Odorant receptors

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of the invention is odorant receptors.

[0004] 2. Background of the Invention

[0005] The detection and discrimination of the multitude of environmental stimuli by the vertebrate olfactory system results from the activation of olfactory neurons within the olfactory epithelium of the nose (reviewed by Shepherd, 1994; Buck, 1996). The first step in olfactory processing resides at the level of the interaction of odorous ligands with odorant receptors. A large multigene family thought to encode odorant receptors was initially identified in the rat (Buck and Axel, 1991). These receptors are predicted to exhibit a seven transmembrane domain topology characteristic of the superfamily of G protein-coupled receptors. The sizes of the receptor repertoires of different vertebrate species are extremely large and are estimated to contain between 100 and 1000 individual genes (Buck, 1996). These observations suggest that the initial step in olfactory discrimination is accomplished by the integration of signals from a large number of specific receptors, each capable of binding only a small number of structurally-related odorants. Consistent with this model, it has been shown that one rat odorant receptor can be activated by 7 to 10 carbon n-aliphatic aldehydes (Zhao et al., 1997; see also Krautwurst et al., 1998; Malnic et al., 1999). In invertebrates, the C. elegans odr-10 gene encodes a G protein-coupled receptor that is sharply tuned to respond to the odorant, diacetyl (Sengupta et al., 1996; Zhang et al., 1997).

[0006] Other olfactory G protein-coupled receptors unrelated to the receptor gene family first described by Buck and Axel (1991) have been identified in the vomeronasal organ (VNO) of mammals (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). The VNO is a specialization of the peripheral olfactory system in higher vertebrates that receives non-volatile pheromonal and non-pheromonal cues (Halpern, 1987). The VNO receptors are encoded by two unrelated gene families; members of the VNR family are localized in a subpopulation of VNO neurons defined by their expression of the G protein alpha subunit, Gai2 (Dulac and Axel, 1995; Berghard and Buck, 1996; Jia and Halpern, 1996), whereas members of the V2R family are expressed predominantly in a separate subpopulation of Gao-expressing cells (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). Interestingly, the V2R receptors are structurally related to the calcium-sensing receptor (CaSR; Hebert and Brown, 1995) and metabotropic glutamate receptor (mGluR; Tanabe et al., 1992) families. While it has been proposed that both classes of VNO receptors comprise pheromone receptors, the actual function of these orphan receptors awaits a direct demonstration of their ligand binding or ligand activation properties.

[0007] As an approach toward identifying ligands for olfactory receptors, we have pursued an expression cloning strategy using the goldfish as a model system. Fish are thought to respond to a smaller range of odorants than terrestrial vertebrates and thus appear to possess a smaller repertoire of odorant receptors (Ngai et al., 1993b). Moreover, the odorants that fish detect are water soluble, and include amino acids (feeding cues), bile acids (nonreproductive social cues with possible roles in migration), and sex steroids and prostaglandins (sex pheromonal cues) (reviewed by Hara, 1994; Sorensen and Caprio, 1998). Electrophysiological studies have defined the sensitivities of fish olfactory systems to specific ligands, demonstrating, for example, thresholds for detection in the picomolar (for sex steroids) to nanomolar (for amino acids) range (Hara, 1994). Thus, the defined properties of odorant-evoked pathways in vivo provide an excellent starting point for the molecular and biochemical characterization of fish odorant receptors.

[0008] In the examples below, we describe the expression cloning of a cDNA encoding a goldfish odorant receptor preferentially tuned to recognize basic amino acids. This cDNA encodes a G protein-coupled receptor that shares significant similarity to receptor families that include the CaSR, mGluR, and V2R class of VNO receptors. Degenerate polymerase chain reaction (PCR) reveals other related sequences that are expressed in the goldfish olfactory epithelium. Together our results indicate that these receptors comprise a family of odorant receptors. Moreover, the characterization of the goldfish amino acid receptor's odorant tuning properties provides critical molecular parameters for considering models of molecular recognition and information coding in the olfactory system.

[0009] Aspects of this invention have been published by Speca et al. (Neuron July 1999;23(3):487-98).

SUMMARY OF THE INVENTION

[0010] The invention provides methods and compositions relating to odorant receptors, including a general expression cloning methodology which is useful for identifying novel G protein-coupled receptors and a novel family of odorant receptors and related nucleic acids, ligands, agonists and antagonists. These compositions provide a wide variety of applications such as screening for related receptors, and by modulating the function of the disclosed receptors by modulating their expression or contacting them with agonists, antagonist or ligands modulating reproductive/sexual and non-sexual social behaviors mediated via the olfactory system, reproductive physiologies and olfactory system regulated feeding behaviors, migratory behaviors and events such as conception, implantation, estrous and menstruation.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0011] The following description of particular embodiments and examples are offered by way of illustration and not by way of limitation. While particularly directed and exemplified often in terms of goldfish R5.24, the following descriptions, including fragment limitations and assay utilizations, also apply to the other disclosed CaSR-like polypeptides and polynucleotides.

[0012] The subject domains provide R5.24 domain specific activity or function, such as R5.24-mediated olfaction, ligand signal transducing or transducing inhibitory activity and/or R5.24-specific binding target-binding or binding inhibitory activity. R5.24-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. The specific binding target may be a ligand, agonist or antagonist, a R5.24 regulating protein or other regulator that directly modulates R5.24 activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a R5.24 specific agent such as those identified in screening assays such as described below. R5.24-binding specificity may be assayed by binding equilibrium constants (usually at least about 107 M−1, preferably at least about 108 M−1, more preferably at least about 109 M−1), by the ability of the subject polypeptides to function as negative mutants in R5.24-expressing cells, to elicit R5.24 specific antibody in a heterologous host (e.g a rodent or rabbit), etc.

