Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M411508200 on December 14, 2004

J. Biol. Chem., Vol. 280, Issue 12, 11807-11815, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/12/11807    most recent
M411508200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shirokova, E.
Right arrow Articles by Krautwurst, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shirokova, E.
Right arrow Articles by Krautwurst, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification of Specific Ligands for Orphan Olfactory Receptors

G PROTEIN-DEPENDENT AGONISM AND ANTAGONISM OF ODORANTS*

Elena Shirokova{ddagger}§, Kristin Schmiedeberg{ddagger}, Peter Bedner¶||, Heiner Niessen¶, Klaus Willecke¶, Jan-Dirk Raguse**, Wolfgang Meyerhof{ddagger}, and Dietmar Krautwurst{ddagger}{ddagger}{ddagger}

From the Departments of {ddagger}Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany, Genetics, University Bonn, Roemerstrasse 164, 53117 Bonn, Germany, **Clinic and Polyclinic for Oral and Maxillofacial Surgery and Plastic Surgery, Charité, Campus Virchow Hospital, Augustenburger Platz 1, 13353 Berlin, Germany

Received for publication, October 8, 2004 , and in revised form, December 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein {alpha}olf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of G{alpha}olf in odorant-specific signaling out of OR. The employment of the non-typical G protein {alpha}15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of G{alpha}15 or G{alpha}olf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A multitude of up to 1000 OR1 (1, 2) and their combinatorial odorant recognition properties (3, 4) have made it difficult to assign ligand profiles to individual OR (5). Few studies have associated single odorants with individual OR by means of adenoviral overexpression assays in vivo (6), single-cell RT-PCR of odorant-responsive OSN (3, 4, 7), and OR gene targeting followed by functional analysis of a single OSN (8). The heterologous expression and functional imaging of some N-terminally tagged recombinant OR in HEK-293 cells have been achieved by forcing them to activate phosphoinositol signaling and Ca2+ release via G protein subunits {alpha}15, 16 (7, 9-11) or other Ca2+ release-activating G proteins (12). However, it is not known whether all of the OR and by which efficacy may couple to G proteins that activate phosphoinositol signaling. The apparent virtues of natural cell receptor systems have been discussed (13), and cell lines derived from OSN expressing the olfactory CNG channel have been reported previously (14-16). However, the expression of exogenous OR in these cells is problematic2 or they appear to express several endogenous OR (15, 16), thus complicating the investigation of specific cognate OR-odorant pairs.

Heterologously expressed OR can trigger odorant-induced cAMP signaling (9, 17). Thus far, however, the functional genomics of OR were hampered by the absence of a generally applicable cell system to study recombinant OR in their olfactory-specific G{alpha}olf/AC type-III/cAMP (18) signaling background.

