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Published as doi: 10.1096/fj.07-9233com.
 (The FASEB Journal. 2008;22:1416-1425.)
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The human vomeronasal type-1 receptor family— detection of volatiles and cAMP signaling in HeLa/Olf cells

Elena Shirokova*,1, Jan Dirk Raguse{dagger}, Wolfgang Meyerhof* and Dietmar Krautwurst*,2

* Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany; and

{dagger} Clinic for Oral and Maxillofacial Surgery and Plastic Surgery, Charité Universitätsmedizin Berlin, Campus Virchow Hospital, Berlin, Germany

2Correspondence: Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany. E-mail: dietmark{at}dife.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human genome harbors 5 remnant genes coding for vomeronasal type-1 receptors, compared with 187 of such receptors in mice. In rodents, vomeronasal type-1 receptors are typically expressed in the vomeronasal organ. They are believed to be highly selective and sensitive pheromone detectors, as may be inferred from one receptor, V1rb2, responding to picomolar concentrations of the mouse pheromone 2-heptanone. Expression patterns, ligands, and signal transduction of human vomeronasal type-1 receptors have, however, remained largely obscure. Our aim was to deorphan and functionally characterize these 5 putative human pheromone receptors. Here, we report functional expression for all 5 receptors in HeLa/Olf cells. The recombinant, N-terminally tagged receptors expressed at the plasma membrane of HeLa/Olf cells and responded differentially to 19 of 140 odorants in a combinatorial way. C9–C10 aliphatic alcohols or aldehydes emerged as the best agonists at submicromolar concentrations above human odorant thresholds. Surprisingly, and in contrast to mouse V1rb2, all human vomeronasal type-1 receptors activated cAMP signaling via G protein {alpha}olf, when expressed in HeLa/Olf cells. While a biological function of human vomeronasal type-1 receptors is still elusive, our data show that their major functional characteristics are similar to those of odorant receptors.—Shirokova, E., Raguse, J. D., Meyerhof, W., Krautwurst, D. The human vomeronasal type-1 receptor family–detection of volatiles and cAMP signaling in HeLa/Olf cells.


Key Words: pheromones • key aroma volatiles • G protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAMMALS HAVE AN EXQUISITE SENSE of smell. This high discriminatory power arises from 4 unrelated multigene families coding for 7 transmembrane helix receptors: 1) olfactory receptors (ORs) of the main olfactory epithelium (MOE) (1) , 2) vomeronasal type-1 receptors (V1Rs) (2) , 3) vomeronasal type-2 receptors (V2Rs) (3 4 5) , and 4) trace amine-associate receptors (6 , 7) . V1Rs and V2Rs are typically expressed in spatially segregated subpopulations of sensory neurons of the vomeronasal organ (VNO), which is a tubular neuroepithelium in the basal region of the nasal septum (8) and thus is an olfactory organ distinct from the MOE, the major site of odorant detection in mammals.

Experiments in mice have added to the notion that the MOE receives mainly volatile signals, whereas the accessory olfactory system, comprising the VNO and the accessory olfactory bulb, is thought to respond largely to nonvolatile social cues (9) . Indeed, experiments with sliced VNO preparations and isolated vomeronasal sensory neurons (VSNs) from mice demonstrated their activation by main histocompatibility complex (MHC) class I peptides (10) . On the other hand, several reports demonstrated ultrasensitive and highly selective detection of single volatile pheromones by VSNs (11 , 12) . VSNs also responded to odorants in the pico- to nanomolar range (13) . Recently, however, a male-specific volatile pheromone from mouse urine, methanethiol, activated mitral cells of the main olfactory bulb via the MOE at a threshold of 10 parts per billion (14) . Moreover, a recent report demonstrated RNA expression of 1 V1R in the MOE of the mouse (15) and nonvolatile MHC class I peptides functioned as olfactory cues in the mammalian MOE at subnanomolar concentrations (16) .

