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• W3041567162 abstract "Pheromones play critical roles in habitat identification and reproductive behavior synchronization in the sea lamprey (Petromyzon marinus). The bile acid 3-keto petromyzonol sulfate (3kPZS) is a major component of the sex pheromone mixture from male sea lamprey that induces specific olfactory and behavioral responses in conspecific individuals. Olfactory receptors interact directly with pheromones, which is the first step in their detection, but identifying the cognate receptors of specific pheromones is often challenging. Here, we deorphanized two highly related odorant receptors (ORs), OR320a and OR320b, of P. marinus that respond to 3kPZS. In a heterologous expression system coupled to a cAMP-responsive CRE-luciferase, OR320a and OR320b specifically responded to C24 5α-bile acids, and both receptors were activated by the same set of 3kPZS analogs. OR320a displayed larger responses to all 3kPZS analogs than did OR320b. This difference appeared to be largely determined by a single amino acid residue, Cys-792.56, the C-terminal sixth residue relative to the most conserved residue in the second transmembrane domain (2.56) of OR320a. This region of TM2 residues 2.56–2.60 apparently is critical for the detection of steroid compounds by odorant receptors in lamprey, zebrafish, and humans. Finally, we identified OR320 orthologs in Japanese lamprey (Lethenteron camtschaticum), suggesting that the OR320 family may be widely present in lamprey species and that OR320 may be under purifying selection. Our results provide a system to examine the origin of olfactory steroid detection in vertebrates and to define a highly conserved molecular mechanism for steroid-ligand detection by G protein–coupled receptors. Pheromones play critical roles in habitat identification and reproductive behavior synchronization in the sea lamprey (Petromyzon marinus). The bile acid 3-keto petromyzonol sulfate (3kPZS) is a major component of the sex pheromone mixture from male sea lamprey that induces specific olfactory and behavioral responses in conspecific individuals. Olfactory receptors interact directly with pheromones, which is the first step in their detection, but identifying the cognate receptors of specific pheromones is often challenging. Here, we deorphanized two highly related odorant receptors (ORs), OR320a and OR320b, of P. marinus that respond to 3kPZS. In a heterologous expression system coupled to a cAMP-responsive CRE-luciferase, OR320a and OR320b specifically responded to C24 5α-bile acids, and both receptors were activated by the same set of 3kPZS analogs. OR320a displayed larger responses to all 3kPZS analogs than did OR320b. This difference appeared to be largely determined by a single amino acid residue, Cys-792.56, the C-terminal sixth residue relative to the most conserved residue in the second transmembrane domain (2.56) of OR320a. This region of TM2 residues 2.56–2.60 apparently is critical for the detection of steroid compounds by odorant receptors in lamprey, zebrafish, and humans. Finally, we identified OR320 orthologs in Japanese lamprey (Lethenteron camtschaticum), suggesting that the OR320 family may be widely present in lamprey species and that OR320 may be under purifying selection. Our results provide a system to examine the origin of olfactory steroid detection in vertebrates and to define a highly conserved molecular mechanism for steroid-ligand detection by G protein–coupled receptors. Bile acids are amphipathic metabolites that are synthesized from cholesterol and function primarily as a surfactant to emulsify dietary fats in the intestine (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51 (19638645): 226-24610.1194/jlr.R000042Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). These metabolites also serve as signals in a wide variety of physiological processes, including cholesterol homeostasis and endocrine regulation in vertebrates (2Gruy-Kapral C. Little K.H. Fordtran J.S. Meziere T.L. Hagey L.R. Hofmann A.F. Conjugated bile acid replacement therapy for short-bowel syndrome.Gastroenterology. 