[0013] Exemplary suitable R5.24 polypeptides (a) SEQ ID NO:02, or a functional deletion mutant thereof or a sequence about 60-70%, preferably about 70-80%, more preferably about 80-90%, more preferably about 90-95%, most preferably about 95-99% similar to the R5.24 sequence disclosed herein as determined by Best Fit analysis using default settings and/or (b) is encoded by a nucleic acid comprising a natural R5.24 encoding sequence (such as SEQ ID NO:01) or a fragment thereof at least 36, preferably at least 72, more preferably at least 144, most preferably at least 288 nucleotides in length which specifically hybridizes thereto. Suitable deletion mutants are readily screened in R5.24 binding or activation assays as described herein. Preferred R5.24 domains/deletion mutants/fragments comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of SEQ ID NO:2 and provide a R5.24 specific activity, such as R5.24-specific antigenicity and/or immunogenicity, especially when coupled to carrier proteins. The subject domains provide R5.24-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to R5.24-specific domains are covalently coupled to keyhole limpet antigen (I<LH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of R5.24-specific antibodies is assayed by solid phase immunosorbant assays using immobilized R5.24 polypeptides. R5.24 specific antigenic and/or immunogenic peptides encompass diverged sequence regions, preferably diverged extracellular or cytosolic regions, as seen in alignments with related sequences human CaSR, Fugu Ca02.1, mouse V2R2 and rat mGluR1.

[0014] Suitable natural R5.24 encoding sequence fragments are of length sufficient to encode such R5.24 domains. In a particular embodiment, the R5.24 fragments comprise species specific fragments; such fragments are readily discerned from alignments. Exemplary such R5.24-1 immunogenic and/or antigenic peptides are shown in Table 1.

1TABLE 1Immunogenic R5.24-1 polypeptides eliciting RS.24-1 specificrabbit polyclonal antibody: R5.24 polypeptide-KLH conjugatesimmunized per protocol described above.R5.24 PolypeptideImmunogenicitySEQ ID NO:02, res. 1-10+++SEQ ID NO:02, res. 29-41+++SEQ ID NO:02, res. 75-87+++SEQ ID NO:02, res. 92-109+++SEQ ID NO:02, res. 132-141+++SEQ ID NO:02, res. 192-205+++SEQ ID NO:02, res. 258-269+++SEQ ID NO:02, res. 295-311+++SEQ ID NO:02, res. 316-330+++SEQ ID NO:02, res. 373-382+++SEQ ID NO:02, res. 403-422+++SEQ ID NO:02, res. 436-442+++SEQ ID NO:02, res. 474-485+++SEQ ID NO:02, res. 502-516+++SEQ ID NO:02, res. 561-576+++SEQ ID NO:02, res. 595-616+++SEQ ID NO:02, res. 640-656+++SEQ ID NO:02, res. 683-697+++SEQ ID NO:02, res. 717-732+++SEQ ID NO:02, res. 768-777+++SEQ ID NO:02, res. 798-813+++SEQ ID NO:02, res. 829-843+++SEQ ID NO:02, res. 844-877+++SEQ ID NO:02, res. 852-875+++

[0015] In one embodiment, the R5.24 polypeptides are encoded by a nucleic acid comprising SEQ ID NO:01 or a fragment thereof which hybridizes with a full-length strand thereof, preferably under stringent conditions. Such nucleic acids comprise at least 36, preferably at least 72, more preferably at least 144 and most preferably at least 288 nucleotides of SEQ ID NO:01. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO4, pH 7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE (Conditions I); preferably hybridizing in a buffer comprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE buffer at 42° C. (Conditions II). Exemplary nucleic acids which hybridize with a strand of SEQ ID NO:01 are shown in Table 2.

2TABLE 2Exemplary nucleic acids which hybridize witha strand of SEQ ID NO:01 under Conditions I and/or II.R5.24 Nucleic AcidHybridizationSEQ ID NO:01, nucl. 1-47+SEQ ID NO:01, nucl. 58-99+SEQ ID NO:01, nucl. 95-138+SEQ ID NO:01, nucl. 181-220+SEQ ID NO:01, nucl. 261-299+SEQ ID NO:01, nucl. 274-315+SEQ ID NO:01, nucl. 351-389+SEQ ID NO:01, nucl. 450-593+SEQ ID NO:01, nucl. 524-546+SEQ ID NO:01, nucl. 561-608+SEQ ID NO:01, nucl. 689-727+SEQ ID NO:01, nucl. 708-737+SEQ ID NO:01, nucl. 738-801+SEQ ID NO:01, nucl. 805-854+SEQ ID NO:01, nucl. 855-907+SEQ ID NO:01, nucl. 910-953+SEQ ID NO:01, nucl. 1007-1059+SEQ ID NO:01, nucl. 1147-1163+SEQ ID NO:01, nucl. 1258-1279+SEQ ID NO:01, nucl. 1375-1389+SEQ ID NO:01, nucl. 1581-1595+SEQ ID NO:01, nucl. 1621-1639+SEQ ID NO:01, nucl. 1744-1755+SEQ ID NO:01, nucl. 1951-1969+

[0016] A wide variety of cell types express R5.24 polypeptides subject to regulation by the disclosed methods, including many neuronal cells, transformed cells, infected (e.g. virus) cells, etc. Ascertaining R5.24 binding or activation is readily effected by binding assays or cells function assays as disclosed herein. Accordingly, indications for the subject methods encompass a wide variety of cell types and function, etc. The target cell may reside in culture or in situ, i.e. within the natural host.

[0017] In another aspect, the invention provides methods of screening for agents which modulate R5.24-ligand interactions. These methods generally involve forming a mixture of a R5.24-expressing cell, a R5.24 ligand and a candidate agent, and determining the effect of the agent on the R5.24-ligand interaction. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

[0018] The amino acid sequences of the disclosed R5.24 polypeptides are used to back-translate R5.24 polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural R5.24-encoding nucleic acid sequences (“GCG” software, Genetics Computer Group, Inc, Madison Wis.). R5.24-encoding nucleic acids are used in R5.24-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, etc.

[0019] The invention also provides nucleic acid hybridization probes and replication/amplification primers having a R5.24 cDNA specific sequence comprising a fragment of SEQ ID NO: 1, and sufficient to effect specific hybridization thereto. Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 nucleotides in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO4, pH 7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE buffer at 42° C. R5.24 nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410). In addition, the invention provides nucleic acids having a sequence about 60-70%, preferably about 70-80%, more preferably about 80-90%, more preferably about 90-95%, most preferably about 95-99% similar to SEQ ID NO: 1 as determined by Best Fit analysis using default settings.