In mice, V1Rs are encoded by 187 potentially functional genes (17 , 18) . In contrast, only 5 potentially functional V1R-related genes (VN1R1–5) have been identified in humans (19) . Do human VN1Rs function as pheromone receptors or odorant receptors and, if so, in which olfactory epithelium? The neuroepithelial character and function of the adult human VNO is still unclear (20) . Interestingly, VN1R1 RNA has been detected in a human MOE biopsy by RT-PCR (reverse transcriptase-polymerase chain reaction) (21) . However, until now, it was not known whether mRNAs of the other 4 VN1Rs also are present in the human MOE. So far, mRNA expression has not been reported on the level of single olfactory neurons, by the in situ hybridization technique, for any of the VN1Rs or any other human olfactory receptor. Moreover, RNA for VN1R1 has been detected in several nonolfactory tissues (21) , raising the possibility of a nonolfactory function of VN1Rs. No ligands have been described for any of the 5 VN1Rs in binding studies or functional expression experiments using heterologous cell systems. Therefore, it has not been known to which signal transduction cascade human VN1Rs couple. Thus, expression and biological function of the human VN1Rs have so far been obscure.

Here, we demonstrate the functional expression of recombinant VN1Rs as well as the identification of their agonists, agonist profiles, and signal transduction pathway in HeLa/Olf cells, a cellular model system that contains canonical olfactory signaling molecules of the cAMP pathway, such as G protein {alpha}olf, adenylyl cyclase type-III (AC-III), and the Ca2+-permeable cyclic nucleotide-gated channel alpha 2 (CNGA2) (22) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular cloning
The full-length coding regions of human VN1Rs were obtained by PCR amplification (Pfu; Promega Corp., Madison, WI, USA) from human genomic DNA using gene-specific primers (Supplemental Table 1). Amplicons of VN1R1 (AF255342), VN1R2 (AF370359), VN1R4 (AY114733), and VN1R5 (AY114735) were cloned EcoRI/NotI, and VN1R3 (AF336873) was cloned MfeI/NotI into pi2-dk(rho-tag(39)), in frame with the coding region for the first 39 amino acids of the bovine rhodopsin (rho-tag(39)), which serves as an N-terminal tag for all full-length OR proteins (22) . The identities of all subcloned amplicons were verified by sequencing (UKEHH, Hamburg, Germany; or MWG, Martinsried, Germany).

Cell culture and transient DNA transfection
HeLa/Olf cell culture and DNA transfection were performed as described earlier (22) . In short, all cell culture media, ingredients, and antibiotics were obtained from Invitrogen/Life Technologies Inc. (Carlsbad, CA, USA) except for G418 sulfate (Calbiochem, EMD Biosciences Inc., San Diego, CA, USA) and Puromycin (Sigma-Aldrich Corp., Seelze, Germany). HeLa/Olf cells were grown in standard Dulbecco modified Eagle medium (DMEM) with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, supplemented with puromycin (1 µg/ml), zeocin (100 µg/ml), and G418 (400 µg/ml), in a humidified atmosphere (37°C, 5% CO2). Cells were transfected with DNA using lipofection (PolyFect; Qiagen, Valencia, CA, USA) and were taken into experiments 40 h post-transfection.

Single-cell Ca2+ imaging
Single-cell Ca2+ imaging was performed as described previously (22) . In short, 40 h post-transfection, cells were loaded (1 h, 37°C) with 4 µM Fura-2-AM (Molecular Probes Inc., Eugene, OR, USA) in serum-free DMEM and transferred into a bath chamber with HBS: 140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 10 mM HEPES, 10 mM glucose (pH 7.4). Fluorescence from single cells was monitored in 10 s intervals at an emission wavelength of 515 nm, after excitation (3–15 ms) with 340 nm and 380 nm, and ratioed (F340/F380). Images of the cells were monitored by an intensified, cooled CCD camera and analyzed offline. Poly-D-lysine and salts were from Sigma. Thapsigargin (1 µM, 30 min; BioTrend/Tocris, Cologne, Germany) blocked receptor-mediated Ca2+ release from IP3-sensitive internal stores (23) . Bath application of 1 mM EGTA (Sigma) blocked Ca2+-influx.

