Characteristic features and ligand specificity of the two olfactory receptor classes from Xenopus laevis
1 University of Hohenheim, Institute of Physiology, 70593 Stuttgart, Germany and
2 Bayer AG, Agricultural Centre, MWF, Geb. 6240, Monheim, Germany
*Author for correspondence (e-mail: breer{at}uni-hohenheim.de)
Accepted June 25, 2001
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Summary |
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Key words: olfactory receptor, Xenopus laevis, sequence analysis, oocyte, heterologous expression.
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Introduction |
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Comparison of the amino acid sequences of olfactory receptors expressed in different vertebrate groups revealed the existence of two classes of olfactory receptors (Freitag et al., 1998); while fish express only class I receptors (Ngai et al., 1993a; Ngai et al., 1993b; Freitag et al., 1998), mammals express class II receptors (Buck and Axel, 1991; Lancet and Ben-Arie, 1993). Olfactory receptor cells of fish respond to water-soluble odorants, i.e. amino acids (Caprio et al., 1989; Restrepo et al., 1990; Kang and Caprio, 1991; Ivanova and Caprio, 1993; Friedrich and Korsching, 1997), whereas olfactory receptor cells of terrestrial vertebrates respond to volatile compounds (Firestein and Werblin, 1989; Kashiwayanagi and Kurihara, 1995; Tareilus et al., 1995; Duchamp-Viret and Duchamp, 1997; Bozza and Kauer, 1998), so it has been assumed that class I receptors may be specialized for the detection of water-soluble odorants, whereas class II receptors recognize volatiles (Freitag et al., 1998; Sun et al., 1999).
Amphibia live in aquatic as well as terrestrial environments, and perform the transition between the aquatic and terrestrial lifestyle during metamorphosis (Nieuwkoop and Faber, 1956). They are capable of detecting both water-soluble and volatile odorants (Elepfandt, 1996). In adult Xenopus laevis this capability is correlated to the functional morphology of the nasal cavity, as olfactory sensory epithelium is located in the water-filled lateral diverticulum (LD) as well as in the air-filled medial diverticulum (MD) (Föske, 1934; Altner, 1962; Weiß, 1986). Molecular studies have revealed the existence of both vertebrate olfactory receptor classes in X. laevis (XOR), and in situ hybridization studies demonstrated that the class I receptors are expressed in the LD, while the class II receptors are expressed in the MD (Freitag et al., 1995; Freitag et al., 1998).
The available sequence data from polymerase chain reaction (PCR) fragments provides only limited information towards the identification of class-specific sequence motifs of both receptor classes from X. laevis. We therefore set out to decipher the complete coding sequences of receptor types from both classes, in order to analyze the peptide sequences of XORs with respect to putative class-specific motifs. In addition, experiments were performed to explore the functional properties of both receptor types by heterologous expression in a species-identical expression system, the Xenopus oocyte.
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Materials and methods |
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Sequence analysis
All sequences were analyzed using the HUSAR 3.0 software package (Heidelberg Unix Sequence Analysis Resources HUSAR, EMBL, Heidelberg, Germany), which is based on the software package 7.2 of the Genetic Computer Group (GCG, Madison, Wisconsin, USA) and the phylogenetic analysis software PHYLIP 3.5c (J. Felsenstein, University of Washington, USA).
Preparation of Xenopus oocytes
Female Xenopus were anaesthetized in a solution containing 0.2% tricain (Sigma) and NaHCO3 (2gl-1). An incision was made in the abdomen, and ovary lobes containing a total of approx. 5001000 oocytes were dissected free. The lobes were cut into small pieces, containing 2050 oocytes each, transferred into OR-Mg medium (82mmoll-1 NaCl, 20mmoll-1 MgCl2, 5mmoll-1 Hepes, 2mmoll-1 KCl, pH 7.5), washed 2x in fresh OR-Mg medium, and digested in collagenase solution consisting of OR-Mg and 2mgml-1 collagenase A (Roche Molecular Biochemicals, Mannheim, Charge 840 892 22) at 18°C for 2-3h, until the oocytes were released and the follicle cells could be removed easily by gentle shaking (Quick and Lester, 1994). After 5 washes in OR-Mg, vital oocytes of stages V and VI were isolated, washed once in ND96 medium [96mmoll-1 NaCl, 5mmoll-1 Hepes, 2mmoll-1 KCl, 1.8mmoll-1 CaCl2, 1mmoll-1 MgCl2, 50µgml-1 Gentamicin (Gibco BRL), pH 7.5], and finally stored in fresh ND96 medium at 18°C.