[0020] The subject nucleic acids are of synthetic/non-natural sequences and/or are recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. The subject recombinant nucleic acids comprising the nucleotide sequence of disclosed vertebrate R5.24 nucleic acids, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, more preferably fewer than 500 bp, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.

[0021] The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of R5.24 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional R5.24 homologs and structural analogs.

EXAMPLES

[0022] We have developed a general method for expression cloning novel G protein coupled receptors as a strategy for identifying vertebrate odorant receptors. The sensitivity and flexibility of this technique allows the activation of multiple G protein pathways to be detected, even when the relevant receptor mRNA constitutes as little as 0.1% of the injected RNA population. Thus, this system facilitates the functional identification of cDNAs corresponding to any G protein coupled receptor for which specific agonists are available. By using our expression cloning approach, we isolated from the goldfish olfactory epithelium a cDNA encoding a receptor that is activated by amino acid odorants. Characterization of this receptor, receptor 5.24, reveals that it is preferentially activated by arginine and lysine and interacts with these compounds with high affinity (Kd=˜100 nM). Other amino acids bind to receptor 5.24 with lower affinity; parameters affecting binding specificity appear to include the structure and/or charge of the side chain's terminal functional moiety, as well as its backbone length. The receptor demonstrates stereospecificity and does not appear to bind amino acid neurotransmitters found in the peripheral olfactory system. The observed affinity of this cloned receptor agrees well with the in vivo threshold sensitivities of the goldfish olfactory system to arginine (ca. 1 nM). However, the cloned goldfish receptor appears to be different from basic amino acid binding sites characterized in isolated fish olfactory cilia, which show 50˜100-fold lower affinities for ligand (e.g., Cagan and Zeiger, 1978).

[0023] It has been suggested that amino acid odorant stimuli are transduced by phospholipase-mediated pathways in fish (Huque and Bruch, 1986; Restrepo et al., 1993). Consistent with these observations, our results demonstrate that odorant activation of the cloned goldfish amino acid receptor leads to increased PI turnover in Xenopus oocytes as well as in mammalian cells. No coupling to Gαs-like pathways is observed even though these these G protein subunits are present in both heterologous cell systems, indicating that the goldfish amino acid odorant receptor and all olfactory CaSR receptors, stimulate PI turnover in vivo. Interestingly, these receptors are expressed in microvillous neurons (see also Cao et al., 1998), which morphologically resemble the sensory neurons of the VNO. Both the VNO as well as fish olfactory microvillous neurons do not appear to express cyclic nucleotide-gated channel alpha subunits (Berghard et al., 1996), which are required for transducing odorant-evoked cAMP elevations into changes in membrane potential in mammalian ciliated olfactory neurons (Brunet et al., 1996).

[0024] Receptor 5.24 shares sequence similarity to previously identified G protein receptors, including the CaSR, mGluR, and V2R families (Nalanishi et al., 1990; Hebert and Brown, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). Additional CaSR-like receptors are also expressed in the goldfish olfactory epithelium. Our results indicate that receptor 5.24 is a member of a multigene family of receptors expressed by olfactory sensory neurons, and together with our biochemical characterization of this receptor provide direct evidence that the family of olfactory CaSR-like receptors are in fact odorant receptors.

[0025] The mammalian V2R receptors have been proposed to constitute a family of pheromone receptors based on their expression in the VNO (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). It should be noted, however, that the VNO—a specialization of the olfactory apparatus in terrestrial vertebrates—receives both pheromonal as well as non-pheromonal cues (Halpern, 1987). While the ligand specificities of the mammalian V2R receptors remain to be demonstrated, our data clearly show that at least one member of the goldfish receptor family, receptor 5.24, is an odorant receptor that recognizes a specific subset of amino acid stimuli. Since amino acid odorants are not pheromones, the family of olfactory CaSR-like receptors, including the mammalian V2R receptors, may in fact function to receive a wide variety of stimuli that includes both pheromonal and non-pheromonal odorants. We disclose that receptors related to receptor 5.24 are used by the fish to detect other amino acid odorants.

[0026] Electrophysiological recordings from isolated salmon olfactory neurons have demonstrated that ˜60% of the cells are sensitive to 0.01-10 μM L-serine (Nevitt and Dittman, 1999). Similarly, single-unit recordings from the catfish olfactory epithelium have shown that ˜40% of the olfactory neurons respond to 100 μM L-arginine (Kang and Caprio, 1995). Multi-unit recordings from the goldfish olfactory epithelium, where activity from 5 cells was detected at each recording site, also suggest that a large fraction of olfactory neurons respond to arginine (25 out of 28 locations with spontaneous activity responded 100 μM L-arginine), but few appeared sensitive to pheromones (e.g., only 25 out of 65 locations responded to 0.1 μM 15-ketoprostaglandin F2a). Thus, the widespread expression of receptor 5.24 mRNA in goldfish olfactory neurons is consistent with electrophysiological recordings which independently suggest that a large fraction of fish olfactory neurons express amino acid odorant receptors.

[0027] The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.

[0028] General Strategy for Expression Cloning of Odorant Receptors. We elected to utilize the goldfish olfactory system, owing to the extensive physiological and behavioral characterization of its responses to both pheromonal and non-pheromonal olfactory stimuli in this species (Sorensen and Caprio, 1998; Sorensen et al., 1998). Our approach was designed to allow for the detection of receptor activation of multiple G protein-mediated pathways—whereas previous studies have demonstrated that odorant-evoked excitatory signaling in mammalian olfactory neurons is mediated exclusively by the intracellular second messenger, cAMP (Brunet et al., 1996), in vitro biochemical studies have suggested that stimulation of phosphatidyl inositol (PI) turnover, resulting in the production of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (IP3), may mediate olfactory signaling in fish (Huque and Bruch, 1986; Restrepo et al., 1993). Thus, for expression cloning of fish odorant receptors it seemed prudent to utilize a system capable of detecting activation of multiple signaling pathways.