1999; 116 (9869597): 15-2110.1016/S0016-5085(99)70223-4Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 3Houten S.M. Watanabe M. Auwerx J. Endocrine functions of bile acids.EMBO J. 2006; 25 (16541101): 1419-142510.1038/sj.emboj.7601049Crossref PubMed Scopus (434) Google Scholar, 4Hofmann A. Hagey L. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.Cell. Mol. Life Sci. 2008; 65 (18488143): 2461-248310.1007/s00018-008-7568-6Crossref PubMed Scopus (625) Google Scholar, 5Yoshida M. Murata M. Inaba K. Morisawa M. A chemoattractant for ascidian spermatozoa is a sulfated steroid.Proc. Natl. Acad. Sci. U. S. A. 2002; 99 (12411583): 14831-1483610.1073/pnas.242470599Crossref PubMed Scopus (150) Google Scholar). These functions of bile acids are mediated by six known receptors, including both G protein–coupled receptors (GPCRs) (6Pols T.W. Noriega L.G. Nomura M. Auwerx J. Schoonjans K. The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation.J. Hepatol. 2011; 54 (21145931): 1263-127210.1016/j.jhep.2010.12.004Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) and nuclear receptors such as the farnesoid X receptor (7Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284 (10334992): 1362-136510.1126/science.284.5418.1362Crossref PubMed Scopus (2145) Google Scholar). In fish species, bile acids, once excreted into the water via the urine (8Zhang C. Brown S.B. Hara T.J. Biochemical and physiological evidence that bile acids produced and released by lake char (Salvelinus namaycush) function as chemical signals.J. Comp. Physiol. B. 2001; 171 (11302533): 161-17110.1007/s003600000170Crossref PubMed Scopus (67) Google Scholar) or feces (9Doyle W.I. Dinser J.A. Cansler H.L. Zhang X. Dinh D.D. Browder N.S. Riddington I.M. Meeks J.P. Faecal bile acids are natural ligands of the mouse accessory olfactory system.Nat. Commun. 2016; 7 (27324439): 1193610.1038/ncomms11936Crossref PubMed Scopus (24) Google Scholar) or from the gills (10Li W. Scott A.P. Siefkes M.J. Yan H. Liu Q. Yun S.-S. Gage D.A. Bile acid secreted by male sea lamprey that acts as a sex pheromone.Science. 2002; 296 (11935026): 138-14110.1126/science.1067797Crossref PubMed Scopus (308) Google Scholar), are known to be potent olfactory stimulants and pheromones. However, little is known about the receptors in the olfactory epithelium that respond to these bile acids. Olfactory detection of bile acids in fishes is highly sensitive and specific (8Zhang C. Brown S.B. Hara T.J. Biochemical and physiological evidence that bile acids produced and released by lake char (Salvelinus namaycush) function as chemical signals.J. Comp. Physiol. B. 2001; 171 (11302533): 161-17110.1007/s003600000170Crossref PubMed Scopus (67) Google Scholar, 11Giaquinto P.C. Hara T.J. Discrimination of bile acids by the rainbow trout olfactory system: evidence as potential pheromone.Biol. Res. 2008; 41 (18769761): 33-4210.4067/S0716-97602008000100005Crossref PubMed Scopus (23) Google Scholar, 12Zhang C. Hara T.J. Lake char (Salvelinus namaycush) olfactory neurons are highly sensitive and specific to bile acids.J. Comp. Physiol. A. 2009; 195 (19137319): 203-21510.1007/s00359-008-0399-yCrossref PubMed Scopus (32) Google Scholar, 13Buchinger T.J. Li W. Johnson N.S. Bile salts as semiochemicals in fish.Chem. Senses. 2014; 39 (25151152): 647-65410.1093/chemse/bju039Crossref PubMed Scopus (51) Google Scholar). This allows fish to discriminate diverse fish bile acids, including 5α and 5β forms, bile alcohol sulfates, and conjugated bile acids (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51 (19638645): 226-24610.1194/jlr.R000042Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Olfactory detection of bile acids has been observed throughout fish taxa, including jawless (14Li W. Sorensen P.W. Gallaher D.D. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.J. Gen. Physiol. 