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Human Molecular Genetics, 2004, Vol. 13, No. 7 771-780
DOI: 10.1093/hmg/ddh086

Amino-acid changes acquired during evolution by olfactory receptor 912-93 modify the specificity of odorant recognition

Isabelle Gaillard1,{dagger}, Sylvie Rouquier1, Alain Chavanieu2, Patrice Mollard3 and Dominique Giorgi1,*

1IGH, CNRS UPR 1142, rue de la Cardonille, 34396 Montpellier cedex 5, France, 2CBS, CNRS-INSERM, 15, avenue Charles Flahault, 34093 Montpellier cedex 5, France and 3CCIPE, INSERM U469, rue de la Cardonille, 34094 Montpellier cedex 5, France

Received December 18, 2003; Revised January 26, 2004; Accepted February 2, 2004

DDBJ/EMBL/GenBank accession nos{ddagger}


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sense of smell in mammals can perceive and discriminate a wide variety of volatile odorants. Odorants bind to specific olfactory receptors (ORs) to initiate an action potential that transduces olfactory information to the olfactory cortex. We previously identified the structural motifs of odorant molecules (aliphatic 2- or 3-ketones) required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in transfected cells. In order to study changes in the specificity of this receptor that might have occurred during evolution, we cloned the orthologous genes from six primate species and pig and assayed the encoded receptors for responses to odorants. Primate OR912-93 orthologs share 88–97% sequence identity. All the receptors responded to 2- and 3-heptanone except the squirrel-monkey OR, which responded only to 3-heptanone, and the human and orangutan ORs, which were not functional. Directed mutagenesis allowed us to convert the squirrel-monkey response to that of the other functional 912-93 ORs by substituting three amino acids in the second extracellular loop. Orangutan and human 912-93 ORs regained function after restoration of the arginine residue in the DRY motif required for G-protein activation. However, the human receptor was constitutively activated in the absence of ligand stimulation. Using natural mutants of the OR912-93 receptor, we provide evidence that squirrel-monkeys evolved towards a restriction of the specificity of this receptor and therefore that slight alterations in the sequence of a receptor can induce subtle changes in recognition specificity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G-protein-coupled receptors (GPCRs) are tranducers of extracellular messages that allow tissues to respond to a wide array of signaling molecules. Vertebrate olfactory receptors (ORs) belong to the GPCR family (class A) that share sequence and structural features, such as seven hydrophobic transmembrane domains separated by extracellular and intracellular loops.

The first critical step in processing of olfactory information consists of binding odorous compounds to ORs, which are expressed in the chemosensory neurons of the nasal neuroepithelium. For many species, the ability to detect the olfactory environment is an essential survival tool in many behavioral processes, including location of food, reproduction or detection of predators. Humans possess a reduced sense of smell compared with rodents or dogs and are considered as microsmates. The reduction of olfactory perception in humans correlates with a high percentage of non-functional OR pseudogenes. The human OR repertoire contains ~1000 genes of which >60% are pseudogenes (1–3), whereas mouse has evolved an OR repertoire of 1200–1500 genes of which approximately only 20% are pseudogenes (4,5). Thus, mouse has three times more potentially functional genes than humans. Humans have also accumulated mutations that disrupt OR coding regions 4-fold faster than any other species, leading to a fraction of OR pseudogenes twice as high as in the non-human primates (6) and suggesting a human-specific process of OR gene pseudogenization.

Different approaches have been used successfully to express olfactory receptors either in situ by adenovirus-mediated transfections (7–9) or in heterologous cell lines (10–13). These studies have permitted the identification of odorants that are specific for given ORs in screening experiments based on calcium imaging techniques. These studies have demonstrated that a given OR recognizes a set of odorants that share common molecular determinants (9,13). Furthermore, odors are specified by a combinatorial receptor code, meaning that each odor is recognized by a specific set of receptors (12,14). Other observations suggest that conserved and orthologous ORs recognize very related ligands (10,12). Nevertheless, it is unclear whether minor structural differences, such as the amino-acid changes that exist among OR orthologs modify the recognition specificity of a particular receptor.

Previously, we isolated the mouse and human OR912-93 coding sequences. OR912-93 defined a new mammalian OR family, because it was highly diverged from all other OR sequences (15). This uniqueness allowed us to isolate this gene in a variety of species. Moreover, we recently identified the odor ligands of mouse OR912-93: aliphatic ketones with a carbon chain length of >4 carbon atoms and a carbonyl group preferentially located on carbon 2 or 3 (C2 or C3) of the carbon backbone. The human ortholog is a pseudogene that contains a nonsense point mutation in the region corresponding to the extracellular amino-terminus of the receptor and is not functional even after correction of this nonsense mutation.