[0029] The Xenopus oocyte provides a powerful method for expression cloning certain G protein-coupled receptors, owing to the ability to detect increases in PI turnover through the IP3-mediated release of Ca2+from internal stores and the subsequent activation of Ca2+-dependent C1-channels (Masu et al., 1987). This cell does not normally exhibit an electrophysiologic response to the activation of Gas (and therefore adenylyl cyclase), however. We therefore engineered the oocyte to provide a robust read-out for this G protein-dependent pathway (Lim et al., 1995). This method relies upon the ectopic expression of Gaolf (a Gas-like isoform highly enriched in olfactory cilia; Jones and Reed, 1989) and G protein-gated inwardly rectifying potassium channels (GIRKs), together with candidate receptors (Lim et al., 1995). Potassium currents can be observed in response to gating of GIRK channels by free G protein bg subunits following their dissociation from activated Gas-like or Gai subunits (Reuveny et al., 1994; Lim et al., 1995). To determine whether this system would be amenable to expression cloning, where a cDNA encoding the receptor of interest comprises only a small fraction of a pool of cDNAs, we injected into oocytes RNA encoding the dopamine D1 receptor together with Gaolf and GIRK RNAs, with the amount of receptor RNA diluted 1,000-fold prior to injection. We could typically elicit robust agonist-dependent currents in receptor-expressing oocytes. These controls indicated that a pool of ˜1,000 cDNA clones containing a single receptor cDNA could still give rise to a detectable signal when expressed and activated in our assay system. Thus, oocytes expressing Gaolf and GIRK provide a means of expression cloning G protein-coupled receptors whose downstream coupling pathways are ambiguous.

[0030] Identification by Expression Cloning of a cDNA Encoding a Goldfish Odorant Receptor. RNA was synthesized from pools of goldfish olfactory cDNA clones (900 individual clones per pool) and injected into Xenopus oocytes together with synthetic RNAs encoding GIRK and Gaolf. Oocytes were then screened for responses upon exposure to odorant cocktails containing amino acids, bile acids, or sex pheromones. Odorants were tested at concentrations 100- to 1,000-fold higher than those required to elicit half-maximal physiological responses in vivo (Caprio, 1978; Sorensen et al., 1987; Sorensen et al., 1988; Michel and Lubomudrov, 1995; see Experimental Procedures); these concentrations did not elicit activity in oocytes injected only with GIRK and G protein RNAs. Oocytes injected with RNA from one pool, pool 19, demonstrated a robust response to amino acids, but not to bile acids or sex pheromones. The response to amino acids was biphasic, beginning with an oscillating inward current above the basal inward K+ current, followed by a decline in inward current to below the basal level. Such a biphasic effect is thought to be caused by Gaq-mediated activation of phospholipase C (responsible for the intial inward current via IP3) and protein kinase C (leading to suppression of the K+ current through phosphorylation of GIRK channel subunits) (Sharon et al., 1997). Indeed, the oscillating inward currents in response to amino acids were still observed in oocytes expressing pool 19 RNA without Gaolf or GIRK RNAs, suggesting that the receptor contained in this pool interacts with a phospholipase-mediated pathway.

[0031] Iterative subdivision of pool 19 by sib-selection allowed the isolation of a single clone encoding a receptor, designated receptor 5.24. Receptor 5.24 responds best to basic L-amino acids, showing roughly equivalent evoked currents upon activation by arginine and lysine, smaller responses to neutral aliphatic L-amino acids (e.g., methionine, isoleucine, threonine, serine, alanine) and little or no response to acidic and aromatic L-amino acids (e.g., glutamate, aspartate, tyrosine, phenylalanine, tryptophan, histidine); the receptor is not activated by D-amino acids (all amino acids referred to hereafter are L-isomers unless stated otherwise).

[0032] High-Affinity Binding of Radiolabeled L-Arginine to the Cloned Goldfish Odorant Receptor. To determine the affinity of the cloned goldfish amino acid receptor for basic amino acids, we characterized the ligand-receptor interaction by radiolabeled ligand binding to receptors expressed in mammalian cells. Human embryonic kidney (HEK) 293 cells were transfected with expression plasmids containing the receptor 5.24 cDNA insert. Membranes prepared from receptor 5.24-expressing cells exhibit saturable binding at concentrations of up to 1 mM 3H-arginine, and the extent of ligand binding is significantly higher than with membranes from control cells. Further analysis of specific binding activity from multiple experiments indicates a receptor with a single binding site (Hill coefficient=0.95±0.07 [mean±SEM], n=4 determinations) with a dissociation constant (Kd) of 121±33 nM arginine (mean±SEM, n=4; range: 52-207 nM). We next wished to determine what second messenger pathway receptor 5.24 couples to in HEK 293 cells. Control cells or cells expressing receptor 5.24 were exposed to varying concentrations of arginine and assayed for the accumulation of IP3 and cAMP. Arginine elicits a specific increase in IP3 in receptor 5.24-expressing cells in a dose-dependent manner. By way of contrast, arginine at 0.1 mM or 10 mM does not cause a detectable change in cAMP levels in these cells, even though activation of b-adrenergic receptors (expressed endogenously by the host cell line) leads to increased cAMP accumulation. These results indicate that, as in Xenopus oocytes, receptor 5.24 preferentially stimulates PI turnover in HEK 293 cells.

[0033] Structure-Activity Properties of Compounds Interacting with the Cloned Goldfish Amino Acid Odorant Receptor. An understanding of how odorant receptors are used to encode olfactory information requires a characterization of the odorant specificities of individual receptor types. We therefore wished to determine the relative specificity of receptor 5.24 for structurally related ligands. Since this receptor binds to arginine with high affinity, we screened other compounds for receptor 5.24 binding by using a 3H-arginine displacement assay. While these assays do not give information regarding whether a compound functions as an agonist, partial agonist, or antagonist, they do nonetheless allow insight into the molecular specificity of the receptor. Briefly, 3H-arginine binding to membranes from receptor 5.24-expressing HEK 293 cells was assayed in the absence or presence of varying concentrations of competitor ligands. Consistent with their profiles of receptor 5.24 activation in Xenopus oocytes, arginine and lysine displace 3H-arginine binding with similar concentration dependencies, showing half-maximal inhibition (IC50) at ˜0.3 and 0.5 mM, respectively (corresponding to inhibition constants or Ki's of 80 and 90 nM; see Table 3). Glutamate, which does not appear to activate receptor 5.24 expressed in Xenopus oocytes, displaces 3H-arginine approximately 80-fold less well than either unlabeled arginine or lysine (IC50=˜20 mM; Ki=6.7 mM). Interestingly, agmatine, a decarboxylated analogue of arginine, displaces 3H-arginine very poorly (Ki>1 mM; see below and Table 4).