1995; 105 (7658193): 569-58710.1085/jgp.105.5.569Crossref PubMed Scopus (163) Google Scholar) and jawed fishes (15Michel W. Lubomudrov L. Specificity and sensitivity of the olfactory organ of the zebrafish, Danio rerio.J. Comp. Physiol. A. 1995; 177 (7636767): 191-19910.1007/BF00225098Crossref PubMed Scopus (71) Google Scholar). A progression has been documented from the internal secretion and detection of the 5α-bile acids of basal vertebrates such as lamprey to the 5β-bile acids of ray-finned fish (16Hagey L.R. Møller P.R. Hofmann A.F. Krasowski M.D. Diversity of bile salts in fish and amphibians: evolution of a complex biochemical pathway.Physiol. Biochem. Zool. 2010; 83 (20113173): 308-32110.1086/649966Crossref PubMed Scopus (102) Google Scholar). Olfactory detection of waterborne bile acids also shows an evolutionary trend in that basal vertebrates release and detect 5α-bile acids and teleost fish release and detect 5β-bile acids (13Buchinger T.J. Li W. Johnson N.S. Bile salts as semiochemicals in fish.Chem. Senses. 2014; 39 (25151152): 647-65410.1093/chemse/bju039Crossref PubMed Scopus (51) Google Scholar). Recently, zebrafish olfactory class A receptors (ORA) that are orthologous to the type 1 mammalian vomeronasal receptors were reported to detect 5β-bile acids (17Cong X. Zheng Q. Ren W. Chéron J.-B. Fiorucci S. Wen T. Zhang C. Yu H. Golebiowski J. Yu Y. Zebrafish olfactory receptors ORAs differentially detect bile acids and bile salts.J. Biol. Chem. 2019; 294 (30833327): 6762-677110.1074/jbc.RA118.006483Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). This finding is consistent with a previous hypothesis that odorant receptors (ORs) and type 1 vomeronasal receptors detect steroids (18Oka Y. Saraiva L.R. Korsching S.I. Crypt neurons express a single V1R-related ora gene.Chem. Senses. 2012; 37 (22038944): 219-22710.1093/chemse/bjr095Crossref PubMed Scopus (60) Google Scholar, 19Bazáes A. Olivares J. Schmachtenberg O. Properties, projections, and tuning of teleost olfactory receptor neurons.J. Chem. Ecol. 2013; 39 (23468224): 451-46410.1007/s10886-013-0268-1Crossref PubMed Scopus (33) Google Scholar, 20Kermen F. Franco L.M. Wyatt C. Yaksi E. Neural circuits mediating olfactory-driven behavior in fish.Front. Neural Circuits. 2013; 7 (23596397): 6210.3389/fncir.2013.00062Crossref PubMed Scopus (63) Google Scholar). Sea lamprey (Petromyzon marinus), a jawless vertebrate animal, provides an advantageous model for matching bile acid pheromones to their olfactory receptors. Although bile acids are known to be highly potent olfactory stimuli in several fish species, only sea lamprey bile acids have been shown unequivocally to be bona fide pheromones (10Li W. Scott A.P. Siefkes M.J. Yan H. Liu Q. Yun S.-S. Gage D.A. Bile acid secreted by male sea lamprey that acts as a sex pheromone.Science. 2002; 296 (11935026): 138-14110.1126/science.1067797Crossref PubMed Scopus (308) Google Scholar). The latter are rigorously defined to be stimuli secreted by an individual and received by a second conspecific individual inducing innate and specific reactions (21Brennan P.A. Zufall F. Pheromonal communication in vertebrates.Nature. 2006; 444 (17108955): 308-31510.1038/nature05404Crossref PubMed Scopus (355) Google Scholar). Many 5α-bile acids and their derivatives, such as petromyzonol sulfate (PZS), petromyzones (PZ), petromyzonamine disulfate, petromyzosterol disulfate, and allocholic acid (ACA) (14Li W. Sorensen P.W. Gallaher D.D. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.J. Gen. Physiol. 1995; 105 (7658193): 569-58710.1085/jgp.105.5.569Crossref PubMed Scopus (163) Google Scholar, 22Li W. Sorensen P.W. Highly independent olfactory receptor sites for naturally occurring bile acids in the sea lamprey, Petromyzon marinus.J. Comp. Physiol. A. 1997; 180: 429-43810.1007/s003590050060Crossref Scopus (68) Google Scholar, 23Li K. Siefkes M.J. Brant C.O. Li W. Isolation and identification of petromyzestrosterol, a polyhydroxysteroid from sexually mature male sea lamprey (Petromyzon marinus L.).Steroids. 2012; 77 (22475879): 806-81010.1016/j.steroids.2012.03.006Crossref PubMed Scopus (19) Google Scholar, 24Li K. Scott A.M. Riedy J.J. Fissette S. Middleton Z.E. Li W. Three novel bile alcohols of mature male sea lamprey (Petromyzon marinus) act as chemical cues for conspecifics.J. Chem. Ecol. 2017; 43 (28634722): 543-54910.1007/s10886-017-0852-xCrossref PubMed Scopus (13) Google Scholar, 25Sorensen P.W. Fine J.M. Dvornikovs V. Jeffrey C.S. Shao F. Wang J. Vrieze L.A. Anderson K.R. Hoye T.R. Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey.Nat. Chem. Biol. 2005; 1 (16408070): 324-32810.1038/nchembio739Crossref PubMed Scopus (200) Google Scholar), as well as sex pheromones, such as 3-ketopetromyzonol sulfate (3kPZS) and 3-ketoallocholic acid stimulate the olfactory epithelia of sea lamprey (26Johnson N.S. Yun S.-S. Thompson H.T. Brant C.O. Li W. A synthesized pheromone induces upstream movement in female sea lamprey and summons them into traps.Proc. Natl. Acad. Sci. 2009; 106 (19164592): 1021-102610.1073/pnas.0808530106Crossref PubMed Scopus (150) Google Scholar, 27Buchinger T.J. Siefkes M.J. Zielinski B.S. Brant C.O. Li W. Chemical cues and pheromones in the sea lamprey (Petromyzon marinus).Front. Zool. 2015; 12 (26609313): 3210.1186/s12983-015-0126-9Crossref PubMed Scopus (62) Google Scholar, 28Buchinger T.J. Wang H. Li W. Johnson N.S. Evidence for a receiver bias underlying female preference for a male mating pheromone in sea lamprey.Proc. R. Soc. B Biol. Sci. 2013; 280 (24068361): 2013196610.1098/rspb.2013.1966Crossref PubMed Scopus (28) Google Scholar). Critically, 3kPZS is a major contributor to the male sea lamprey sex pheromone mixture that induces specific mating-related behavioral responses in adult female sea lamprey. It is the first-ever vertebrate pheromone biopesticide registered by the United States Environmental Protection Agency. Another advantage of the sea lamprey model is that its genome encodes one of the smallest known odorant receptor repertoires among all vertebrate genomes sequenced (29Libants S. Carr K. Wu H. Teeter J.H. Chung-Davidson Y.-W. Zhang Z. Wilkerson C. Li W. The sea lamprey Petromyzon marinus genome reveals the early origin of several chemosensory receptor families in the vertebrate lineage.BMC Evol. Biol. 2009; 9 (19646260): 18010.1186/1471-2148-9-180Crossref PubMed Scopus (53) Google Scholar), providing a less daunting process to match a pheromone to its cognate receptor. Here we report the identification of two highly related sea lamprey odorant receptors that respond to 3kPZS and numerous 3kPZS analogs that control sea lamprey migration and nest attraction behavior. We further identified a single amino acid residue in the second transmembrane domain (TM2) that is critical to the regulation of the strength of signaling. Our results provide a system to examine the origin as well as molecular mechanisms of olfactory detection and potential novel modulators of steroid pheromone signaling in vertebrates. We used the previously reported 27 OR gene sequences identified in the P. marinus primary genome assembly version 2.0 as query sequences (29Libants S. Carr K. Wu H. Teeter J.H. Chung-Davidson Y.-W. Zhang Z. Wilkerson C. Li W. The sea lamprey Petromyzon marinus genome reveals the early origin of several chemosensory receptor families in the vertebrate lineage.BMC Evol. Biol. 2009; 9 (19646260): 18010.1186/1471-2148-9-180Crossref PubMed Scopus (53) Google Scholar). Using these sequences, we remined sequences encoding ORs from the improved genome assembly version 7.0 (30Smith J.J. Kuraku S. Holt C. Sauka-Spengler T. Jiang N. Campbell M.S. Yandell M.D. Manousaki T. Meyer A. Bloom O.E. Morgan J.R. Buxbaum J.D. Sachidanandam R. Sims C. Garruss A.S. et al.Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.Nat. Genet. 2013; 45 (23435085): 41510.1038/ng.2568Crossref PubMed Scopus (474) Google Scholar) using BLASTN searches. An additional 13 intact single-exon OR genes were further identified, which increased the number of OR genes encoded in the lamprey genome to 40. Neighbor-joining, maximum parsimony, and maximum likelihood (ML) analyses of the predicted amino acid sequences indicated that these 40 genes clustered into one well-supported ingroup that separated well from the outgroup (Fig. S1). Consistent with previous phylogenetic analyses (31Niimura Y. Evolutionary dynamics of olfactory receptor genes in chordates: interaction between environments and genomic contents.Hum. Genomics. 