Ca2+-FLIPR assay
FLIPR assays were performed as described previously (22) . In short, 40 h post-transfection, cells were loaded with 4 µM FLUO-4/AM (Molecular Probes) and 0.04% Pluronic F-127 (Molecular Probes) in HBS (see above), but with 20 mM HEPES and 2.5 mM probenecid. After loading, cells were washed twice with HBS by an automated plate washer (Denley Cellwash, Labsystems, Franklin, MA, USA) and transferred to the FLIPR (Molecular Devices Corp., Sunnyvale, CA, USA). Fluorescence from the 96 wells were monitored simultaneously at an emission wavelength of 515 nm, after excitation with 488 nm. Fluorescence data were collected at 0.25 Hz, 48 s before and 5–10 min after stimulation, and analyzed offline. Odorants were from Sigma-Aldrich, or Givaudan (Duebendorf, Switzerland).

Data analysis
In the FLIPR screening experiments, agonist response amplitudes were determined from the peak stimulated fluorescence of the solvent control- or mock-transfected-subtracted and baseline-corrected traces and averaged over 4–5 wells expressing the same receptor and receiving the same stimulus. For control, we applied each odorant concentration to mock-transfected cells. EC50 values were derived from fitting the function f(x) = (a–d)/[1+(x/C)nH] + d to the data by nonlinear regression, where a = minimum, d = maximum, C = EC50, and nH = Hill coefficient.

Immunocytochemistry
Experiments were performed as described previously (22) . Plasma membrane expression of N-terminally tagged rho-tag(39)-VN1Rs was assessed using the primary anti-rhodopsin antibody B6-30 (24) (1:1000, 1 h at RT) on goat serum-blocked cells (5%, 30 min), and nonpermeabilized (4% paraformaldehyde, 15 min at RT) and permeabilized (4% paraformaldehyde, 15 min at RT, acetone/methanol 1:1, –20°C, 3 min) cells; 2% serum was present throughout. Labeled VN1R protein was visualized using an Alexa-488-coupled secondary antibody (1:200, goat-anti-mouse; Molecular Probes), and confocal microscopy (Leica TCS SP2 Laser Scan, 100x HCX PL APO oil immersion; Leica Microsystems, Wetzlar, Germany).

cAMP assay
cAMP assays were performed as described previously (22) . HeLa/Olf cells (106 cells/well in 6-well plates) were used nontransfected or were transfected with 1.5 µg of receptor DNA. After 40 h, cells were preincubated with IBMX (Calbiochem), a blocker of phosphodiesterase (100 µM, 30 min), and simultaneously exposed to odorants or isoproterenol and forskolin (2 min) (25) . The cAMP levels were assayed with the 125I-labeled cAMP assay system (Amersham Radiochemicals, GE Healthcare, Freiburg, Germany) in triplicate determinations. Values are given as fold stimulation over basal.

[35S]GTP{gamma}S binding assay
[35S]GTP{gamma}S binding assays were performed as described previously (22) , with modifications that include a G-protein immunoprecipitation step (26) . HeLa/Olf cells were transfected with 6 µg of receptor DNA or empty vector in 10-cm dishes. For the binding reaction, cell membrane protein (50 µg) was incubated for 15 min at 37°C in a binding buffer (10 mM HEPES, pH 7.4, 3 mM MgCl2, and 50 mM NaCl) that included 0.05 µM [35S]GTP{gamma}S and 3 µM GDP in a total volume of 100 µl. Basal condition was determined in the absence of an agonist. Parallel assays containing unlabeled GTP{gamma}S (10 µM) were used to define nonspecific binding. The reactions were stopped by the addition of ice-cold binding buffer and centrifuged at 16,000 g at 4°C for 30 min to pelletize the protein. After centrifugation, the pelleted protein was resuspended in the solubilization buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM EDTA, and 0.5% Triton-X), which included 1 µg anti-rabbit G{alpha}s/{alpha}olf (Calbiochem); G{alpha}i1, G{alpha}i2, or G{alpha}i3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); and 20 µl protein A-conjugated agarose beads, and rotated overnight at 4°C. The beads were washed 3x with solubilization buffer, and the bound radioactivity was measured in a liquid scintillation counter. Under these conditions, nonspecific binding was typically <10% of the total. The nonspecific binding was subtracted, and the basal value was set at 100%. Each data point within an experiment was determined from duplicate samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human VN1Rs respond to specific combinations of volatiles
RNA for VN1Rs has been detected in the human MOE and other tissues (21) (Supplemental Fig. 1). Can human VN1Rs be activated by volatile compounds? If so, do VN1Rs perform as many ORs, displaying combinatorial odorant detection? Or are they highly selective for a rather limited set of volatiles?