Here, we demonstrate stable expression of canonical olfactory signal transduction molecules together with individual OR in the human HeLa/Olf cell line. This allows odorant/OR-activated intracellular cAMP signaling as well as cyclic nucleotide-induced Ca2+ influx through CNGA2 that can be monitored with conventional single-cell and high-throughput Ca2+ -imaging methods. We demonstrate the central role of G protein {alpha}olf and show evidence for G protein-dependent and ligand-selective agonism and antagonism of odorants at OR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Cloning—The 93 chimeric OR tested here were taken from a collection of chimeric OR (10) but were exclusive of the 80 OR that had been tested in that study. cDNA encoding the bovine olfactory CNGA2 channel (BTCAMPGC, X55010 [GenBank] ) was inserted into the vector pcDNA3.1/Zeo(+). To obtain the coding region of the olfactory human guanine nucleotide-binding G protein {alpha} (hG{alpha}olf, GNAL: GenBankTM accession number NM002071), we first isolated total RNA from human surgical olfactory epithelium biopsies with TRIzol (Invitrogen) and we then isolated mRNA with Micro-FastTrack 2.0 (Invitrogen) and synthesized first-strand cDNA using ImProm-II (Promega) reverse transcriptase. We PCR-amplified and subcloned hG{alpha}olf into the cytomegalovirus promoter-driven expression cassette pi2-dk based on plasmid pIRES2-EGFP (Clontech) but lacking the DNA coding for an internal ribosome entry site and the enhanced green fluorescent protein part. We subcloned G{alpha}15 (Gna15, GenBankTM accession number NM010304) and mOR912-93 (Olfr154) from their original plasmids (for pBSG15, see Ref. 19 and supplemental material; for mOR912-93, see Ref. 11) into the EcoRV/XbaI sites of pcDNA3 (Invitrogen) or the EcoRI/NotI sites of pi2-dk(Rho-tag(39)), respectively. We PCR-amplified the full-length coding regions of Ors6 (GenBankTM accession number NM020289), Ors86 (Olfr586, GenBankTM accession number NM147111), and Olfr43 (GenBankTM accession number XM111129) from mouse (C57BL/6J) genomic DNA with Pfu (Promega) or PfuUltra (Stratagene). Amplicons were subcloned into pi2-dk(Rho-tag(39)). This expression vector provides the first 39 amino acids of the bovine rhodopsin (Rho-tag(39)) as an N-terminal tag for all of the full-length OR. We cloned the full-length coding region of mouse Olfr49 (I-C6, GenBankTM accession number NM010991), mouse and rat Olfr41 (mI7, GenBankTM accession number NM010983; rI7, GenBankTM accession number NM_031710 [GenBank] ) (10), and human OR17-40 (OR3A1) EcoRI/NotI into pi2-dk(Rho-tag(39)) from the original plasmids. The identities of all of the subcloned amplicons were verified by sequencing (UKEHH, Hamburg, Germany) (for details and primer sequences, see supplemental material).

Cell culture and Transient DNA Transfection—All of the cell culture media, ingredients, and antibiotics were obtained from Invitrogen with the exception for G418 sulfate (Calbiochem) and puromycin (Sigma). HeLa cells were grown in standard DMEM with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere (37 °C, 5% CO2). Before transfection, cells were seeded in black wall/clear bottom 96-well plates (Molecular Devices) for FLIPR, coated with poly-D-lysine (10 µg/ml), and cultured to a preconfluent monolayer. Cells were transfected with DNA using lipofection (PolyFect, Qiagen) and, 40 h posttransfection, the cells were taken into experiments (for stable cell lines, see supplemental material). Usually, ~5% OR-transfected cells show a signal in anti-Rho-tag immunocytochemistry. However, 10-40% cells usually respond in single-cell Ca2+-imaging experiments, presumably because of coupling of cells via Cx-43.

Stable Cell Lines: Establishment of the HeLa/CNGA2 Cell Line—The HeLa-Cx43 cell line expressing Cx-43 (20) was cultured in standard DMEM supplemented with 1 µg/ml puromycin. For transfection, cDNA encoding the bovine olfactory CNGA2 subunit (BTCAMPGC, X55010 [GenBank] (21)) was inserted into the vector pcDNA3.1/Zeo(+). HeLa-Cx43 transfectants were stably transfected by lipofection (Tfx-50 Reagent, Promega). 48 h posttransfection, 100 µg/ml zeocin was added to the medium. Clones were picked after 3-4 weeks and grown under selective conditions. The presence of Cx-43 proved beneficial for obtaining clones that stably express the wild-type homomeric olfactory CNGA2 channel.3 Cx-43 is expressed in OSN and passes second messengers such as inositol 1,4,5-trisphosphate, Ca2+, and cyclic nucleotides. In HeLa cells that are connected by Cx43, a noxious overload by Ca2+ of individual cells may be prevented, thus increasing the number of surviving stable transfectants. Thus we refrained from using a mutant homomeric CNGA2 channel that was described to have a similar low EC50 for cAMP as the heteromeric wild-type channel. An increased sensitivity for cyclic nucleotides may result in an increased resting conductance of CNG channels, which in the long run might lead to noxious Ca2+ concentrations, hampering the establishment of stable cell lines (for reference to Cx-43 and CNGA2, see supplemental material).