2009; 4 (20038498): 107-11810.1186/1479-7364-4-2-107Crossref PubMed Scopus (79) Google Scholar), sea lamprey OR genes separated into two distinct clades, type 1 and type 2 ORs. In particular, the four ORs that formed the type 2 clade in our study were identical to the four ORs placed in the type 2 clade in the previous study (31Niimura Y. Evolutionary dynamics of olfactory receptor genes in chordates: interaction between environments and genomic contents.Hum. Genomics. 2009; 4 (20038498): 107-11810.1186/1479-7364-4-2-107Crossref PubMed Scopus (79) Google Scholar). These four ORs belonged to the η group of type 2 ORs in vertebrates (31Niimura Y. Evolutionary dynamics of olfactory receptor genes in chordates: interaction between environments and genomic contents.Hum. Genomics. 2009; 4 (20038498): 107-11810.1186/1479-7364-4-2-107Crossref PubMed Scopus (79) Google Scholar). In addition, sea lamprey type 1 ORs segregated into two major classes, class I and class II, with characteristics similar to those of mammalian class I and class II ORs, respectively. We were successful in cloning 32 of 40 ORs from sea lamprey genomic DNA. The clones were inserted into the pCMV-N-Rho eukaryotic expression vector (Fig. S2), in which the first 21 amino acid residues of bovine rhodopsin were introduced at the N terminus. The Rho tag was reported to increase the efficiency of OR cell plasma membrane targeting (32Krautwurst D. Yau K.-W. Reed R.R. Identification of ligands for olfactory receptors by functional expression of a receptor library.Cell. 1998; 95 (9875846): 917-92610.1016/S0092-8674(00)81716-XAbstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). We selected HEK293T as our heterologous expression system, as it has been widely used for functional expression of ORs from mammalian systems (33Zhuang H. Matsunami H. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.Nat. Protoc. 2008; 3 (18772867): 1402-141310.1038/nprot.2008.120Crossref PubMed Scopus (131) Google Scholar). We co-transfected an accessory protein construct (mRTP1s) and EGFP with sea lamprey ORs into HEK293T cells. At 48 h post-transfection, the cell-surface expression of ORs was visualized using an antibody against the N-terminal Rho tag (Fig. 1A). Receptors located on the cell surface showed the ring expected for plasma membrane fluorescence and intracellular staining appearing in puncta near the nucleus. Ultimately, 22 of 32 ORs showed a detectable expression on the plasma membrane (Fig. 1B). A subset of both type 1 and type 2 ORs were robustly expressed and localized on the plasma membrane. We used the cAMP-responsive CRE-luciferase reporter OR activation system (33Zhuang H. Matsunami H. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.Nat. Protoc. 2008; 3 (18772867): 1402-141310.1038/nprot.2008.120Crossref PubMed Scopus (131) Google Scholar) to measure responses of lamprey ORs to 3kPZS and PZS. The 22 surface-expressed Rho-tagged ORs were transfected into HEK293T cells along with mRTP1s, Gαolf, and the reporter pGL4.29. The 22 OR-transfected cell preparations were then exposed to two concentrations of 3kPZS (10 and 100 μm). Only OR320a and OR320b showed responses in the luciferase assay (Table S1). Both OR320a and OR320b are type 2 ORs. These two receptors were further examined for their responses to PZS, an analog of 3kPZS and potent odorant in sea lamprey (14Li W. Sorensen P.W. Gallaher D.D. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.J. Gen. Physiol. 1995; 105 (7658193): 569-58710.1085/jgp.105.5.569Crossref PubMed Scopus (163) Google Scholar). PZS also activated both OR320a and OR320b, at a potency similar to that of 3kPZS (Table S1). Because the CRE-luciferase reporter can be activated by kinases other than protein kinase A, we excluded the possibility that another signaling pathway was involved by directly measuring cAMP production using a time-resolved FRET cAMP assay. Rho-tagged OR320a and OR320b were co-transfected along with mRTP1s and Gαolf into HEK29T cells, which were then stimulated with different concentrations of 3kPZS and PZS. Both OR320a and OR320b induced cAMP production upon exposure to 3kPZS (Fig. 2A) and PZS (Fig. 2B) in a concentration-dependent manner. OR320a responded strongly to both 3kPZS (EC50 = 0.86 μm (CI 0.13–5.8 μm); n = 3) and PZS (EC50 = 0.62 μm (CI 0.2–1.9 μm); n = 3) (values are mean (95% CI)). Compared with