Injection and electrophysiological measurements
The coding sequence of the olfactory receptors was amplified with specific primers and PWO polymerase under standard conditions. The primers contained appropriate restriction sites, allowing a directional cloning of the receptors into special expression vectors, pGemHe (Liman et al., 1992) or pcDNA3.1(+) (Invitrogen, Gronigen, The Netherlands). Thereafter the orientations and sequences were controlled by sequencing the complete clone. The pGemHe plasmids were subsequently linearized at the 3' end with appropriate restriction enzymes, transcribed in the sense orientation with a Cap-RNA-Kit as described by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany) using T3 polymerase, the cRNA resuspended in RNase-free water and the concentration set to 100ngµl-1. To ensure that no degradation had occurred during preparation and handling, each RNA was finally analyzed on an agarose gel. The procedure for the GIRK1 (Accession number P48549), GIRK4 (Accession number P48544), green fluorescent protein (GFP, derived from pGreen Lantern, Gibco BRL), and the prostanoid receptor EP3F (accession number HS13214; Schmid et al., 1995) were equivalent. For the cRNA of the cDNA-library of the olfactory epithelium from Xenopus laevis a total of 10µg of the library DNA in the pBK-CMV-phagemid was isolated by IVE, linearized at the 3' end in independent reactions using five different restriction enzymes (ApaI, KpnI, XbaI, NotI and XhoI), the cRNA prepared as described, and dissolved at a concentration of 500ngµl-1 in RNase-free water. In most experiments 50nl (about 5ng) of the receptor, Girk1, GIRK4, EP3F or GFP cRNA solution, or 150nl (approximately 75ng) of the cDNA library cRNA solution, were injected into the cytoplasm of the oocytes. Alternatively, 20nl of plasmid DNA (100ngµl-1) were injected directly into the nucleus of the oocyte. After an incubation period of 47 days in 35mm culture dishes (TC-Dish 35x10, Nunc, Denmark) at 18°C in batches of 2050, the oocytes were transferred individually into the recording chamber, and clamped in the two-electrode voltage-clamp mode at +30mV or -80mV with an oocyte amplifier (Turbo-TEC03, NPI electronics, Germany). The oocytes were continuously superfused with frog Ringer solution (hNa; 115mmoll-1 NaCl, 2.5mmoll-1 KCl, 1.8mmoll-1 CaCl2, 10mmoll-1 Hepes, pH 7.2), Rimland-Ringer solution (115mmoll-1 KCl, 2.5mmoll-1 NaCl, 1.8mmoll-1 CaCl2, 10mmoll-1 Hepes, pH 7.2) or Rimland with barium (3mmoll-1 or 0.3mmoll-1, respectively) by gravity flow, stimulated with odorants diluted in these solutions, and the resulting currents measured as described (Methfessel et al., 1986; Stühmer et al., 1992). For the functional expression studies of Xenopus olfactory receptors (XORs) a total of 155 oocytes were analyzed. Of these, 39 cells were water-injected control oocytes and 116 cells were expressing XORs (N=105) or the olfactory cRNA (N=11).
Stimulations
The oocytes were stimulated with complex stimulants, such as extracts of the fish food tetramarine (Tetra, Germany), minced beef heart (1:100; filtered through a 0.45µm membrane, Nytran, Schleicher Schüll), coffee aroma (stock solution 1:10 in DMSO; final concentration 1:500 to 1:1000, Nestlé, Mainz, Germany), or cocktails of amino acids.
Tetramarine extract, prepared at a concentration of 33mgml-1 in H2O, stirred for 3h at 4°C, then filtered through a 0.45µm membrane and diluted to a final concentration of 0.333.3mgml-1, caused general reactions in all oocytes; it was therefore used as a test for vitality after each series of stimulation in each oocyte. For stimulation of transfected and water-injected oocytes a selective fraction of tetramarine was prepared, consisting of small (<3kDa), heat-stable, water-soluble compounds.