Here, we use an expression system in heterologous cells (13) to characterize significant specificity changes of OR912-93 orthologs from eight different species. OR912-93 from mouse, pig, marmoset, gibbon and chimpanzee respond to 2- and 3-aliphatic ketones, whereas the squirrel-monkey receptor displays a restricted specificity and responds only to the 3-aliphatic ketone. Site-directed mutagenesis of the squirrel-monkey OR912-93 sequence was performed to characterize the amino acids involved in the modification of specificity. We show that the orangutan and human OR912-93 genes, even after correction of the human nonsense mutation, are not functional under the same assay conditions used for the other orthologues. We identify a single amino-acid change in the conserved DRY motif that is responsible for the loss of function in both species.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence analysis of OR912-93 from pig, squirrel-monkey and marmoset
We previously showed that mouse OR912-93 responded specifically to 2- and 3-ketones, whereas the human ortholog was non-functional even after correction of a unique nonsense mutation (13). In order to evaluate how the ligand specificity of this receptor might have changed during evolution, we cloned the orthologs of this gene in pig, marmoset and squirrel-monkey by PCR using genomic DNA. Orthologs in other species (human, mouse, chimpanzee, gorilla, orangutan, gibbon) had been cloned in previous studies (15). Every ortholog was sequenced and cloned in vector pRK5 for expression analysis.

Sequence comparisons revealed that OR912-93 is conserved among species (Fig. 1 and Table 1), i.e. primates share 88–97% of amino-acid sequence identity (ASI) whereas mouse and primates are distantly related with only ~66–70% ASI. Nevertheless, the mouse and human genes are thought to be true orthologs because (1) they are located in synteny regions on mouse chromosome 2E1 and on human chromosome 11q12.3, and (2) the region containing the transmembrane domains is 74% identical between mouse and primates with domains such as TM2, TM6 and TM7 presenting respectively 80, 100 and 85% of protein sequence identity. Figure 1A shows the amino-acid changes that could affect receptor specificity, and Figure 1B shows the phylogenetic analysis of the receptor.



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  		Figure 1. Sequence comparison of OR912-93 receptors. (A) Protein sequences were aligned using ClustalW. Regions of identity are shaded, and aminoacids specific to squirrel-monkey are circled. The single human nonsense codon is represented by an asterisk. The seven transmembrane domains are represented by horizontal lines on top of the alignment. (B) Phylogenetic tree of the receptor sequences deriving from species used in this study.

 

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  		Table 1. Amino-acid sequence identity percentages shared by the OR912-93 receptors isolated from the species examined in this study
 
Functional in vitro expression of the OR912-93 orthologs and ligand characterization
We used an approach similar to the method described by Krautwurst et al. (10). Each ortholog was transfected as previously described (13) in HEK-293 cells together with G{alpha}15 and G{alpha}q0 subunits that are known to couple a number of receptors to phospholipase C. In this reporter system, activation of the receptor leads to an increase of [Ca2+]i which is measured at the cellular level by the intensity of light emission of the Ca2+-sensitive dye FURA-2 (13,16). We checked that the receptors were correctly addressed to the cell surface by immunolocalization of the Myc tag as previously described (13). Transfection efficiencies were typically 60% (the percentage of cells expressing GFP from a cotransfected vector). Approximately 25% of the GFP-positive cells expressed a detectable amount of ORs in the membrane, indicating that ~15% of all cells expressed a potentially functional receptor in these assays. Each receptor was first assayed using the primary pool of odorants as previously described (12). 2-Heptanone was identified as the prototype ligand for all the species except human, orangutan and squirrel-monkey (data not shown). Since 2- and 3-ketones were identified as the ligands for the mouse receptor, in subsequent experiments we tested only 2- and 3-heptanone with the receptors from the other species using a 10–3 M concentration, as commonly used in such studies (7,9). HEK-293 cells expressing OR912-93 from pig, marmoset, gibbon and chimpanzee are activated by both 2- and 3-heptanone. While the mouse receptor displayed a better capacity for binding for the two ketones, which trigger indistinguishable responses, the other species showed a preference for 3-heptanone. This difference is particularly striking in the case of squirrel-monkey, where no rise in intracellular Ca2+ was observed upon stimulation with 2-heptanone, while stimulation with 3-heptanone was comparable to that of the other species (Fig. 2). The human ortholog was previously corrected by mutagenesis for its unique nonsense mutation (TAA, stop to GAA, Glu) in the extracellular N-terminal end (13). Nevertheless, no responses were obtained to either 2- or 3-heptanone (Fig. 2) with either the nonsense-corrected human receptor and the native orangutan receptor, suggesting that both receptors are non-functional as a consequence of a few amino-acid changes.