OR320a, OR320b responded with a markedly lower potency and modestly lower efficacy to both 3kPZS (EC50 > 30 μm; n = 3) and PZS (EC50 = 8.9 μm (CI 2.8–28 μm); n = 3). These data confirmed that OR320a and OR320b are activated by 3kPZS and PZS and regulate the cAMP signaling pathway. The expression patterns of OR320a and OR320b genes were confirmed by absolute real-time PCR analysis. We found that or320a and or320b were expressed primarily in olfactory epithelium and expressed at low level in brain in adult sea lamprey (Fig. S3); they were not observed in intestine, gills, and kidney. A library of natural and potential sea lamprey odorants composed of trace amines, amino acids, and steroids, including α- and β-bile acids (10Li W. Scott A.P. Siefkes M.J. Yan H. Liu Q. Yun S.-S. Gage D.A. Bile acid secreted by male sea lamprey that acts as a sex pheromone.Science. 2002; 296 (11935026): 138-14110.1126/science.1067797Crossref PubMed Scopus (308) Google Scholar, 14Li W. Sorensen P.W. Gallaher D.D. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.J. Gen. Physiol. 1995; 105 (7658193): 569-58710.1085/jgp.105.5.569Crossref PubMed Scopus (163) Google Scholar), was screened against OR320a and OR320b using the CRE-luciferase assay to determine the activation spectra of OR320a and OR320b. OR320a and OR320b were not activated by trace amines or amino acids. Among the 44 bile acids tested, OR320a was activated by 3kPZS and nine analogs at 10 μm and by 10 analogs at 100 μm (Table S2). OR320b was activated by 3kPZS and four analogs at 10 μm and by 10 analogs at 100 μm (Table S2). Although spermidine at 100 μm elicited a 10-fold increase in luciferase activity over the control (Table S2), the increase represented a false positive based on subsequent experiments that examined the concentration-response relationships. The sea lamprey bile acid, ACA, weakly activated OR320a and OR320b. Because the concentration-response curve for ACA on OR320a was not strong enough to determine efficacy, competition between ACA and 3kPZS for OR320a was measured. As expected, ACA inhibited the response of 10 μm 3kPZS with an IC50 of 0.26 μm (Fig. S4). The signal did not go all the way to baseline, suggesting that ACA is an allosteric inhibitor. All active ligands for OR320a and OR320b appear to be 3kPZS analogs, with a C24 5α skeleton and a sulfate conjugation at C-24 (Table S2). It was notable that bile acids with a C24 5β skeleton, including TCL, TLS, TDC, CA, GCA, TCD, DOC, TLC, GLC, and LCS, did not activate OR320a and OR320b. The C24 5β skeleton is characterized by an A ring of the steroid nucleus that bends away, forming an L-shaped molecule, which differs from the C24 5α skeleton that forms a somewhat flat molecule. Also, compounds without a C24 skeleton, such as testosterone sulfate, testosterone, and androstenedione, did not activate OR320a and OR320b. The activation spectra of OR320a and OR320b were thus limited to the C24 5α-bile acid profile. We measured concentration-response relationships for all eleven 3kPZS analogs using CRE-luciferase assays to determine the functional groups critical to C24 5α-bile acid potency for OR320a and OR320b. We found that OR320a and OR320b were activated by all 10 analogs in a concentration-dependent manner (Fig. 3). The sea lamprey mating pheromone 3kPZS, as well as PZS, 3-keto-1-ene PZS, and 3-keto-4-ene PZS, were the most potent ligands, with EC50 values ranging from 0.26 to 8.3 μm and Emax values ranging from a maximum luciferase activity of 0.76 to 1.2 (normalized to the OR320a response to 10 μm 3kPZS). Five other sea lamprey bile acids, PZ, 3kPZ, DKPES (3,12-diketo-4,6-petromyzonene-24-sulfate), 3.12-diketo-4-ene PZS, and 3.12-diketo-1.4-diene PZS, were partial agonists with higher EC50 values that ranged from 1.8 to 27 μm and lower Emax values that ranged from normalized luciferase activity of 0.16 to 0.64. For OR320b, EC50 values of PZ and 3kPZ were undeterminable (>10 μm). A comparison of the structures of the 11 active ligands indicated that the C-12 hydroxyl and C-24 sulfate play critical roles in OR320a and OR320b activation. For the analogs sharing identical functional groups at C-3, C-7, and C-24, those with a