Cocktails of amino acids were prepared as follows. The amino acids were dissolved as stock in double-distilled H2O, 0.1moll-1 NaOH or 0.1moll-1 HCl at a concentration of 100mmoll-1 and stored at 20°C. The amino acids were then diluted to concentrations of 0.103mmoll-1 each in normal frog Ringer solution, and pH adjusted to 7.2. The amino acids were used either as a cocktail of 22 amino acids (alanine, arginine, asparagine, aspartate, cysteine, cystine, glutamate, glutamine, glycine, histidine, hydroxy-proline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine, valine) or as cocktails of amino acids with particular physicochemical characteristics (Kang and Caprio, 1991), including long-chain neutral (LCN), short-chain neutral (SCN), basic, acidic and other amino acids. The human prostanoid receptor EP3F was stimulated with 1µmoll-1 prostaglandin E2 (in RimlandRinger solution).
Western blots
To test the oocyte system for expression of olfactory receptor protein (OR), the Xenopus receptor XB107 was cloned into an expression vector containing in-frame the nucleotide sequence of an N-terminal Flag-Tag (Hopp et al., 1988; Chubet and Brizzard, 1996), producing a peptide representing a highly efficient epitope. With a specific antibody (Anti-Flag-M1 monoclonal antibody, Eastman Kodak Company, Rochester, NY, USA) the receptor protein could be made visible. Three oocytes per batch were solubilized in solubilization buffer (50mmoll-1 imidazole, 100mmoll-1 NaCl, 3mmoll-1 EGTA, 5mmoll-1 EDTA, 0.1moll-1 PMSF, 0.02% sodium azide, 1% sodium deoxycholate, 20mmoll-1 Tris-HCl, pH 7.5), homogenized for 10min in a water bath with ultrasound at room temperature and then incubated on ice for 1h. The samples were centrifuged at 14000r.p.m. at 4°C (Eppendorf, 5417R), the pellet of undissolvable material discarded, 5x sample buffer (625mmoll-1 Tris-HCl, pH 6.8, 50% glycerol, 0.05% Bromophenol Blue, 5% SDS, 7.5mmoll-1 DTT) added to the supernatant and these samples stored at 4°C (Breer and Benke, 1986) until use.
One-dimensional SDSPAGE gel electrophoresis was performed essentially as described by Laemmli (Laemmli, 1970). The gels were blotted onto nitrocellulose membranes using a semidry blotting sytem (Pharmacia, Freiburg, Germany), stained with Ponceau S, dried and stored at 4°C until use. For western blot analysis non-specific binding sites were blocked with 5% non-fat milk powder (Nutraflor, Dietmannsriel, Germany) in TBST (10mmoll-1 Tris-HCl, pH 8.0, 150mmoll-1 NaCl, 0.05% Tween 20). The blots were incubated overnight at 4°C with a specific antibody (Anti-Flag M1 monoclonal antibody, Eastman Kodak Company, Rochester, NY, USA) at a concentration of 1:5000 and after three washes with TBST a horseradish peroxidase-conjugated anti-mouse IgG (1:10000 in TBST with 3% milk powder) was applied. Following three washes an TBST an ECL-System was used to visualize bound antibody as described by the manufacturer (Amersham).
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Results |
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Functional characterization of odorant receptors
To study functional determinants of the two olfactory receptor classes from Xenopus, and to explore a possible functional divergence, olfactory receptors from both classes were expressed in the Xenopus oocyte as a species-identical expression system. As a first step towards establishing the oocyte system for the expression of XORs, expression efficiency of oocyte batches was monitored using GFP (Fig.4A). Only oocytes that have been transfected successfully show fluorescence. In addition, the G-protein-activated, inwardly rectifying potassium channel subunits GIRK1 and GIRK4 (Kubo et al., 1993; Doupnik et al., 1995; Kaprivinsky et al., 1995) were coexpressed with the G-protein-coupled receptors (GPCRs) in order to allow an electrophysiological monitoring of the activity elicited via the intrinsic Gq/11 pathway, which results in a Ca2+-dependent chloride current (Miledi et al., 1987; Guttridge et al., 1995), and the G
o/i pathway, which causes an increase in the potassium current via the coinjected GIRKs (Kubo et al., 1993). Coinjection of the GIRKs and the human prostanoid receptor EP3F, which is known to couple to the G
i subunit of heterotrimeric G proteins (Schmid et al., 1995), resulted in a significant potassium current in ooctyes stimulated with 1µmoll-1 prostaglandin E2 (data not shown). As a first step towards the functional analysis of XORs in oocytes, the translation of XOR protein was assessed by western blot analysis. An immunoreactive protein of apparent molecular mass 29kDa was visualized on western blots (Fig.4B), indicating a significant expression of receptor protein.