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  		Figure 2. Screening of the response of OR912-93 from different species to 2- and 3-ketones (heptanone). (A) Fura-2-loaded cells were transfected and tested as previously described (13). Each odorant was bath-applied at 10–3 M. Columns show the mean+SEM (two to five independent experiments with n=102–397). (B) Stimulation of the squirrel-monkey receptor with 3-heptanone (left) or 2-heptanone (right). The Ca2+ response is representative of responsive cells. The times of each bath-application are indicated by vertical arrows. (C) Stimulation of the marmoset receptor as in (B).

 
Characterization of the specificity of squirrel-monkey OR912-93
Stimulation of squirrel-monkey OR912-93 by 3-heptanone led to an increase of [Ca2+]i, whereas no signal was recorded in response to 2-heptanone (see above). We verified that this difference in receptor recognition was not due to the carbon chain length of the 2-ketone by assaying responses to 2-butanone, 2-pentanone, 2-hexanone, 2-nonanone and 2-decanone under the same conditions used for 2-heptanone (application of ligand at 10–3 M for 5 s). No Ca2+ response was observed to any of these odorants (data not shown), indicating that position of the carbonyl group on the carbon 3 of the carbon skeleton is essential for aliphatic ketone recognition by squirrel-monkey OR912-93. To identify the amino acids involved in this specificity, we first used the mouse receptor as a reference. Mouse and squirrel-monkey receptors share 70% ASI (Fig. 1 and Table 1), and only nine amino-acid changes differentiate the squirrel-monkey receptor from the other species (Figs 1 and 3). Asn65 and Leu69 are not present in the construct (see Materials and Methods) and therefore were not investigated. Cys229 and Leu235 are located in the third intracellular loop and although they can modify the three-dimensional structure of this part of the receptor, they are not thought to be involved in ligand recognition. In contrast, Val114, Thr139, Cys165, Ile173 and Ala199 are located in TM3, TM4, the second extracellular loop and TM5, respectively, and are more likely to be involved in ligand recognition (Fig. 3). These latter five amino acids were replaced separately by mutagenesis to the corresponding mouse residues (Fig. 1) in order to generate five receptor mutants (mutants A–E) that were tested in functional assays (Fig. 4). First, none of these mutants displayed significant differences in response to 3-heptanone relative to the wild-type receptor. Second, although mutants D (Ile173->Leu) and E (Ala199->Leu) led to an increase of [Ca2+]i in a very low and insignificant percentage of cells (4.6 and 1.3%, respectively) upon stimulation by 2-heptanone, none of the five mutants could efficiently restore response to the 2-ketone.



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  		Figure 3. Schematic representation of the squirrel-monkey OR912-93 receptor. The seven transmembrane domains are depicted as embedded in the cell membrane. Subset of residues labeled for reference. Squirrel monkey-specific amino acids relative to the other species analyzed in this study are shown in open circles. The aminoacids most likely to be involved in the differential recognition for 2- and 3-ketones (in extracellular loop E2 and the beginning of TM5) are indicated by arrows.

 


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  		Figure 4. Mutagenesis analysis of the squirrel-monkey and orangutan receptors. The protocol is the same as described in Figure 2. For each mutant, two to five independent experiments with n=118–396 were conducted. Substitutions were performed as shown in Figure 3. For squirrel-monkey, single mutations (mutants A–E) were constructed using the mouse aminoacids as a reference (Fig. 1). To minimize the sequence differences between species, the triple mutant (F), corresponding to mutants C+D+E, was constructed using the corresponding marmoset amino acids (Fig. 1). Comparison of squirrel-monkey wild-type and mutant F receptors indicates that the three amino-acid changes are sufficient to restore ligand specificity similar to that of marmoset (Fig. 2). The wild-type orangutan receptor is not functional; directed mutagenesis of His122->Arg restores the DRY motif that is necessary for G-protein activation and makes the receptor functional upon stimulation by both 2- and 3-heptanone.

