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Characteristic features and ligand specificity of the two olfactory receptor classes from Xenopus laevis

Mario Mezler1,2, Jörg Fleischer1 and Heinz Breer1,*

1 University of Hohenheim, Institute of Physiology, 70593 Stuttgart, Germany and
2 Bayer AG, Agricultural Centre, MWF, Geb. 6240, Monheim, Germany



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  		Fig. 1. Alignment of olfactory receptors from Xenopus leavis. Alignment of the deduced amino acid sequences for ten olfactory receptors from Xenopus laevis. Identical amino acid residues are highlighted in black, residues present in >80% of the sequences are highlighted in dark grey, those with >60% conservation in light grey. Putative transmembrane domains (TM) are highlighted by black bars, and extracellular domains (EC) and intracellular domains (IC) are indicated. The putative N-linked glycosylation site is marked by an arrowhead. Additional class-specific amino acids within the amino acid chain are highlighted by asterisks. Class I receptors: XB107, 239, 238, 242; class II receptors: XB178, 180, 177, 350, 352 and 154.

 


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  		Fig. 2. Identity dendrogram and phylogenetic relationships between divergent olfactory receptor sequences. (A) The complete deduced amino acid sequences of the ten XORs (see Fig.1) were aligned in Pileup, and an identity dendrogram was constructed. Two classes of receptors are clearly distinguishable: class I, comprising the receptors XB107 to XB242 and class II, including the receptors XB178 to XB154. (B) Phylogenetic tree of the amino acid sequences of ORs from different species. The open-reading frames (ORFs) of 100 known olfactory receptors were initially aligned together with the ten XOR-receptors. 14 representative receptors from chicken (GGcor7b) (S. Nef, I. Allaman, E. De Castro and P. Nef, unpublished material) (GGcor1) (Leibovici et al., 1996), rat (RNor18, RNor5) (Raming et al., 1993) (RNscrd9, RNscrg14) (Walensky et al., 1998), mouse (MMor3) (Nef et al., 1992), human (HS05691) (J. E. Lamerdin, P. M. McCready, E. Skowronski, A. W. Adamson, K. Burkhart-Schultz, L. Gordon, A. Kyle, M. Ramirez, S. Stilwagen, H. Phan et al., unpublished material) (HS1740) (Ben-Arie et al., 1994), catfish Ictalurus punctatus (ICtordb,d,e) (Ngai et al., 1993a), zebrafish Danio rerio (DRorp2-3) (Barth et al., 1997) and the ten receptors from Xenopus (XB, this study) together with the receptor LFor1 from the lamprey Lampetra fluviatilis (Freitag et al., 1999) as outgroups were then realigned and analyzed by parsimony analysis with the PHYLIP package (Joseph Felsenstein, Version 3.5c, 1995). The bootstrap-support of 100 replicates is indicated.

 


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  		Fig. 3. Sequence analysis of XORs. (A) Amino acid substitutions in receptor sequences of class I receptors. Substituted amino acids are highlighted in black. Transmembrane domains are shaded in grey. A high number of amino acid substitutions can be found in the TM4, EC2 and TM5 domains (not shown). (B) Class-specific sequence motifs in ORs from Xenopus laevis. XB107, XB239, XBb238, XB242 represent class I receptors, whereas XB178, XB180, XB177, XB350, XB352 and XB154 are class II receptors. The extracellular domains 2 (EC2) and 3 (EC3), as well as the intracellular domains 2 (IC2) and 3 (IC3) are shown. Class-specific amino acid locations, constituting physico-chemically comparable amino acids, are highlighted in black (class I-specific) and light grey (class II-specific). Connecting transmembrane regions, as well as N- and C-terminal domains, are omitted and symbolized by dashes.

 


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  		Fig. 4. Establishing the oocyte expression system. (A) Expression of GFP in Xenopus oocytes. Oocytes were injected with cRNA for GFP (1), or with water (2). Left, cells under normal light; right, fluorescence microscopy. (B) Expression of receptor XB107 in Xenopus oocytes. Oocytes were injected with an expression vector encoding receptor XB107 with an N-terminal Flag-Tag (lane 1) or water (lane 2) and incubated at 18°C for 7 days. The oocyte proteins were subjected to western blot analysis. The positions of molecular mass marker proteins are shown.

 


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  		Fig. 5. Functional expression of both classes of XORs. (A) Oocytes expressing a representative class I receptor (XB107) and (B) cells expressing a representative class II receptor (BX154) were stimulated with tetramarin fractions (water extract, upper traces), coffee aroma (middle traces) or frog-Ringer solution (lower traces). Cells expressing XB107 responded with a typical current upon stimulation with the water extract, but showed no response with coffee aroma (A). (B) Oocytes expressing a representative class II receptor (XB154) did not react to water extract, but showed a strong outward current upon stimulation with coffee aroma. Neither cell type responded to the control-stimulation with frog-Ringer solution (Ringer). In all cases current plots were done at a holding potential of +30mV; the times of stimuli are indicated by bars. Please note the different scaling in 5A and B.

 


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  		Fig. 6. Response of class I XOR expressing oocytes to stimulation with different amino acid fractions. Cells expressing the class I receptor XB107 were stimulated with 22 different amino acids (amino acid mix, A), long-chain neutral amino acids (B) and basic amino acids (C). Clear responses are seen in A and B, while the cells did not react to basic amino acids (C) or other amino acid fractions (see text). The concentration of single amino acids was 200µmoll-1 each in all cases. As in Fig.5 the holding potential was +30mV; times of stimuli are indicated by bars.

 

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