism of TAARs binding to their ligands is rarely studied.The evolution of TAARs may also reflect changes in the chemical communication of animals in social interactions.Previous studies have revealed that TAAR family can be divided into three clades,including Clade I,II and III.However,the evolutionary origin of each clade of TAARs remains unclear.In addition,there are a group of Taar-like genes in sea lamprey,which encode protein called TAAR like(TARLs)due to the lack of TAAR conserved motif.However,the function of these sea lamprey TARLs are unknown.Therefore,in this study,we "de-orphanized" TAARs of different species,analyzed the structural basis of amine identification,and further discussed the evolutionary origin of TAARs and TARLs.We performed ligand screening on 125 TAARs across 11 species including teleost,reptiles and mammals,and ligands for 21 TAARs have been identified.Through multiple sequence alignment and point mutation analysis,we found amino acid residue at positions 5.39 of the fifth transmembrane domain critical for TAAR4-phenethylamine interaction and TAAR5-trimethylamine interaction.In addition,phylogenetic analysis of 2737 TAARs,TARLs and biological amine receptors in 228 species showed that TAARs can be divided into four branches,Clade Ia,Ib,II and III.Shark TAARS1 were the TAARs closest to the common ancestor of Clade Ia.The coelacanth TAARC1 were the closest receptors to the common ancestor of Clade Ib.TAARC2 were the TAARs closest to the common ancestor of Clade II.The TAAR closest to the common ancestor of Clade III were the TAAR13 s.What's more,we discovered that the TARLs formed a separate branch together with 5-hydroxytryptamine receptor 4(5-Htr4)family.The functional assays of TARLs showed that sea lamprey TARLs recognized volatile small molecular amines similar to olfactory TAARs.Bony fish TARLs had a ligand-recognizing pattern similar to TAAR1,suggesting that TARLs are olfactory chemoreceptors for the detection of volatile amines.In summary,our study includes identification of orphan TAARs across multiple species,analysis of characteristics of their binding ligands and discussion about the evolutionary origin of TAARs of each clade.Additionally,we found that TARLs,which were evolutionarily separated from TAARs,can function as chemoreceptors.Our research enriches the knowledge of the TAAR olfactory subsystem.