 
We next determined whether a combination of these five amino-acid changes is responsible for the observed difference in recognition of 2-heptanone between the mouse and squirrel-monkey receptors by constructing multiple mutants. Given that squirrel-monkey is closer to marmoset than mouse (the receptors share 92.5% ASI) and that the marmoset receptor responds to both 2- and 3-heptanone as mouse does, we chose to mutate the five squirrel-monkey-specific residues to the corresponding marmoset residues (Fig. 1). Mutant F contains the three amino-acid changes most likely to be involved in ligand binding (Cys165->Arg; Ile173->Val; Ala199->Val), and mutant G (data not shown) contains all five substitutions, i.e. the same mutations as mutant F plus Val114->Leu and Thr139->Arg (Figs 3 and 4). Both mutants confer the squirrel-monkey receptor responsivity to 2-heptanone. These results suggest that the combination of the three substitutions of mutant F are involved in the structure of the binding pocket and are responsible for the detection of 2-heptanone (Fig. 4). It is likely that similar results could be obtained by substituting the same three amino acids with the corresponding mouse residues, but this mutant was not assayed.

To estimate whether these three amino acids are involved in the binding site, we built a three-dimensional model of the squirrel-monkey receptor. Cys165 and Ile173 are located in the E2 loop, and Ala199 is found at the beginning of TM5 (Fig. 3). The side-chains of Ile173 and Ala199 are located at interacting distances (<5 Å) from the putative binding pocket (Fig. 5), a situation which is similar to that found in bovine rhodopsin. Cys165 is more likely to be involved in a disulfide bond with Cys97 (rather than with Cys179) that connects TM3 and the E2 loop. However, models generated with this disulfide bridge did not affect the structure of the E2 loop.



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  		Figure 5. Three-dimensional model of squirrel-monkey OR912-93. The model was constructed using the programs Modeller version 4.0 and Discover3 in Insight-II (Accelrys). The predicted squirrel-monkey OR912-93 transmembrane helix (blue) was aligned to the transmembrane helix of bovine rhodopsin (PDB ID code 1L9H). The putative binding site (red) was determined using the Binding_Site module of Insight-II. The three aminoacids in the extracellular loop E2 that are responsible for the receptor response to 2-heptanone are indicated in red (Ala199), yellow (Ile173), and green (Cys165). (A) Larger view of the E2 region containing Ala199, Ile173 and Cys165. Ala199 and Ile173 are located at interacting distances from the putative binding site (<5 Å), whereas Cys165 is located at a non-interacting distance, but could be involved in a disulfide bond with Cys97 (green). (B) The same region but with the amino acids found in the marmoset receptor (Val199, Val173 and Arg165) which recognizes both 2- and 3-heptanone.

 
Characterization of the orangutan and human receptors
Although the orangutan gene and the nonsense-corrected human gene contain open reading frames (ORFs), the encoded receptors do not respond to either 2- or 3-heptanone. An amino-acid change in the motif involved in coupling G-proteins (15) differentiates these two species from the others, and we suspected that this change might be responsible for the lack of response. The DRY motif (C-terminus of TM3, Fig. 1) is replaced by DHY in orangutan and DCY in humans. In order to characterize the importance of this substitution, we mutated His122 (orangutan) and Cys123 (human) to Arg and assayed the modified receptors upon stimulation with 2- and 3-heptanone. The orangutan DRY-receptor displayed a response to the two ketones similar to that of the other functional primate receptors, indicating that the DRY motif is essential for signal transduction in this assay (Fig. 4).

Surprisingly, the human DRY-receptor induced spontaneous and periodic [Ca2+]i rises in the absence of stimulation by 2- or 3-heptanone in ~46% of GFP-positive cells, indicating constitutive G{alpha} coupling (Fig. 6A). If cells were transfected using normal conditions, but without G{alpha} subunits, this spontaneous response was abolished (Fig. 6C). The rise in [Ca2+]i is the result of a release of intracellular stores of Ca2+, since similar results were observed using Ca2+-free medium (Fig. 6B). To confirm this result, cells were incubated for 20 min in Ca2+-free medium, but in the presence of 1 µM thapsigargin, a blocker of Ca2+-ATPase found in the endoplasmic reticulum. The release of intracellular calcium was blocked and the spontaneous response was abolished (Fig. 6D). These experiments suggest that the rise of [Ca2+]i involves the mobilization of intracellular stores. Finally, since G{alpha}-proteins are known to couple a wide range of receptors to phospholipase C (PLC), we used U73122 (at 5 µM for 20 min), a specific inhibitor of PLC (11), to block the production of inositol triphosphate (IP3) before recording the variations of [Ca2+]i. Under such conditions, no response was obtained (Fig. 6E).