3TABLE 3Binding Affinity of the Receptor for Amino Acids,Amino Acid Derivatives and Neurotransmitters. Binding affinity(Ki) was determined by the ability of the individualamino acids to displace 3H-L-arginine binding from membranesprepared from HEK 293 cells expressing receptor 5.24.AMINO ACID CLASSAMINO ACIDKi (μM)Basic side chainL-Arginine0.08L-Lysine0.09Sulfur-containing side chainL-Cysteine0.53L-Methionine0.81Amide side chainL-Glutamine0.32L-Asparagine2.1Acidic side chainL-Glutamate6.7L-Aspartate27Long aliphatic side chainL-Isoleucine2.2L-Leucine4.4L-Valine6.2Short aliphatic side chainL-Serine2.8L-Threonine3.2L-Glycine3.9L-Alanine5.6L-Proline58Aromatic side chainL-Tryptophan4.1L-Phenylalanine5.8L-Histidine13L-Tyrosine16Arginine/Lysine derivativesAgmatine>1000L-Citruline0.96L-Ornithine1.00L-Homoarginine1.63L-NAME1.02Cadaverine>1000Putrescine>1000Neurotransmittersγ-Aminobutyric Acid (GABA)>1000Taurine>1000Carnosine>1000

[0034]

4

TABLE 4

Odorant Cocktails Used for Screening

the Goldfish Olfactory cDNA Library in

Xenopus Ooctyes

Amino Acids/Bile Acids

L-Amino Acids
Serine, Alanine, Methionine, Glutamic

(Final concentration 50 mM)
Acid, Arginine, Glutamine, Lysine,

Histidine

Bile Acids
Taurocholic Acid, Taurolithocholic Acid

(Final concentration 1 mM)
Sulfate, Taurodeoxycholic Acid,

Taurochenodeoxycholic Acid, Glycocholic

Acid, Prostaglandins/Sex Steroids

Prostaglandins
Prostaglandin F2a, 15-Ketoprostaglandin

(Final concentration 100 mM)
F2a

Sex Steroids
17,20 b-dihydroxy-4-pregnen-3-one, 17,20

(Final concentration 10 mM)
b-dihydroxy-4-pregnen-3-one Sulfate

[0035] We performed comparative binding studies for 20 naturally occurring amino acids as well as amino acid analogues and neurotransmitters (Tables 3 and 4). A number of trends are evident from this analysis. First, receptor 5.24 apparently is tuned to recognize amino acids containing basic R group side chains; of the 20 amino acids tested, arginine and lysine display the highest affinities. In addition, 10- to >100-fold lower affinities are observed with arginine and lysine analogues, and no specific interactions could be detected for the amino acid neurotransmitters g-amino butyric acid (GABA), camosine, or taurine (Ki>1 mM), which are present in the peripheral olfactory system (Nicoll, 1971; Collins, 1974; Margolis, 1974). Second, the side chain's terminal functional group is an important parameter in determining specificity. Substitution of the basic side chain with R groups containing amide (glutamine, asparagine), sulfur-containing (cysteine, methionine), or carbamyl (citrulline) moieties results in a ˜4- to ˜25-fold decrease in affinity. Amino acids with side chains containing terminal amide or sulfur-containing groups in general demonstrate higher affinity than those with aliphatic side chains lacking these structures. Substitution with acidic side chains (glutamate, aspartate) results in a large (˜80- to ˜300-fold) decrease in affinity. Amino acids containing cyclized (proline) or aromatic (tryptophan, phenylalanine, histidine, tyrosine) side groups in general interact with receptor 5.24 with low affinities. Third, specificity is based in part on the R group's carbon backbone length, as illustrated by comparing lysine (Ki=0.09 mM) vs. omithine (Ki=1.0 mM; backbone shorter than lysine's by one carbon) and arginine (Ki=0.08 mM) vs. homoarginine (Ki=1.6 mM; backbone longer than arginine's by one carbon). Tuning of this receptor based on carbon chain length appears to be sharper than has been observed for a cloned rat odorant receptor, which shows a somewhat broad response profile for 7-, 8-, 9-, and 10-carbon n-aliphatic aldehydes (Zhao et al., 1997). Finally, the a-carboxylic acid moiety is critical for receptor binding, as agmatine and cadaverine (decarboxylated analogues of arginine and lysine, respectively) show essentially no binding to receptor 5.24 (Ki 's >1 mM, or greater than 10,000 times the Kd for arginine or lysine). However, the interaction with the carboxylic acid is probably not dependent on the negative charge per se, since the carboxy-methylated arginine analogue, L-NAME, binds the receptor with modest affinity (Ki=1.0 mM).

[0036] The Goldfish Amino Acid Odorant Receptor Belongs to a Family of CaSR-Like Receptors. The sequence of the goldfish receptor 5.24 cDNA predicts a protein with seven membrane spanning regions preceded by a 566 amino acid N-terminal extracellular domain. Receptor 5.24 exhibits significant similarity to previously identified G protein-coupled receptors, including the CaSR (Hebert and Brown, 1995), the family of mGluR receptors (Tanabe et al., 1992), the family of vomeronasal V2R receptors (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997), a family of olfactory CaSR- and V2R-related receptors found in the puffer fish, fugu (Naito et al., 1998) and goldfish (Cao et al., 1998), and two putative taste receptors found in the mouse (Hoon et al., 1999). Receptor 5.24 shares between 25 and 33% amino acid sequence identity with these receptors, showing a somewhat greater degree of similarity with human and fugu CaSR sequences. In spite of this weak homology to the CaSR and mGluR sequences, receptor 5.24 is not activated by calcium or glutamate.