To investigate activation by odorants and a possible specificity of VN1Rs for certain volatiles, we used as a cellular model system HeLa/Olf cells which contain canonical olfactory signaling molecules, such as G{alpha}olf, AC-III, and CNGA2. We established expression of N-terminally rhodopsin-tagged (22) VN1Rs at the plasma membrane level of HeLa/Olf cells (Fig. 1 ). Comparing the averaged fluorescence in immunocytochemistry stainings of nonpermeabilized vs. permeabilized HeLa/Olf cell populations expressing rho-tag(39)-VN1Rs, rho-tag(39)-V1rb2, or rhodopsin, suggested: 1) plasma membrane expression of rho-tag(39)-VN1Rs, rho-tag(39)-V1rb2, and rhodopsin, and 2) a similar fraction of fluorescence in nonpermeabilized compared with permeabilized cells for rho-tag(39)-VN1Rs and rho-tag(39)-V1rb2. Using FLIPR calcium imaging technology on FLUO-4-loaded HeLa/Olf cells (22) , we then screened all human VN1Rs against an initial collection of 46 volatiles from different chemical classes, which included some putative pheromones, such as androstenol and androstenone (Table 1 ). We found that 9 odorants activated Ca2+ signals in rho-tag(39)-VN1R-transfected HeLa/Olf cells in a combinatorial way (Fig. 2 ). However, VN1Rs responded differentially and specifically to mostly aliphatic aldehydes or alcohols. We did not record receptor-dependent Ca2+ signals on application of a variety of acids, esters, lactones, pyrazines, steroids, amines, and camphor (Fig. 2 ; Table 1 ). To obtain information about the size and the functional group that would determine the highest efficacy of a given odorant that elicited responses in the initial screening, we established concentration-response relations, systematically varying the carbon chain length and functional groups of C8–C11 aliphatic aldehydes and alcohols, and obtained receptor-specific EC50-ranking odorant profiles (Fig. 3 A, B; Table 2 ). In addition, we established concentration-response relations for {alpha}- and β-ionone, linalool, (–)-carveol, (–)-myrtenal, and, as a measure of odorant efficacy, determined their EC50 values (Table 2) . We further screened all human rho-tag(39)-VN1Rs against an additional set of 94 odorants. At 30 µM, 5 odorants, {alpha}-hexyl-cinnamic aldehyde, methyl cinnamate, phenoxanol, jasmone-cis, and methylbenzodioxepinone, specifically activated VN1R1–4 but not VN1R5 (Table 2) . To confirm the odorant specificity of VN1Rs for aliphatic aldehydes or alcohols of certain chain lengths, we measured odorant-induced [35S]GTP{gamma}S binding to membrane preparations of VN1R-transfected HeLa/Olf cells before and after immunoprecipitation with an antibody directed against G proteins {alpha}olf/{alpha}s (Fig. 3C ).


Figure 1
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  		Figure 1. Visualizing plasma membrane expression of rho-tagged receptors in HeLa/Olf cells by anti-rho-tag immunocytochemistry. A) Confocal fluorescence images of B6–30 antirho-tag/Alexa488-labeled, nonpermeabilized HeLa/Olf cells, transfected with DNA coding for rho-tag(39)-VN1R1–5 and -V1rb2, or empty vector (mock). B) Densitometric analysis (Tina software, raytest) of fluorescence signals from confocal images of HeLa/Olf cells expressing rhodopsin or full-length rho-tag(39)-VN1R1–5 and -V1rb2. Fluorescence signals from entire single cells were normalized to cell area and background subtracted. Data are fluorescence from a population of nonpermealized cells (filled bars, representing plasma membrane expression of receptors) in percentage of fluorescence from a population of permeabilized cells (open bars), given as mean ± SD from at least 55 cells for each condition.