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  		Figure 6. Mutagenesis of the human OR912-93 receptor leads to a constitutively activated receptor. Two amino acids were changed in the native receptor sequence: a nonsense mutation at position 11 changed to Glu11, and Cys123 changed to Arg123 in the DRY motif. Although the receptor was still non-functional after correcting the nonsense mutation (13), correction of both mutations made the receptor constitutively functional. (A) HEK293 cells were cotransfected with human OR912-93, G{alpha}15 and Gq0G{gamma}. Rises in [Ca2+]i were obtained in the absence of stimulation by odorant ligands. (B) The same experiment as described in (A), but using Ca2+-free medium, showing that the rise in Ca2+ is the result of the release of intracellular stores. (C) The activity is abolished when cells are transfected with OR912-93 only (without G-proteins). (D) The same experiment as described in (B), but performed in the presence of 1 µM thapsigargin, which blocks the release of intracellular calcium. (E) The same experiment as described in (A), but performed in the presence of 5 µM U73122, a specific inhibitor of phospholipase C that blocks the production of IP3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The use of functional assays has allowed researchers to characterize the specificity of a number of olfactory receptors both in vivo and in vitro (7,9,10,13,14,17). We studied OR gene 912–93 in primates and mouse (15,18) to show that it was probably a recent pseudogene in some primate species since it contained a unique nonsense mutation in humans and a frameshift in gorilla, whereas the other species contained an open reading frame. We showed that the mouse receptor is specific for 2- and 3-aliphatic ketones, while the human gene is non-functional even after correction of its nonsense mutation, suggesting that it accumulated other deleterious mutations (13). Here, we have cloned the pig, marmoset and squirrel-monkey orthologs in order to study the evolution of the ligand specificity of the encoded receptors together with that of the human, mouse, gibbon, orangutan and chimpanzee receptors. The human and orangutan receptors showed no calcium rise upon stimulation with ketones. The squirrel-monkey receptor responded exclusively to 3-heptanone, whereas receptors from all the other tested species responded to both 2- and 3-heptanone. A combination of three amino-acid changes, corresponding to the residues we predicted were most likely to be involved in the binding site, allowed the squirrel-monkey receptor to respond to both 2- and 3-heptanone in a manner similar to that of the marmoset receptor. The study of receptor OR912-93 exemplifies the evolution of the olfactory receptor repertoire. Although the size of the functional repertoire decreased during primate evolution (19), a number of genes such as OR912-93 have evolved either towards extinction in the case of hominids (human and gorilla in this study) or towards a more restrained or specialized specificity as in the case of squirrel-monkey. It is worth noting that squirrel-monkey belongs to the new-world monkey clade that possesses an OR repertoire with a low pseudogene fraction comparable to mouse, and is likely to be a macrosmate species that relies on olfaction (19). The reason for this specialization is unknown but in most species OR912-93 can bind both 2- and 3-heptanone, two chemicals that exhibit quite different odors; 2-heptanone is described as the Roquefort cheese odor (produced by Penicillium roqueforti) whereas 3-heptanone is a sweet odor.

Knowledge of the three-dimensional structure of olfactory receptors is crucial for the interpretation of structure-function data. However, with the exception of the rhodopsin light receptor, the structures of GPCRs are unknown. In order to evaluate the role of these three aminoacids in the choice of ligand, we built a three-dimensional model of squirrel-monkey OR912-93 based on the bovine rhodopsin three-dimensional structure. The bovine rhodopsin three-dimensional structure (20) corresponds to its basal state in which retinal is covalently bound. Upon ligand binding, GPCRs adopt different conformational states with TM rearrangements and a spatial reorganization of extracellular domains, especially the second extracellular loop (21). Thus, rhodopsin-based homology models cannot be directly used for docking purpose of agonists but are useful tools to interpret results from site-directed mutagenesis experiments. Data obtained from the three-dimensional model support the hypothesis that these three aminoacids are responsible for the responsivity to the 2-ketone. However, in the absence of structural data of ORs in the model, it is not possible to decipher whether the effect of the three amino-acid substitutions that differentiate squirrel-monkey from marmoset is direct by interacting with the ligand or indirect through a reorganization of the structure of the binding site.

Functional assays show that in the case of squirrel-monkey, the receptor structure allows the binding of the carbonyl group branched on a carbon backbone with a two-carbon end only, while in the other cases the receptor may bind ketones with either a one- or a two-carbon end.