Establishment of the HeLa/CNGA2/Rho-tag(39)-Olfr49 (HeLa/Olfr49), HeLa/CNGA2/hG{alpha}olf (HeLa/Olf), and HeLa/CNGA2/G{alpha}15 (HeLa/15) Cell Lines—Before transfection with Olfr49 or hG{alpha}olf, cells were plated in 100-mm dishes at a density of 1.6 x 106 cells and incubated overnight. HeLa/CNGA2 cells were transfected with expression plasmids carrying the coding regions for Rho-tag(39)-Olfr49, hG{alpha}olf, or G{alpha}15 by calcium phosphate precipitation. HeLa/Olfr49 cells, HeLa/Olf cells, and HeLa/15 cells were then obtained by selection of clonal populations, resistant to 800 µg/ml G418 and responsive to (-)citronellal, or isoproterenol, respectively, and confirmed by RT-PCR or immunocytochemistry. Clonal lines were maintained in standard DMEM supplemented with puromycin (1 µg/ml), zeocin (100 µg/ml), and G418 (400 µg/ml). We observed stable expression of hG{alpha}olf or G{alpha}15 up to passage 10 (HeLa/Olf cells and HeLa/15 cells) or passage 15 for Rho-tag(39)-Olfr49 (HeLa/Olfr49 cells).

Single-Cell Ca2+ Imaging—Odorant/OR-induced changes in the intracellular concentration of cyclic nucleotides modulate a Ca2+-influx through CNGA2 that can be monitored in Ca2+-imaging experiments. Single-cell Ca2+-imaging was performed as described previously (10) but with the following modifications. We used the monochromator-based Till Photonics imaging system in combination with an inverted Olympus IX50 microscope equipped with a UApo/340 x40 1.35 oil immersion lens. 40 h posttransfection, cells were loaded (1 h/37 °C) with 4 µM Fura-2/AM (Molecular Probes) in serum-free DMEM and transferred into a bath chamber with a 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 and 380 nm and ratios calculated (F340/F380). Images of the cells were monitored by an intensified cooled CCD camera and analyzed off-line. We applied isoproterenol (10 µM) at the end of each experiment to stimulate endogenous {beta}-AR, proving that the G protein-dependent signal transduction cascade was functional. Odorants (Supplemental Table 2), poly-D-lysine, probenecid, salts, buffers, and db-cAMP (membrane-permeable, used at 1 mM) were from Sigma. Thapsigargin (BioTrend/Tocris; 1 µM, 30 min) blocked receptor-mediated Ca2+ release from inositol 1,4,5-trisphosphate-sensitive internal stores (22). Bath application of 1 mM EGTA (Sigma) blocked Ca2+ influx.

Ca2+-FLIPR Assay—Cells were loaded with 4 µM FLUO-4/AM and 0.04% Pluronic F-127 (both from 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) and transferred to the FLIPR (Molecular Devices). The FLIPR integrates an argon laser excitation source, a 96-well pipettor, and a detection system utilizing a CCD imaging camera. Equipment for the FLIPR system was obtained from Molecular Devices. 50 µl of 3x concentrated agonists were delivered within 2 s simultaneously to all of the wells containing 100 µl of HBS. Fluorescence emissions from the 96-wells were monitored simultaneously at an emission wavelength of 515 nm after excitation with 488 nm (F488). Fluorescence data were collected at 0.25 Hertz, 48 s before and 5-10 min after stimulation, and analyzed off-line.

Data Analysis—In the FLIPR-screening experiments, agonist response amplitudes were determined from the peak-stimulated fluorescence of the solvent control- or mock-transfected-substracted and base-line-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 and curves were derived from fitting the function f(x) = (a - d)/(1 + (x/C)nH) + d to the data by non-linear regression with a = minimum, d = maximum, C = EC50, and nH = Hill coefficient.

Immunocytochemistry—Plasma membrane expression of N-terminally tagged Rho-tag(39)-OR was assessed using the primary anti-rhodopsin antibody B6-30 (1:1000, 1 h at room temperature or 4 °C overnight) (10) in goat serum-blocked cells (5%, 30 min) and non-permeabilized (4% fresh paraformaldehyde, 15 min at room temperature) and permeabilized (4% fresh paraformaldehyde, 15 min at room temperature, acetone/methanol 1:1, -20 °C, 3 min) cells. 2% serum was present throughout. Labeled OR 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).

cAMP Assay—HeLa/CNGA2/Rho-tag(39)-Olfr49 cells (106 cells/well in 6-well plates) were used as non-transfected or were transfected with 1.5 µg of rat G{alpha}olf or rat G{alpha}s DNA using PolyFect. After 40 h, cells were preincubated with IBMX (Calbiochem), a blocker of phosphodiesterase (100 µM, 30 min), and exposed to (-)citronellal or isoproterenol (2 min). The cAMP levels were assayed with the 125I-labeled cAMP assay system (Amersham Biosciences) in triplicate determinations. Values are given as fold stimulation over basal.

