| The sea lamprey(Petromyzon marinus)is an agnathan vertebrate species with a welldeveloped olfactory system.This sensory system detects odorants that inform critical behaviors,such as foraging,migrating and mate searching,throughout the larval,juvenile,and adult stages of sea lamprey.The sea lamprey genome encodes one of the smallest repertoires of olfactory receptors among sequenced vertebrate genomes,and contains genes for odorant receptors(ORs),trace amine-associated receptors(TAARs)and vomeronasal type 1 receptors(V1Rs).In this dissertation study,I used a heterologous CRE-luciferase screen system to match odorants to sea lamprey olfactory receptors,and investigated the mechanisms whereby the receptors and ligands interact.1.Sea lamprey olfactory receptor-ligand pair screeningI mined the sea lamprey genome to define its olfactory receptor gene repertoire,and found 40 OR,31 TAAR,and 3 V1 R genes.I cloned c DNAs of 32 OR,26 TAAR,and 3 V1 R genes,and inserted them into a heterologous expression vector with Rho-tag,which were transfected into the HEK293 T cell line along with m RTP1s(mouse receptortransporting protein 1 short).A total of 46 receptors were targeted to the plasma membrane and subsequently screened against 104 compounds including sea lamprey pheromones,amino acids,trace amines and bile acids.The receptor activity was reported with the CRE-luciferase system.In this initial screen,OR320 a and OR320 b responded to sea lamprey sex pheromone 3-keto-petromyzonol sulfate(3k PZS)and analogues whereas TAAR348 responded to a biogenic polyamine,spermine,and trace amines.In addition,several receptors showed constitutive activity.2.Interaction of sea lamprey pheromone 3k PZS with OR320 a and OR320bIn both CRE-luciferase assays and c AMP assays,OR320 a and OR320 b were activated by 3k PZS through the c AMP signal pathway,indicating that OR320 a and OR320 b are cognate olfactory receptors of 3k PZS.Structure-activity relationship was analyzed by measuring responses of OR320 a and OR320 b to 20 analogues of 3k PZS.Compounds that substitute –OSO3,-OH,-OSO3 or –OH with-OH?OSO3?-OH or-OSO3,respectively,on C3,C7,C12 and C24-positions,respectively,yielded higher activities on OR320 a and OR320 b.3k PZS analogue C1,which has all these four characteristics,is the full agonist of OR320 a and OR320 b.Moreover,OR320 b displayed weaker responses to all 3k PZS analogues than OR320 a,especially to the analogues with –OSO3 on C12 position.Through transmembrane(TM)domain switches and single-site mutations,I found that the 79 th amino acid residue on TM2 plays a deterministic role in the function difference between OR320 a and OR320 b by affecting their affinities to the ligands.OR320 a and OR320 b are the first cognate steroid sex pheromone receptors identified in vertebrates,providing further evidence for pheromone detection by odorant receptors in vertebrate animals.3.Functional comparison of spermine receptors Pm TAAR348 and Lm TAAR348Sea lamprey TAAR348(Pm TAAR348)responded to tyramine,phenylethylamine,?-phenylethylamine and spermine in CRE-luciferase assays.In both CRE-luciferase and c AMP assays,spermine activated Pm TAAR348 in a classical concentration dependent manner,which indicates Pm TAAR348 is the cognate receptor for spermine and provides the olfactory receptor of spermine in an animal.Taar348 was also cloned from Korean lamprey(Lethenteron morii)which is a landlocked species in China.Lm TAAR348 proved to be a constitutively active receptor without stimuli,and Pm TAAR348 was not.It was the 340 th amino acid residue on C-terminal that plays a critical role in the difference of constitutive activity between Lm TAAR348 and Pm TAAR348.These results implicate a possibility of different behavior responses induced by spermine between sea lamprey and Korean lamprey.4.The constitutively active olfactory receptors of sea lampreyThe high levels of basal signal in seven sea lamprey olfactory receptors,namely 5904.OR327,6425.OR330 A,3721.TAAR365,7446.TAAR346 A,9665.TAAR351,9755.TAAR355 B and 9755.TAAR355 A,indicated they are constitutively active.Their constitutive activities were Golf independent,suggesting that they may be involved in axon targeting of olfactory sensory neurons in the sea lamprey.To carry out a preliminary test of this hypothesis,we started with TAAR365,a highly constitutively active receptor.HEK293 T cells that expressed TAAR365(1ng/?l)48 hours post-transfection showed morphological changes.As pseudopodia extend longer and thinner,the pseudopodia of adjacent TAAR365 expressing cell contacted and fused with each other.In contrast,the HEK293 T cells expressing OR331,which does not display constitutive activities,at equal expression level with TAAR365,showed no morphological changes.These results showed that the constitutively active olfactory receptors changed the shape of HEK293 T cells,indicating a potential to affect axon targeting if expressed in olfactory sensory neurons of sea lamprey.Finally,none of the constitutively active olfactory receptor responded to 3k PZS or spermine in CRE-luciferase assays,and the constitutive activities could be inhibited by a candidate inverse agonist 171-IDE-048-1,which provides a basis to further study the function of constitutive active receptors in the sea lamprey olfactory system.In conclusion,we de-ophanized sea lamprey sex pheromone 3k PZS to its cognate olfactory receptors,OR320 a and OR320 b,providing the first cognate steroid sex pheromone receptors identified in vertebrates.We identified TAAR348 as a spermine olfactory receptor in sea lamprey,the first olfactory receptor identified for spermine,an odorant detected by many vertebrate species.We explored the potential of the constitutively active receptors to affect axon targeting in olfactory sensory neurons.Taken together,these results provide a foundation to study the link between olfactory neural system and motor neural circuit in sea lamprey,and the mechanism of pheromone signaling transduction,offering a useful model for understanding olfactory receptor function in vertebrates. |
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