Neither the orangutan nor the nonsense-corrected human receptors responded to odorant stimulation under the same experimental conditions. We demonstrated that the change of an Arg (R) residue to either His (H) or Cys (C) in the DRY motif at the end of TM3 was responsible for this phenotype by hindering G-protein coupling. In rhodopsin-like G-protein-coupled receptors, efficient signal transduction is conditioned on the presence of this Arg residue (22,23). The activation of 7TM-receptors is associated with a conformational change allowing interaction with G-protein and subsequent signal transduction (24). Directed mutagenesis studies showed that the substitution of this Arg led to important perturbations in signal transduction (25–27). Together, these observations suggest that ORs that do not contain an Arg residue in the DRY motif could be non-functional or poorly activatable. For example, human OR912-93 that contains a DCY motif is not activatable even upon stimulation with 10–2 M ketone, whereas the orangutan receptor that contains a DHY motif is slightly activatable under the same conditions (data not shown). There is no example in the literature of a functional OR without the Arg residue in the DRY motif. These observations imply that non-synonymous substitutions could have altered the function of ORF-containing OR genes and that the number of OR pseudogenes in humans could be higher than the count based on ORF disruption alone. Indeed, Young et al. (4) reported that the Arg residue is found in the DRY motif of 98% of mouse ORs (from a total of 866 ORF-containing genes), but only in 89% of human sequences (347 ORF-containing genes). Thus, the number of functional human OR genes could be 11% lower than the theoretical number of 347 and could be rather around 300–310. Also, the human receptor is very similar to the functional chimpanzee receptor whereas the orangutan receptor is closer to the functional gibbon receptor (Fig. 1). Orangutan represents the asian branch of the evolutionary tree. Except for the mutation of the Arg122 of the DRY motif, orangutan and humans accumulated species-specific mutations, i.e. orangutan possesses four specific mutations that are Trp102->Arg, Phe200->Leu, Cys235->Tyr, Ser267->Cys, and humans possess five specific mutations that are described below. It is therefore likely that this receptor lost function independently in both species. Similarly, the gorilla receptor described in Rouquier et al. (15) is non-functional since it contains a frameshift mutation between residues Pro184 and Leu185, and four specific mutations that are Met60->Leu, Cys113->Ser in TM3, and Asn156->Tyr and Thr157->Ser in TM4, whereas the DRY motif is intact. It is therefore possible that such mutations, especially Cys113->Ser and Asn156->Tyr, may alter the structure and the specificity of recognition of the receptor.

Interestingly, correction of both the nonsense mutation (TAA, stop->GAA, Glu) and the non-synonymous substitution DCY->DRY made the human receptor both functional and constitutively activated. This result suggests that, once inactivated by the first deleterious mutations, the resulting pseudogene randomly accumulated a number of non-synonymous mutations rendering it permanently activated, presumably by permitting constitutive G{alpha} coupling. The human receptor contains six specific substitutions compared to the other species we have evaluated: Thr72, Asp112, Glu117, Pro177, His262 and Thr266 (Fig. 1). The substitution His->Pro177 in the third extracellular loop could be involved in the binding site, and the substitutions Glu->Asp112 and Ala->Glu117 are located in the vicinity of the DRY motif. These three substitutions could induce structural changes resulting in a conformation that allows the receptor to couple G{alpha} proteins constitutively without the need for ligand binding. Recently, Wang et al. (28) hypothesized that modifications of the structure of extracellular loop 2 would lead to structural changes of intracellular loop 2 triggering G-protein activation. However, mutagenesis of these three substitutions in other receptors will be required to confirm this hypothesis. Constitutive (agonist- or ligand-independent) activity of GPCRs is a well-documented phenomenon that can occur either naturally and lead, in some cases, to human diseases or artificially by mutagenesis experiments (29). For example, constitutive receptors for gonadotropin, TSH, LH and FSH (30 and references therein) or histamine H1-3 (31) have been characterized. Mutations leading to constitutive activity can be widespread, but a number are located in the second and third intracellular loops (30,32,33). However, how these mutant receptors recapitulate the structural modifications of the ligand-induced active conformation of the native receptor is still unknown (34).