Western Blot Analysis—Crude membrane preparations of HeLa/CNGA2 and HeLa/Olf cells were cholate-extracted (1%) in the presence of 2 µg/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml pepstatin A (all from Sigma). 19 µg of protein of each preparation were run in 11% SDS-PAGE and blotted onto nitrocellulose using a Sammy Dry Blotter (Schleicher & Schüll). Blots were blocked and incubated 2 h with a polyclonal anti-G{alpha}s antibody (C-18, Santa-Cruz Biotechnology, 1:200), which is directed against the C-terminal amino acids and also recognizes G{alpha}olf. Chemiluminescence of peroxidase-coupled secondary antibody (anti-rabbit IgG, 1.5 h) was visualized by Hyperfilm-ECL exposure (7 min).

[35S]GTP{gamma}S Binding Assay—HeLa/15 cells and HeLa/Olf cells were grown in a 10-cm dish and transfected with 6 µg of Ors6 plasmid or empty vector using PolyFect. After 42 h, cells were harvested by manual scraping and the [35S]GTP{gamma}S binding assay was performed as described by Bidlack and Parkhill (23) with modifications. The membrane protein concentration was 80 µg per experiment, and the agonist stimulation was measured as triplicates in a Beckmann scintillation counter "LS6500." The Whatman GF/B glass fiber filters were shortly preincubated with 20 mM Tris-HCl, 10% polyethylene glycol, 10 mM MgCl2, pH 7,4. Basal [35S]GTP{gamma}S binding was determined in the absence of an agonist. The nonspecific binding was subtracted, and the basal value was set at 100%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We initially established the human HeLa/CNGA2 cell line stably expressing Cx-43 (20) and the olfactory homomeric CNGA2 channel (21) as a Ca2+-influx reporter for receptor-induced changes in the concentration of intracellular cyclic nucleotides. The presence of Cx-43 proved to be beneficial for obtaining stable transfectants for CNGA2. We confirmed the expression of mRNA for the CNGA2 channel in HeLa/CNGA2 cells by RT-PCR (Fig. 1) and functional expression of CNGA2 in single-cell Ca2+-imaging experiments (Fig. 2a, left panel, and d and e).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.
Characterization of HeLa/CNGA2 cells. RT-PCR products from HeLa/CNGA2 mRNA using gene-specific primers for the olfactory CNGA2 channel subunit and all nine types of human AC (ACI-IX) (upper panel). Expected sizes of RT-PCR products are shown as base pairs within parentheses: ACI (266); ACII (148); ACIII (292); ACIV (166); ACV (176); ACVI (242); ACVII (210); ACVIII (245); and ACIX (180). Lower panel, reverse transcriptase omitted; M, marker sizes (bp).

 