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  		Table 1. Panel of odorants used for the initial screening of hVN1R1–5 in HeLa/Olf cells


Figure 2
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  		Figure 2. Screening of VN1R1–5-transfected HeLa/Olf cells against odorants. A) Calcium imaging using FLIPR technology on HeLa/Olf cells transfected with DNAs of rho-tag(39)-VN1R1–5, each tested with 46 different odorants (10 µM, Table 2 ). Gray shading = mock. *VN1R-dependent responses to odorants. X-Y-scaling: 3 min, 4500 fluorescence counts. B) Odorants that elicited VN1R-dependent responses in A. Some signals were not considered to be entirely receptor-dependent responses because they were not consistently negative in mock-transfected cells (compare B10 for all VN1Rs) or activated mock cells in other screening experiments (data not shown).


Figure 3
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  		Figure 3. Specific activation and agonist profiles of VN1Rs. A, B) Specificity of aliphatic aldehydes and alcohols determined in Ca2+ imaging experiments using FLIPR technology on HeLa/Olf cells transfected with DNAs of full-length rho-tag(39)-VN1Rs. Black = aldehydes; gray = alcohols; carbon chain lengths = C7–C11. A) Traces represent mock-subtracted Ca2+ signals averaged from triplicate determinations, in response to 1 µM of aliphatic odorants. F = 500 fluorescence units. B) Schematic representation of the EC50-ranking odorant profiles of VN1Rs (Table 2) . C) VN1R-dependent and aliphatic odorant-induced [35S]GTP{gamma}S binding to G proteins depended on the odorant’s functional group and concentration. Note the same percent [35S]GTP{gamma}S binding to membrane preparations of VN1R-transfected HeLa/Olf cells before (open bars) and after immunoprecipitation with an antibody directed against G proteins {alpha}olf/{alpha}s (filled bars), suggesting exclusive coupling of all VN1Rs to G{alpha}olf, G{alpha}s. Data are given as mean ± SD from 2 independent experiments with duplicate determinations. *Odorant-induced responses are significantly different from those in mock-transfected cells (P<0.05, Student’s t test).


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  		Table 2. EC50-ranking odorant profiles for hVN1R1–5 in HeLa/Olf cells

Mouse V1rb2 attenuates cAMP signaling via G{alpha}i2,3 in HeLa/Olf cells
Sensory neurons in the 2 best-investigated olfactory organs of rodents, the MOE and VNO, express distinct signaling molecules (27) . Coexpression of V1rs with signal-transducing G{alpha}i2 proteins in a segregated subpopulation of sensory neurons of the mouse VNO (28) has spurred speculation about their functional coupling. However, a direct participation of G{alpha}i proteins in V1R signaling has never been demonstrated (29) . In HeLa/Olf cells, RNA for G{alpha}i1–3 proteins is present (unpublished results). Here we show that the only functionally identified pheromone receptor from mouse, V1rb2 (11) , 1) activated [35S]GTP{gamma}S binding to G proteins G{alpha}i2 and G{alpha}i3 (Fig. 4 A), 2) decreased a cAMP production (Fig. 4D ), and 3) attenuated a cAMP signaling-induced Ca2+ influx (Fig. 4E ), when expressed as rho-tag(39) receptor in HeLa/Olf cells and on stimulation with its agonist 2-heptanone. The effect of 2-heptanone on Ca2+ influx into V1rb2-transfected HeLa/Olf cells was concentration-dependent, with an EC50 of 8.3 ± 2.9 pM (Fig. 4I ). Blocking the coupling of V1rb2 with G{alpha}i proteins by PTX prevented the 2-heptanone-induced attenuation of the cAMP-dependent Ca2+ influx (Fig. 4E ).