We present here the first case of a constitutively activated olfactory receptor that displays periodic calcium signals similar to isolated signals produced with normal receptors upon stimulation with specific odorant ligands. Moreover, this particular receptor displays a transfection efficiency >45%, and compared with the 15% usually obtained for functional receptors, it would obviate the ligand-application and calcium-imaging steps to permit direct measurement of IP3 levels. The use of such a receptor could be a valuable tool to study membrane addressing, G protein coupling, or receptor dimerization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation and expression of chimeric receptors
OR912-93 full-length sequences were cloned previously (15,18): human [accession number AF045576, OR5G1P or OR5G5P in the HORDE nomenclature (http://bioinformatics.weizmann.ac.il/HORDE/), and localizing to chromosome 11q12.3], chimpanzee (Pan troglodytes), orangutan (Pongo pygmaeus), gibbon (Hylobates lar; accession numbers AF045577, 045579-80) and mouse (Mus musculus domesticus; accession number AF146372, ORL782, Olfr154, and localizing to chromosome 2E1). Marmoset (Callithrix jacchus), squirrel-monkey (Saimiri boliviensis) and pig (Sus scrofa) OR912-93 partial sequences were cloned by PCR on genomic DNA as described in Rouquier et al. (19) using consensus degenerate internal primers Z93-1F (5'-CAG/TC/TTG/CCA/GA/GA/GTC/TG/TTCCTC/TTTCC-3') located at the predicted border of the extracellular N-terminal end and the first transmembrane domain 1 (amino acids QLRVFLF in the human sequence) and Z93-1R (5'-GTCCTGA/TG/TG/TAA/TG/TGCATCTTTG-3') located in the intracellular C-terminal end (KDAIHR). Therefore, the receptor sequence from these three species lacks both ends (Fig. 1). PCR products were subcloned in the TA cloning vector (Invitrogen). Recombinant clones were identified by PCR and five to ten different clones per species were sequenced.

All the receptor sequences were cloned into the pRK5 mammalian expression vector as described previously (13). Chimeric receptor cassettes for heterologous expression of human and mouse OR912-93 were already constructed (13). A 904 bp EcoRI–StuI restriction fragment corresponding either to a peptide from aminoacid 11 (Glu) to amino acid 312 (Lys) for orangutan and gibbon, or to amino acid 313 (Lys) for chimpanzee was subcloned into the EcoRI–StuI-digested human OR912-93 expression vector (13). These constructs contain a Kozak sequence, a modified influenza haemagglutinin signal (IHS) sequence, and the Myc tag coding sequence for the docapeptide MEQKLISEEDLN in frame with the corresponding OR912-93 full-length sequence.

The TM2-TM7 moiety of marmoset, squirrel monkey and pig OR912-93 were amplified by PCR from the cloned genes using species-specific primers: (Marmoset–squirrel-93F, 5'-GATATCTGCTTCTCCTCAGTTGTG-3'; squirrel-93R, 5'-CCGCGGTATAAAACACAGATACCAGTT-3'; marmoset-93R, 5'-CCGCGGTGTAAAGCACAGATACCACTT-3'; pig-93F, 5'-GATATCTGCTTCTCCTCGTCGTG-3'; pig-93R, 5'-CCGCGGTGTAAAAGACAGACACCACTT-3'). The resulting PCR products were EcoRV–SacII-digested and subcloned in frame into the human OR912-93 vector as described in Gaillard et al. (13). Previous work has shown that TM2-TM7 moiety of an OR confers the same specificity as the entire receptor (10,35).

Site-directed mutagenesis
The mutations were generated by PCR-mediated mutagenesis using Pfu polymerase (Stratagene). Mutants were verified by sequencing on both strands. Oligonucleotides are named according to the amino-acid change, i.e. V114L is Valine 114 mutated to Leucine. The following sense and antisense primers were used: squirrel monkey, V114L-1F, 5'-GCAGAGTGTTTCCTCTTGGCGTCCATG-3'; 1R, 5'-CATGGACGCCAAGAGGAAACACTCTGC-3'; T139R-2F, 5'-GCAATGTCCCAGAGACTCTGCATCCAG-3'; 2R, 5'-CTGGATGCAGAGTCTCTGGGACATTGC-3'; C165V-3F, 5'-CACAAATGCATTTGTTCTCCCTTTTTG-3'; 3R, 5'-CAAAAAGGGAGAACAAATGCATT-3'; C165R-4F, 5'-CACAAATGCATTTCGTCTCCCTTTTTG-3'; 4R, 5'-CAAAAAGGGAGACGAAATGCATTTGTG-3'; I173L-5F, 5'-GTGGCCCTAATCTCATCAATCATTTC-3'; 5R, 5'-GAAATGATTGATGAGATTAGGGCCAC-3'. I173V-6F, 5'-GTGGCCCTAATGTCATCAATCATTTC-3'; 6R, 5'-GAAATGATTGATGACATTAGGGCCAC-3'; A199L-7F, 5'-CTTAATAAGTTGGCACTTTTCATTATG-3'; 7R, 5'-CATAATGAAAAGTGCCAACTTATTAAG-3'; A199V-8F, 5'-CTTAATAAGTTGGCAGTTTTCATTATG-3'; 8R, 5'-CATAATGAAAACTGCCAACTTATTAAG-3'; Orangutan and human: H122R/C123R-9F, 5'-GTCCATGGCCTATGACCGCTATGTGGC-3'; 9R, 5'-GCCACATAGCGGTCATAGGCC ATGGAC-3'.