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 2.
OR signaling stably reconstituted in HeLa/CNGA2 cells. a, left panel, single-cell Ca2+ imaging of Fura-2-loaded HeLa/CNGA2 cells stimulated with db-cAMP. Experiments were done in the presence of thapsigargin and IBMX. Averaged traces of 20 cells within the camera field. We observed no db-cAMP-induced Ca2+ influx in cells lacking CNGA2 (data not shown). a, right panel, HeLa/CNGA2 cells with G{alpha}olf but lacking Rho-tag(39)-Olfr49. (-)Citronellal ((-)c-al) was 10 µM. Averaged traces of 36 cells within the camera field. b, confocal fluorescence images of anti-Rho-tag/Alexa-488-labeled non-permeabilized HeLa/Olfr49 cells (right panel), demonstrating expression of Rho-tag(39)-Olfr49. Control (HeLa/CNGA2) cells lacking Rho-tag(39)-Olfr49 are shown on the left panel. Scale bar, 8 µm. c-e, single-cell Ca2+ imaging of Fura-2-loaded HeLa/CNGA2, stimulating endogenous {beta}-AR with 10 µM isoproterenol (Iso) (left panels), or HeLa/Olfr49 cells, stimulating stably expressed Rho-tag(39)-Olfr49 with 10 µM (-)c-al (right panels). Cells were transiently transfected with mock DNA (c) or DNA for G{alpha}s (d) or G{alpha}olf (e). Shown are averaged traces from all of the responsive cells within the camera field (number of responders/total cells): (d) {beta}-AR (22/74) and Olfr49 (20/71); (e) {beta}-AR 14/68 and Olfr49 (11/61); and all of the cells in c. f, cAMP production (fold stimulation over basal) in HeLa/Olfr49 cells transfected with either G{alpha}olf or G{alpha}s DNA, IBMX-preincubated, and stimulated for 2 min with isoproterenol (left panel), (-)citronellal (right panel), or both at 3 µM. All of the differences in cAMP production are significant at p < 0.05. Data are means ± S.D. from two independent experiments.

 
A further characterization of HeLa/CNGA2 cells by RT-PCR revealed endogenous expression of mRNA for ACIII, ACVI, ACVII, and ACIX from the nine known ACs (Fig. 1) as well as for G{alpha}s but not G{alpha}olf (Fig. 3a). In these cells, the {beta}-AR agonist isoproterenol was not able to induce a {beta}-AR-stimulated Ca2+ influx (Fig. 2c, left panel). However, when we transfected HeLa/CNGA2 cells with DNA coding for G proteins {alpha}s or {alpha}olf, isoproterenol elicited a Ca2+ influx that could be antagonized with EGTA (Fig. 2, d and e, left panels).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 3.
Expression of G{alpha}olf in HeLa/Olf cells. RT-PCR products from HeLa/CNGA2 mRNA (a) and HeLa/Olf cells (b) using gene-specific primers for the human G proteins {alpha}s and {alpha}olf are shown. -RT, omitting reverse transcriptase; M, marker sizes (in base pairs). c, Western blot analysis of HeLa/CNGA2 cells ({alpha}s) and HeLa/Olf cells ({alpha}s + {alpha}olf). Anti-{alpha}s antibody recognizes both G proteins {alpha}s and {alpha}olf at 45 kDa. The 41-kDa band may be the result of degradation or improper protein expression.

 
To study OR/odorant-induced cAMP signaling and Ca2+-influx, we established stable and functional expression in HeLa/CNGA2 cells of Rho-tag(39)-Olfr49 (HeLa/Olfr49) (Fig. 2, b-e, right panels) for which (-)citronellal has been identified as a cognate odorant (10). We observed a receptor-dependent and (-)citronellal-induced Ca2+ influx in HeLa/Olfr49 cells that were transfected with DNA coding for either G protein {alpha}olf or G protein {alpha}s (Fig. 2, d and e, right panels). (-)Citronellal did not induce Ca2+ signals in HeLa/CNGA2 cells lacking Rho-tag(39)-Olfr49 (Fig. 2a, right panel) or cells that were not transfected with G proteins {alpha}olf or {alpha}s (Fig. 2c, right panel).

We compared the efficacy of both G proteins in OR and {beta}-AR signaling in HeLa/Olfr49 cells, employing a cAMP assay (Fig. 2f). The isoproterenol-induced cAMP production was higher in cells transfected with G{alpha}s, whereas the (-)citronellal-induced cAMP production was higher in G{alpha}olf-transfected cells. These results indicated first that an overexpression of signal transducing G protein appears necessary for functional OR expression in HeLa/CNGA2 cells and second that an odorant/OR-induced cAMP production was most efficient in G{alpha}olf-transfected cells. Therefore, we established stable expression of human G{alpha}olf in HeLa/CNGA2 cells (HeLa/Olf). As a result, the amount of G{alpha}olf/G{alpha}s was ~2.5-fold higher in HeLa/Olf cells as compared with the endogenous G{alpha}s level in HeLa/CNGA2 cells (Fig. 3).