Figure 4
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  		Figure 4. The signal transduction pathways of human VN1Rs and mouse V1rb2 in HeLa/Olf cells. A–C) Odorant (10 µM)-induced [35S]GTP{gamma}S binding in membrane preparations of HeLa/Olf cells transfected with DNA for mouse receptor rho-tag(39)-V1rb2 (A), human rho-tag(39)-VN1R1 (B), and mouse OR rho-tag(39)-Olfr49 (C) was determined before and after immunoprecipitation with antibodies directed against G-proteins {alpha}olf, {alpha}s, {alpha}i1, {alpha}i2, or {alpha}i3. Data are given as mean ± SD from 2 independent experiments with duplicate determinations. *Odorant-induced responses are significantly different from those in mock-transfected cells (P<0.02, Student’s t test). D) cAMP production (fold stimulation over nonforskolin conditions) in HeLa/Olf cells, transfected with rho-tag(39)-V1rb2, rho-tag(39)-VN1Rs1–5, or rho-tag(39)-Olfr49. Cells were IBMX-preincubated (100 µM, 30 min) and stimulated simultaneously with forskolin (10 µM) and increasing concentrations of odorants. Note: stimulation of adenylyl cyclase by forskolin was necessary to detect the inhibitory effect of V1rb2 on the cAMP production. 2h = 2-heptanone; c-al = (–)-citronellal; black = aldehydes; gray = alcohols; carbon chain lengths = C9–C10; mock = cells transfected with plasmid lacking any receptor coding region; b = basal; all odorants at 10 µM. Data are given as means ± SD from 2 independent experiments with triplicate determinations. Significantly different from forskolin stimulation alone at P ≥ 0.05. E–G) Single-cell Ca2+ imaging of Fura-2-loaded HeLa/Olf cells, transfected with mouse rho-tag(39)-V1rb2 (E), human rho-tag(39)-VN1Rs (F), rho-tag(39)-Olfr49 (G), or mock (H). Cells were pretreated (30 min) with, and experiments performed in the presence of thapsigargin (1µM), to exclude Ca2+ release from intracellular stores. E) 2-heptanone(h)-induced Ca2+ signals were recorded from cells pretreated (15 min) with, and in the presence of forskolin (15 µM), to prestimulate adenylyl cyclase. Dotted line: note the absence of 2-heptanone-induced Ca2+ signals under PTX (1 µg/ml, 2 h) in V1rb2-transfected cells. (F, G) All G{alpha}s/G{alpha}olf-mediated Ca2+ signals were recorded under PTX and thapsigargin. EGTA (1 mM) confirmed an odorant-induced Ca2+ influx. Odorants were 10 nM (E) or 10 µM (F, G). Averaged traces from all responsive cells within the camera field (number of responders/total cells), or all cells in mock: E), V1rb2, (13/25); V1rb2/PTX, all; F) VN1R1, (19/49); VN1R2, (30/69); VN1R3, (27/43); VN1R4, (12/52); VN1R5, (22/48); G) Olfr49 (8/22). One of the 2 independent experiments. I) Concentration-response relation of 2-heptanone applied to HeLa/Olf cells transfected with DNA coding for rho-tag(39)-V1rb2. Cells were pretreated (30 min) with, and experiments performed in the presence of thapsigargin (1 µM), to exclude Ca2+ release from intracellular stores. 2-heptanone-induced Ca2+ signals were recorded from HeLa/Olf cells pretreated (10 min) with, and in the presence of forskolin (10 µM), to prestimulate adenylyl cyclase. Data are given as mean ± SD from 2 independent experiments. IC50 is 8.3 ± 2.9 pM. J) Model of the mouse V1rb2 vs. human VN1R signal transduction in HeLa/Olf cells.