C-12 hydroxyl group activated OR320a and OR320b more potently, with higher Emax and lower EC50 values, than the analogs with a C-12 carbonyl group (Table 1). In addition, for the analogs sharing identical functional groups at C-3, C-7, and C-12, those with a C-24 sulfate group activated OR320a and OR320b more potently, with higher Emax and lower EC50 values, than the analogs with a C-24 hydroxyl or carboxyl group (Table 1). Furthermore, for the analogs sharing identical functional groups at C-7, C-12, and C-24, those with a C-3 hydroxyl group activated OR320a and OR320b more potently, with higher Emax and lower EC50 values, than the analogs with a C-3 carbonyl group (Table 1). These comparisons define clear structure-activity relationships for OR320a and OR320b activation by C24 5α-bile acids.Table 1Structure-activity relationship at carbon positions 3, 12, and 24 of 3kPZS analogs Open table in a new tab We next sought to determine the transmembrane domain(s) and amino acid residue(s) that play critical roles in the differential response of OR320a and OR320b to 3kPZS and its analogs. OR320a and OR320b share 92.8% amino acid identity, with only 22 amino acid residues that differ (Fig. 4A). Because OR320a and OR320b have virtually identical specificity but different sensitivity to the same set of analogs, this receptor pair offers an opportunity to decipher the molecular mechanism for receptor sensitivity at the olfactory epithelium. We further reasoned that, because three transmembrane domains, TM3, TM4, and TM6, were identical between OR320a and OR320b, these three regions could not explain the different sensitivity between the two receptors. To narrow down our search, we separated each receptor sequence into three regions. The first region contained the N terminus, TM1–3, and ICL2. The second region included TM4, TM5, and ICL3. The remaining sequence, TM6, TM7, and the C terminus, was assigned as the third region. We replaced each of the three regions of OR320a with the corresponding region of OR320b while maintaining the remaining sequence intact. Conversely, we replaced each of the three regions in OR320b with those of OR320a. We first assessed the surface expression level of the six chimeras. All constructs showed expression in a similar fraction of cells (Fig. S5; one-way ANOVA, F = 0.5, p = 0.7). Subsequently, we compared responses of all receptor chimeras to 3-keto-1-ene PZS and DKPES using CRE-luciferase assays (Fig. 4, B and C). These two ligands were chosen because their Emax differed the most for both OR320a and OR320b. The receptor chimeras in which the first region of OR320a (TM1–3; e.g. ABB) was substituted into the intact OR320b (e.g. BBB) showed lower EC50 and higher Emax values. We therefore decided to focus on this region thereafter. There are nine amino acid residues that differ between OR320a and OR320b in the first region. We sequentially replaced each of the nine residues and measured the response of each mutant to 3-keto-1-ene PZS and DKPES. There is no significant difference among the mutated receptors' membrane expression (Fig. S6; Kruskal–Wallis test, p = 0.17). Among the nine residues on OR320a, only one mutation (OR320a-C79Y) significantly affected the receptor EC50 to 3-keto-1-ene PZS based on the Kruskal–Wallis ANOVA test (Fig. 5A; EC50, p <0.05, Dunn's multiple-comparison test, p <0.05; Emax, p >0.05). The OR320a-C79Y responses to 3-keto-1-ene PZS were comparable with that of WT-OR320b (Mann–Whitney test; EC50, p = 0.85; Emax, p > 0.9) but were weaker than those induced by the other eight mutants of OR320a and WT-OR320a. OR320a-C79Y showed the lowest Emax to DKPES, even weaker than WT-OR320b (Fig. 5B, EC50, unpaired t tes" @default.
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• W3041567162 date "2020-08-01" @default.
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• W3041567162 title "Two highly related odorant receptors specifically detect α-bile acid pheromones in sea lamprey (Petromyzon marinus)" @default.
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• W3041567162 doi "https://doi.org/10.1074/jbc.ra119.011532" @default.
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