[0037] The N-terminal extracellular domain of the mGluR is required for glutamate binding (O'Hara et al., 1993; Takahashi et al., 1993), and this region of the metabotropic as well as ionotropic glutamate receptors shows significant sequence similarity with bacterial periplasmic amino acid binding proteins (Nakanishi et al., 1990; O'Hara et al., 1993). Molecular modeling of the mGluR1 N-terminal domain based on the bacterial protein structures suggests that serine 165 and threonine 188 may interact with glutamate by coordination of the amino acid ligand's a-amino and a-carboxyl moieties; conservative mutations at these positions in mGluR1 results in a significant reduction in agonist binding (O'Hara et al., 1993). Interestingly, the corresponding two residues are conserved in receptor 5.24 (serine 152 and threonine 175), but not in the CaSR or in every V2R sequence. These residues in the goldfish receptor probably serve to coordinate the high affinity binding of amino acid odorants.

[0038] A Family of Olfactory CaSR-Like Receptors Related to the Goldfish Amino Acid Odorant Receptor. Recent work by others has identified members of a family of CaSR-like receptors that show similarity to receptor 5.24 and are specifically expressed in the goldfish olfactory epithelium (Cao et al., 1998). To identify additional receptor sequences in this gene family, we performed PCR on goldfish olfactory cDNA using degenerate primers based on motifs conserved among the N-terminal regions of receptor 5.24, CaSR, mGluR, and V2R sequences. Subcloning and DNA sequencing of the resulting PCR products revealed numerous CaSR-like sequences (SEQ ID NOS:3-6) that can be grouped into 5 distinct subfamilies. Within this portion of the N-terminal domain, the goldfish CaSR-like olfactory receptor subfamilies exhibit between 20 and 43% amino acid identity. Receptor 5.24 is the most divergent member of the group of receptors identified thus far, showing 20 to 27% similarity with the other sequences in this region. The receptor 5.24 full length cDNA detects 1-2 bands in genomic DNA blots, suggesting that this gene exists as a single copy in the goldfish genome.

[0039] In addition, full-length CaSR-like sequences are readily isolated from cDNA libraries using the foregoing techniques. For example, full, native length CaSR-like protein sequences SEQ ID NOS:7-8, 10 and 12 are encoded by full length goldfish cDNAs. Natural coding sequences for SEQ ID NOS:10 and 12 are shown as SEQ ID NOS:9 and 11, respectively. Furthermore, heterologous CaSR-like sequences are readily isolated using these techniques. For example, a zebrafish protein shown to be functionally and structurally similar to goldfish 5.24 (ca. 70% amino acid identity)is shown as SEQ ID NO: 14 (the natural coding sequence is shown as SEQ ID NO:13).

[0040] Expression Patterns of Goldfish Olfactory CaSR-Like Receptors. Analysis of receptor 5.24 expression by RNA blots revealed that the mRNA encoding this receptor is expressed in olfactory epithelium but not in brain, kidney, liver, muscle, ovary, intestine, or testis. The receptor 5.24 probe also detects at high stringency an MRNA in skin from the trunk, gill, lips, tongue, and palatal organ. Similar RNA blot analysis with probes for receptor 5.3 and receptor 3.13 indicate that these genes are expressed exclusively in olfactory epithelium.

[0041] RNA in situ hybridizations were performed to determine the expression patterns of the goldfish olfactory CaSR-like sequences in the olfactory epithelium. We probed tissue sections with an 35S-labeled antisense RNA probe corresponding to the N-terminal extracellular domain of receptor 5.24. This is the most divergent region of this class of receptor, and therefore is expected to anneal only to receptor 5.24 under the stringent conditions of hybridization used in these experiments. We found receptor 5.24 MRNA expressed widely over the apical and medial portions of the olfactory sensory epithelium-regions which contain the olfactory sensory neurons. In situ hybridizations using a digoxigenin-labeled probe confirm that receptor 5.24 is expressed in a large fraction of cells in the neuronal layers of the sensory epithelium. These observations are consistent with electrophysiological recordings which suggest that roughly half of the olfactory neurons in fish can respond to amino acid stimuli (Kang and Caprio, 1995; Nevitt and Dittman, 1999). In situ hybridizations using a probe for receptor 5.3 indicate that this MRNA is also localized to a large subset of cells. In contrast to the broad patterns of receptor 5.24 and receptor 5.3 expression, other CaSR-like receptors are expressed in punctate patterns within the olfactory epithelium. Probes for receptor 3.13, receptor 9, and receptor 10 subfamily members hybridize to a small subset of cells (ca. 1˜5% each).

[0042] We noticed that signal strengths for receptor 5.24 in situ hybridizations were consistently weaker than for the other receptors. This appears to be a peculiarity of the receptor 5.24 MRNA and not due to a low level of its expression, as screening of the goldfish olfactory cDNA library with olfactory CaSR-like sequences reveals that these RNAs are expressed at roughly equivalent levels (each sequence is represented at between 1 in ˜100,000 clones [receptors 5.24 and 5.3] to 1 in 600,000 clones [receptor 10.8]).

[0043] Cells expressing the goldfish CaSR-like receptors localize more apically than we typically observe for olfactory neurons expressing the olfactory cyclic nucleotide-gated ion channel (Goulding et al., 1992; see below) or odorant receptors belonging to the family originally described by Buck and Axel (1991). The fish olfactory epithelium contains two major classes of sensory cells, the ciliated and microvillous neurons, that are segregated along the apical-basal axis (Yamamoto, 1982). The microvillous cells reside in the apical portion of the epithelium in a zone above and distinct from the ciliated neurons, whose cell bodies lie medially. Previous in situ hybridization studies have shown that the class of receptors originally described by Buck and Axel (1991), as well as the cyclic nucleotide-gated ion channel, are expressed in the medially-disposed ciliated olfactory neurons in fish (Goulding et al., 1992; Ngai et al., 1993a; Ngai et al., 1993b). Thus, the goldfish CaSR-like receptors are probably expressed in microvillous olfactory neurons (see also Cao et al., 1998).