Human VN1Rs activate cAMP signaling via G proteins {alpha}olf/{alpha}s in HeLa/Olf cells
Can human VN1Rs be functionally expressed in HeLa/Olf cells in a similar way as V1rb2, such that on stimulation with an agonist they interact with G{alpha}i proteins, which then attenuate cAMP signaling by inhibiting an AC activity? Our experiments, so far, rather suggested an activation of the cAMP signaling pathway in HeLa/Olf cells by all human rho-tag(39)-VN1Rs. Interestingly, and in contrast to mouse V1rb2, all 5 human VN1Rs employed the canonical OR signal transduction (Fig. 4J ), as inferred from 1) odorant-induced [35S]GTP{gamma}S binding to G proteins G{alpha}olf/s (Figs. 3D , 4B) , 2) odorant-induced cAMP production (Fig. 4D ), and 3) odorant-induced and PTX-insensitive Ca2+ influx into rho-tag(39)-VN1R-transfected HeLa/Olf cells (Fig. 4F ). Moreover, the intrinsic activity of all rho-tag(39)-VN1Rs in HeLa/Olf cells was comparable with that of an olfactory receptor, rho-tag(39)-Olfr49, at each station of the signaling cascade (Fig. 4C, D, G ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our study, we observed that each human rho (39) -tagged recombinant VN1R responded to several odorants, when expressed in HeLa/Olf cells. Moreover, a given odorant activated several VN1Rs. For instance, C9 aldehyde activated 4 of 5 VN1Rs. Increasing the number of different volatiles in our screening experiments increased the number of VN1R-activating odorants even further. Replacing the initial 46 odorants, which yielded 9 responders, by another set of 94 different substances resulted in 5 additional odorants that were active on some, but not all, VN1Rs. Combinatorial odorant detection at the receptor level is a functional attribute typical for ORs (29 , 30) and is in contrast to an ultrasensitive and highly selective pheromone detection of a rather limited set of chemical cues, as has been suggested for V1r-expressing sensory neurons of the mouse VNO (11 , 12) .

In our hands, human rho-tagged VN1Rs responded differentially and specifically to the functional groups and chain lengths of odorants. For instance, 3 of 5 VN1Rs responded selectively to long-chain aliphatic aldehydes but not to aliphatic alcohols of the same chain length. Our data demonstrate the absolute requirement of the aldehydic group for activity at VN1R1, VN1R3, and VN1R4, and the alcohol moiety for activity at VN1R2. However, the aldehyde carbonyl was not, by itself, sufficient for the activity of these 3 receptors, as several other aldehyde-containing compounds, such as (–)-myrtenal or {alpha}-hexylcinnamic aldehyde were inactive at VN1R3 and VN1R4. Similarly, the hydroxyl group was necessary for an activation of VN1R2-transfected cells but not sufficient alone to activate this receptor because linalool and (–)-carveol did not activate VN1R2. Overall, we demonstrate in our study different VN1R-specific, EC50-ranking, and thus concentration-dependent odorant profiles, suggesting that each VN1R has its own specific function in detecting certain volatiles. However, because some human ORs can detect, for example, aliphatic aldehydes (31) , with similar efficacies as the VN1Rs, their biological significance in detecting these odorants has yet to be defined.

Do these volatiles have a pheromonal function in humans? As yet, no bioassay-guided study has led to the isolation of true human pheromones (32) . The most critical attribute of a putative human pheromone is that it must emanate from humans. Long-chain aliphatics, indeed, are volatiles in human skin emanations (33) but also are key aroma odorants in food (34) . In HeLa/Olf cells, the concentration-response relations of the most potent odorants activating VN1Rs, such as nonanal and decanal, are in the nanomolar to micromolar range. Their rank order of potency in HeLa/Olf cells is in line with the ranked human odor threshold concentrations for C8–10 aldehydes (34) . However, a pheromonal function of aliphatic aldehydes or alcohols on humans has yet to be proven.

In mice, V1rs supposedly activate via G{alpha}i2/β2/{gamma}8 a phospholipase Cβ2/IP3 pathway and the TRPC2 channel (35 , 36) . In humans, TRPC2 is not functional (37) and any vomeronasal pheromone signal transduction will be impaired. Any VN1R using the TRPC2-directed signaling pathway, if not used in additional physiological processes, would be released from functional constraints. These findings, together with the low number of functional VN1R genes in humans compared with other species, have been interpreted to suggest that the human VN1Rs may not possess physiological functions but rather may simply be relics of an ongoing pseudogenisation process (31 , 38) .