Culture and transfection
HEK-293 cells were grown and maintained at 37°C in DMEM (ICN Pharmaceuticals Inc., CA, USA) supplemented with 10% fetal calf serum, 0.3 g/L L-glutamine, 0.05 g/l streptomycin and 50 000 IU/l penicillin. Electroporation was performed as described in Gaillard et al. (13). OR construct DNAs were cotransfected with G{alpha}15 and the mutated G{alpha}qo subunits that have been shown to couple a wide range of receptors to phospholipase C (10,13,36–38). Transfection efficiencies were estimated by cotransfection with a GFP reporter plasmid. Immunolocalization of tagged receptors were performed 48–72 h after transfection as previously described (13).

Calcium imaging
Transfected cells were washed once in Locke buffer (NaCl 140 mM, KCl 5 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, CaCl2 1.8 mM, glucose 10 mM, and HEPES 10 mM, pH 7.2) and loaded with 2.5 µmol/l of Fura-2AM and 0.02% Pluronic F-127 (Fura-2/AM and Pluronic, Molecular Probes Inc., Eugene, OR, USA) for 20 min at 37°C. The loading solution was removed and the cells were incubated in Locke buffer for 30 min to allow cleavage of the AM ester.

Only cells showing both GFP and FURA-2 fluorescence were recorded with an intensified cooled charge-coupled device camera (PentaMAX Gen Iv; Princeton Instruments, Trenton, NJ, USA). Camera acquisition rate was 200 ms/frame. The F360/F380 emission ratio, which is an image of [Ca2+]i was measured for 120 s. [Ca2+]i changes were acquired with Metafluor (Universal imaging Corp. West Chester, PA, USA) and analyzed with IgorPro 3.16 software (Wavemetrics Inc., Lake Oswego, OR, USA).

The test odorants were locally pressure-ejected for 5 s with a picospritzer apparatus. The flow rate was adjusted to allow renewal of the bath solution within 10 s. Odorant solutions were prepared as 1 M stocks in dimethyl sulfoxide (DMSO), conserved under nitrogen, and then diluted with Locke buffer. All the solutions were used within 1 h. 3-Heptanone, 4-heptanone, 2-octanone, 2-nonanone, 2-decanone, 2-hexanone and 2-pentanone were provided by Lancaster Synthesis Ltd; the other odorants were purchased from Aldrich.

Multiple alignments, phylogenetic tree and molecular modeling
Multiple alignment was performed using ClustalW version 1.8 (39). A phylogenic tree was constructed using the neighbor-joining method based on the number of amino-acid substitutions. Pairwise sequence comparisons were performed with GAP from the GCG package (Wisconsin Package, version 8). OR912-93 sequences were aligned with the bovine rhodopsin sequence (Swiss-Prot database accession number P02699) using ClustalW. Alignments were refined with the TopPred membrane-spanning regions prediction program (40). Using Modeller 4.0 (http://bioserv.cbs.cnrs.fr/) (41,42), the coordinates of squirrel-monkey OR912-93 were then assigned to those of the bovine rhodopsin three-dimensional structure (code PDB, 1L9H, chain A) (20). No disulfide bond was added to the model. The three-dimensional structure was refined with 1000 iterations of steepest descent minimization (Insight II/Discover3 program, Accelrys, San Diego, CA, USA) with the {alpha}-carbon trace fixed. The second extracellular (E2) loop of squirrel-monkey OR912-93 has more residues than bovine rhodopsin. In order to optimize the conformation of this loop, a 200 ps molecular dynamic calculation was performed allowing atoms of the E2 loop to be flexible, while keeping the rest of the receptor fixed. Finally, the model was submitted to several steps of minimization with the backbone fixed. Then, using the Binding_Site module of Insight II, the putative binding site for odorous compounds was located by searching the cavities within the squirrel-monkey receptor, as already described for other receptors (43,44).


   ACKNOWLEDGEMENTS
 
We are grateful to Barbara Trask, Jean-Philippe Pin and Rosemary Kiernan for critical comments on the manuscript, and to Patrick Atger for artwork.


   FOOTNOTES
 
* To whom correspondence should be addressed. Email: giorgi{at}igh.cnrs.fr

{dagger} Present address: INRA, IPV, 2 place Viala, 34000 Montpellier, France. Back

{ddagger} AY230213–15. Back


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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