[0044] Localization of Receptor 5.24 mRNA Expression in Non-Olfactory Tissue. The expression of receptor 5.24 mRNA in external epithelia raises the question as to whether this receptor might be playing a chemosensory function outside of the olfactory system. These epithelia contain both taste buds as well as solitary chemosensory cells; both of these systems are sensitive to amino acid stimuli in fish (Sorensen and Caprio, 1998), although facial nerve recordings in goldfish indicate that arginine is a poor taste stimulus in this species. We therefore performed additional in situ hybridizations to determine whether receptor 5.24 is indeed expressed in these chemosensory systems. Exemplary data showed representative tissue sections of gill rakers that were hybridized with a digoxigenin-labeled receptor 5.24 probe. The rakers are non-respiratory structures associated with the gill arches and are covered with an epithelium containing taste buds and solitary chemosensory cells. Examination of these tissue sections as well as numerous others similarly hybridized with the receptor probe indicates that this sequence is expressed widely in the overlying epithelium, but is distinctly excluded from taste buds. In addition, the epithelial cells expressing receptor 5.24 are far too numerous to be accounted for solely by the solitary chemosensory cells, which are relatively rare and dispersed in the epithelium (hence the term “solitary;” Sorensen and Caprio, 1998). Thus, these results argue against a role for receptor 5.24—an odorant receptor tuned to recognize basic amino acids —in non-olfactory chemosensory transduction.

[0045] Experimental Procedures: Expression Cloning. Poly(A)+RNA was prepared from adult male goldfish olfactory rosettes. CDNA was synthesized using an oligo(dT) primer and double stranded DNA was ligated directionally into pSPORT-1 plasmid (Life Technologies, Inc.) via 5′ Sal 1 and 3′ Not I restriction sites. Ligation reactions were introduced into E. coli by electroporation. Plasmid DNA was prepared from pools of 900-1000 clones, linearized with Not I, purified, and used as template for in vitro transcription with T7 RNA polymerase. For production of cRNAs encoding G protein and GIRK subunits, cDNAs for Gaolf (Jones and Reed, 1989) and the GIRK subunits Kir 3.1 (Reuveny et al., 1994) and Kir 3.4 (Ashford et al., 1994) were amplified by PCR using Pfu polymerase and subcloned into the RNA expression vector, pGEMHE (Liman et al., 1992). Following in vitro transcriptions, cRNAs were precipitated in LiCl and resuspended in water.

[0046] Oocytes were removed from anesthetized Xenopus laevis and treated with collagenase. Forty nanograms of cRNA from each cDNA library pool (˜40 pg cRNA/clone) was injected per oocyte, together with cRNAs encoding Gaolf and the GIRK subunits Kir 3.1 and Kir 3.4 (˜30 pg each). Approximately 30 pools were assayed before these primary screens were halted. Injected oocytes were incubated at 17° C. for over 80 hours prior to electrophysiological recordings. Recordings were performed by two-electrode voltage clamping using an Axoclamp-2A amplifier (Axon Instruments) or a Dagan Calif.-1 amplifier (Dagan Corp.). Data acquisition and analysis were performed using pCLAMP software (Axon Instruments). Membrane potential was held at −80 mV. For trials involving GIRK, oocytes were first perfused with Na-MBSH (88 mM NaCl, 1 mM KCl, 2.4 MM NaHCO3, 10 mM Hepes, pH 7.5, 0.82 mM MgSO4[7H20], 0.33 mM Ca[NO3]2[4H20], 0.41 mM CaCl2[2H20]), and then switched into K-MBSH, which contains elevated K+(88 mM KCl, 1 mM NaCl) until the basal current had stabilized (˜45 seconds) before challenging with agonist. Recordings on oocytes injected with receptor 5.24 in the absence of Gaolf and/or GIRK subunits were performed in Na-MBSH. Oocytes were exposed to solutions containing different agonists by switching bathing solutions with an 8 channel valve (Hamilton).

[0047] DNA sequencing was carried out with a Pharmacia AlfExpress sequencer. Sequences were analyzed using MacVector software.

[0048] Mammalian Cell Culture and DNA Transfections. For cell transfections, the receptor 5.24 cDNA insert was subcloned into two expression vectors: CMVI, which utilizes a human cytomegalovirus (CMV) immediate early promoter-enhancer plus the CMV intron A sequence to drive expression, and 608RX-2.2L, which is similar to CMVI except the cDNA insert is followed by an internal ribosome entry site (IRES)—enhanced green fluorescent protein (EGFP) coding sequence; 608RX-2.2L also contains a puromycin resistance gene. For transient assays, HEK 293 cells expressing the SV40 large T antigen (293 TSA cells) were transfected with the CMVI-receptor 5.24 or CMVI control plasmid using lipofectamine (Life Technologies) and harvested at 48 hours for membrane preparations. For stable cell lines, 293-TSA cells were transfected with the 608RX-2.2L-receptor 5.24 plasmid or a control 608RX-2.2L plasmid and selected in puromycin. Colonies showing high levels of EGFP fluorescence (as judged by epifluorescence microscopy) were picked, expanded, and screened for 3H-L-arginine binding (see below). In five independent receptor 5.24-transfected stable cell lines, receptor densities (Rt) varied between 0.5 and 6.0 pmol L-arginine binding sites/mg membrane protein. In four independent 608RX-2.2L-transfected control cell lines, EGFP expression was comparable to receptor 5.24-transfected cells, but L-arginine binding was indistinguishable from untransfected cells (Rt=0.01-0.03 pmol/mg). All competition binding assays and signal transduction studies were performed using the stable cell line 5.24-20 (Rt=2.0 pmol/mg) and a control cell line (2.2-9) stably transfected with vector alone (Rt=0.03 pmol/mg).

[0049] Membrane Preparations and Ligand Binding Assays. Membranes for ligand binding assays were prepared by washing confluent cells three times with phosphate buffered saline (PBS), detaching cells with a PBS-based enzyme-free dissociation solution, and resuspending cells in ice-cold 5.0 mM Hepes, pH 7.4, 1.0 mM EDTA, 1.0 μg/ml leupeptin, 0.5 mM PMSF. All subsequent manipulations were performed on ice. After a 30 min incubation, the cell suspension was homogenized and centrifuged at 100,000×g for 30 min. Cell membrane pellets were washed twice by resuspension and centrifugation in binding buffer (20 mM Hepes, pH 7.4, 1.0 mM EDTA, 2.0 mM MgCl2, 1.0 μg/ml leupeptin, 0.5 mM PMSF), resuspended in binding buffer, and frozen at −80° C.