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Discussion |
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The functional analysis of ORs expressed in Xenopus oocytes showed that receptors of each class did indeed detect different categories of odorants. While class I receptors responded to complex mixtures of water-soluble substances as well as to amino acids, class II receptors responded to mixtures of hydrophobic compounds. Some of the class I receptors from Xenopus may thus be amino acid receptors. However, in fish, receptors with sequence similarities to calcium-sensing receptors (CaSR) and metabotropic glutamate receptors (mGluR) act as receptors for amino acids (Speca et al., 1999). It is conceivable that vertebrates possess different types of receptors that interact with this group of ligands. The two types of putative amino acid receptors, expressed in different vertebrate orders, could be tuned to different amino acids, or display high or low affinities for a given amino acid. Alternatively, it is conceivable that the two receptor types are expressed in different sensory cells, e.g. one receptor type in pheromone-sensing cells, in the fish olfactory epithelium, and the other receptor type in sensory cells detecting general odors.
Previous studies have shown that the length of the extracellular loop EC3 diverges between the two OR classes (Freitag et al., 1998). The present study underlines this trait. Significant differences regarding class-specific, particularly charged amino acids, were found both in this region and in the EC2 domain. Furthermore, throughout the XOR sequences of both classes, amino acids in certain positions, e.g. in TM4 between XB178 and XB180, and also in TM3 and TM5, show high variability. This observation agrees with a recent report that describes a highly variable region in the proposed inner cleft between the transmembrane domains TM4 and TM5, marking a complementarity-determining region (CDR), which seems to be specific for olfactory receptors (Sharon et al., 1998). While this highly variable region within the internal faces of the transmembrane domains TM3 to TM5 might reflect the structural diversity of appropriate odorous ligands (see also Floriano et al., 2000), the distinct class-specific motifs within the extracellular loops EC2 and EC3 (Fig.3B) may determine which general physico physico-chemical features a compound must have to act as an appropriate ligand for a class I or class II receptor.
For rhodopsin-like GPCRs the intracellular domains IC2 and IC3 are responsible for G-protein coupling (Wess, 1997; Wess, 1998), and a distinct pattern of charged amino acids in these domains is considered to be fundamental for the G-protein specificity of a given GPCR (Iismaa et al., 1995; Ji et al., 1998). Interestingly, in XORs, characteristic patterns of charged amino acids were found within domains IC2 and IC3 for each of the two receptor classes. This could imply that each of the two receptor classes couples to a distinct G-protein subtype. This idea fits in with the results of in situ hybridization experiments that demonstrated a colocalization of Go1 -subunits and class I XORs in the LD, whereas a novel Gs-like
-subunit is colocalized with class II XORs in the MD (Mezler et al., 2001).
Taken together our results demonstrate that the two receptor classes identified in Xenopus laevis show class-specific sequence motifs in both the putative ligand-binding, and the possible G-protein interaction domains. Furthermore, we could demonstrate that class I and class II XORs are activated by water-soluble and volatile ligands, respectively. Future studies combining detailed sequence comparisons of both receptor classes with functional expression studies of a large array of ORs from both classes in one standardized expression system are necessary to find the structural basis for divergent receptor function, not only for ligand binding but also G-protein coupling. If this were followed by a mutagenic approach to these receptors and advanced molecular modeling studies (Floriano et al., 2000), a much more complete picture of olfactory receptor functioning might emerge. Use of the Xenopus oocyte system to express both classes of receptors from a single species, Xenopus laevis, may prove an ideal model system in which to shed new light on the structural features of receptor proteins interacting with distinct chemical compounds.
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Acknowledgments |
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The GenBank accession numbers of the sequences reported here are: XB3, AJ249488; XB6, AJ249489; XB107, AJ249404; XB238, AJ250750; XB239, AJ250751; XB242, AJ250752; XB154, AJ250753; XB177, AJ250754; XB178, AJ250755; XB180, AJ250756; XB350, AJ250757; XB352, AJ250758.
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