However, in this study, we demonstrate for the first time, and at each level of the signal transduction cascade, the employment of different signal transduction pathways of mouse and human VN1-type receptors, as shown by [35S]GTP{gamma}S binding to immunoprecipitated G-protein {alpha}-subunits, AC activity, and Ca2+ influx measurements.

By first demonstrating inhibitory coupling to the cAMP pathway of mouse V1rb2 via G{alpha}i2,3 proteins, our experiments validate the HeLa/Olf cell system beyond cAMP signaling of ORs (22) as an expression system for V1rs. The EC50 for the mouse pheromone 2-heptanone attenuating a cAMP-dependent Ca2+ influx into V1rb2-transfected HeLa/Olf cells (8.3 pM) lies within the same concentration range as the reported EC50 (140 pM) for 2-heptanone on isolated V1rb2-expressing mouse vomeronasal sensory neurons (11) . However, this EC50 is at least 3 orders of magnitude lower than the EC50 for 2-heptanone on mouse OR, mOR912–93 (Olfr154) measured in HeLa/Olf cells (22) , or HEK-293 cells (39) , suggesting different concentration ranges for pheromone vs. odorant detection. Our findings are in line with earlier observations where volatile odorants activated only AC-inhibiting G{alpha}i proteins in membrane preparations from mouse VNO (40) .

Surprisingly, and in sharp contrast, all human rho(39)-tagged VN1Rs activated cAMP production and Ca2+ influx in a PTX-independent way via AC-stimulating G{alpha}olf, G{alpha}s proteins, thus behaving like ORs. Interestingly, a recent study in mice suggested that signaling through AC-III in the MOE is obligatory for male sexual behavior, male-male aggressiveness, and the detection of some pheromones (41) .

It may be anticipated that, at the receptor level, both odorant as well as pheromone detection can be achieved by human ORs or VN1Rs. In mice, the volatile compound 2-heptanone can activate mOR912–93 (Olfr154) as well as V1rb2. An interpretation of pheromone vs. odorant detection by the brain will depend on the topographical projections of receptor-expressing neurons to specialized brain areas (42) . In mice, V1rs are typically expressed in the VNO and project their axons to a specialized structure in the brain, the accessory olfactory bulb (42 , 43) . It is not known whether human VN1Rs, which share as little as 15% amino acid identity with ORs, project their axons to specialized brain areas distinct from those of ORs. Given the existence of thousands of possible chemical cues, we may not have identified the best agonists for the human VN1Rs yet. However, so far, and in contrast to the mouse pheromone receptor V1rb2, all human VN1Rs show major functional attributes of odorant-detecting ORs: 1) mRNA expression in the human MOE, 2) combinatorial odorant detection in HeLa/Olf cells above human thresholds and at thousandfold higher concentrations than 2-heptanone acting on mouse V1rb2, and 3) OR-typical cAMP signaling via G{alpha}olf/G{alpha}s. Further experiments will answer the question whether the human MOE may have adopted pheromone sensor function by a subpopulation of VN1R-expressing OSNs that may project their axons to specialized brain areas. A nonolfactory related, yet unknown, biological function of VN1Rs cannot be ruled out at present.


   ACKNOWLEDGMENTS
 
We thank Paul A. Hargrave, Department of Ophthalmology, University of Florida (Gainesville, FL, USA), for B6-30 anti-rhodopsin antibody. We thank Boris Schilling, Givaudan (Duebendorf, Switzerland), for a set of odorants. We are grateful to T. Rathjen for expert technical assistance. We thank K. Schmiedeberg, P. Shi, and F. Stähler for helpful discussions. E.S. was supported by a grant of the Potsdam University Graduate Promotion Program and the Federal Ministry of Education and Science (BMBF). This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft to D.K. (Kr-1548).


   FOOTNOTES
 
1 Current address: Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada.

Received for publication June 19, 2007. Accepted for publication November 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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