[0050] Saturation binding assays were performed using 20-25 μg of membrane protein in a final volume of 100 μl binding buffer and increasing concentrations of [2,3-3H] L-arginine (specific activity=40 Ci/mmol; Dupont NEN). Non-specific binding was measured in the presence of 500 μM unlabeled L-arginine. Competition assays were performed with 500 nM [2,3-3H] L-arginine and increasing concentrations of competitor ligands. All incubations were performed at 4° C. for 60 min and terminated by rapid filtration through Whatman GF/C filters pretreated with ice cold 0.1% polyethyleneimine. Filters were washed three times with 4.0 ml ice cold 20 mM Hepes, pH 7.4, and retained radioactivity was measured by scintillation counting. All experiments were performed in triplicate and repeated at least twice. Data were analyzed by nonlinear curve fitting using Origin software (Microcal). The concentration of competitor which caused 50% inhibition of 3H-L-arginine binding (IC50) was determined by non-linear curve-fitting; inhibition constants were calculated according to the equation Ki=(IC50)/(1+[3H-arginine]/Kd).

[0051] IP3 and cAMP Measurements. For measurements of IP3 and cAMP, cell lines 5.24-20 and 2.2-9 (negative control) were plated at densities of 3.5×105 cells/well in 24 well plates and incubated overnight in DMEM containing 10% FBS. All subsequent manipulations prior to reaction termination were performed at 37° C. in 5% CO2. Prior to odorant exposure, confluent cells were washed twice with PBS to remove free amino acids and incubated for 3 hours in amino acid-free media (Earle's Balance Salt Solution containing 0.1% BSA) to allow for recovery from potential desensitization of receptors or downstream signaling pathways. For IP3 assays, cells were washed once with PBS, pre-incubated for 15 min in PBS containing 10 mM LiCl, and then exposed to odorants in 400 μl PBS/10 mM LiCl for 30 min. IP3 levels were determined by a radioreceptor competition assay (Dupont NEN). For cAMP measurements, after the 3 hour amino acid-free preincubation cells were washed once with PBS, preincubated for 15 min in PBS containing 0.5 mM IBMX, and exposed to odorants in 400 μl PBS/0.5 mM IBMX for 30 min. cAMP levels were determined by radioimmunoassay (DuPont NEN).

[0052] PCR. PCR was performed to identify additional CaSR-like odorant receptor cDNAs. Three degenerate oligonucleotide primers were designed based on an alignment of receptor 5.24, mGluR (Duvoisin et al., 1995), human CaSR (Garrett et al., 1995), and mammalian V2R2 (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997) sequences:

[0053] Primer A: corresponding to amino acids 211-215 in receptor 5.24;

[0054] Primer B: corresponding to amino acids 518-514 in receptor 5.24;

[0055] Primer C: corresponding to amino acids 755-751 in receptor 5.24.

[0056] Nested PCR was performed on plasmid library pools containing approximately 20,000 clones. DNA from each library pool was used as template for a primary PCR reaction using a 5′T7 primer with Primer C. Primary PCR reactions were separated on a 1% agarose gel and products between 1-4 kb were excised, eluted, and used as template for a secondary PCR reaction using Primer A and Primer B. Secondary PCR products were electrophoresed on a 1% agarose gel and fragments of ˜1 kb were subcloned into the TA plasmid vector (Invitrogen) and sequenced.

[0057] RNA Blot Analysis and In Situ Hybridizations. The distribution of receptor 5.24 mRNA in goldfish tissues was determined by RNA blot analysis, using 32P-labeled antisense RNAs as probes at high stringency (Ambion). One-half microgram of poly(A)-enriched RNA from each goldfish tissue analyzed was electrophoresed under denaturing conditions, blotted to a nylon membrane, and probed with a 600 nt RNA probe corresponding to amino acids 389-600 of receptor 5.24. Since the full-length receptor 5.24 cDNA appears to recognize a single gene in genomic DNA blots, and sequences encoding this region comprise the most divergent portion of this class of receptors, this probe most likely is entirely specific for receptor 5.24 RNA under stringent hybridization conditions. As a control, the membrane was subsequently hybridized to a goldfish b-actin RNA probe.

[0058] RNA in situ hybridizations were performed on 20 mm-thick fresh frozen tissue sections from adult goldfish olfactory rosettes, essentially as described (Barth et al., 1996; Barth et al., 1997). Slides were hybridized with 35S-labeled (107 cpm/ml) or digoxigenin-labeled (1 mg/ml) probes at 60-65° C. for a minimum of 16 hours and washed in 0.2×SSC at 65° C. Slides hybridized with 35S probes were additionally treated with 20 mg/ml RNase A, rewashed in 0.2×SSC at 65° C., dehydrated, dipped in Kodak NTB-2 emulsion, exposed for 14-28 days at 4° C., developed, and counterstained with toluidine blue. Digoxigenin-labeled probes were visualized with an alkaline phosphatase-conjugated anti-digoxigenin antibody and chromogenic development in NBT/BCIP.

[0059] For receptor 5.24 localization, a ˜1.8 kb Pst I fragment corresponding to the first 600 amino acids of the full-length receptor was subcloned into pBluescript and used for in vitro transcription of 35S- and digoxigenin labeled RNA probes. Digoxigenin-labeled probes for receptors 3.13 and 5.3 were synthesized from the cloned −1 kb PCR inserts derived from these receptors' N-terminal domains. To identify members of the receptor family initially identified by Buck and Axel (1991), degenerate reverse transcription PCR was carried out on goldfish olfactory RNA, as described previously (Barth et al., 1996). One goldfish clone from this class of receptors, designated D1/113-6, was used to synthesize digoxigenin-labeled RNA probes. Cells expressing the olfactory cyclic nucleotide-gated channel were localized with a digoxigenin-labeled probe synthesized from a ˜2.5 kb full-length zebrafish cDNA (see Barth et al., 1996).

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[0127] All publications and patent applications cited in this specification and all references cited therein are herein incorporated by reference as if each individual publication or patent application or reference were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

	Number	Date	Country
Parent	09619353	Jul 2000	US
Child	10151208	May 2002	US

Odorant receptors

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