At the level of OR-odorant interaction, combinatorial coding is commonly accepted as inferred from often broadly tuned OSN (3, 4). However, until now, only 14 OR gene sequences were published from one (3) but not the other (4) of the two comprehensive studies that deduced OR sequences from hundreds of single-cell RT-PCR experiments with mouse OSN that had responded to a chemically related set of odorants. Neither of these studies used the deduced OR sequences to confirm the odorant responsiveness of their gene products by means of EC50-ranking odorant profiles in functional expression studies. For two of the deduced putative OR, Ors6, and Ors86, we now have determined EC50 values in HeLa/Olf cells for their specific agonists, nonanedioic acid and nonanoic acid, respectively (Figs. 4 and 5 and Table 1), that reflect their odorant specificities as observed in OSN (see Table 1) (3).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 4.
The odorant specificity of Ors6 is determined by the G protein {alpha}olf. Single-cell Ca2+ imaging from odorant-stimulated HeLa/15 (a) and HeLa/Olf (b) cells (left panels) transiently transfected with DNA for Ors6 is shown. 8-ac, octanoic acid; 9d-ac, nonanedioic acid. Concentration of odorants was 10 µM (a and b, lower panel) and 30 µM (b, upper panel). G{alpha}15-mediated Ca2+ release signals (a) were recorded under EGTA to exclude Ca2+ influx. Ca2+ influx signals (b) were recorded under thapsigargin to exclude Ca2+ release from intracellular stores. Dotted line, mock-transfected cells. Shown are averaged traces from all of the responsive cells within the camera field (number of responders/total cells and all cells in mock): a, upper left, 18/119; a, lower left, 8/114; b, upper left, all; b, lower left, 12/21. Similar results were observed in two independent experiments. a and b, right panels, confocal fluorescence images of anti-Rho-tag/Alexa-488-labeled non-permeabilized HeLa/15 and HeLa/Olf cells, demonstrating plasma membrane expression of Rho-tag(39)-Ors6. Control, no primary antibody. Scale bar, 8 µm. c and d, concentration-response relations of octanoic acid () and nonanedioic acid ({circ}) on Ca2+ release in HeLa/15 cells (c) or Ca2+influx into HeLa/Olf cells (d), both expressing Rho-tag(39)-Ors6. e and f, [35S]GTP{gamma}S binding assay with HeLa/15 cells (e) or HeLa/Olf cells (f) that were either mock-transfected (open bars) or transfected with DNA coding for Rho-tag(39)-Ors6 (filled bars). Iso, isoproterenol. c-f, data are means ± S.D. from two independent experiments.

 



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 5.
Octanoic acid acts as an antagonist on Ors6 and Ors86 in HeLa/Olf cells. Concentration-response relations of nonanedioic acid (9d-ac) (a) and nonanoic acid (9-ac) (b) versus Ors6 and Ors86, respectively, in the absence (filled symbols) or presence (open symbols) of 3 µM octanoic acid. Data were calculated as means ± S.D. from at least two independent FLIPR experiments. EC50 values: , 0.5 ± 0.03 µM (n = 3); {circ}, 1.6 ± 0.3 µM (n = 2); {blacksquare}, 3.3 ± 0.5 µM (n = 3); {square}, >100 µM (n = 2). c and d, cAMP production (fold stimulation over basal) in HeLa/Olf cells transfected with either Ors6 or Ors86 DNA, IBMX-preincubated, and stimulated with increasing concentrations of 9d-ac (c) or 9-ac (d) in the absence (filled bars) or presence (open bars) of 3 µM octanoic acid. Data are means ± S.D. from two independent experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Ligand profiles for known ORs in OSN, HeLa/Olf, and HeLa/15 cells

Data are given as single concentrations or EC50 values as mean ± S.D. with numbers of experiments within parentheses. With the exception of Olfr43, the historical names of ORs are listed with their references. ND, no data.

 
Historically, we (Supplemental Fig. 1) and others4 have observed that some OR, when expressed in HEK-293 cells together with G{alpha}15, displayed an odorant specificity dramatically different from that observed in isolated OSN expressing the same OR. To investigate the influence of non-typical G protein {alpha}15 on the EC50-ranking odorant profiles of OR, we established stable expression of G protein {alpha}15 (19) in HeLa/CNGA2 cells (HeLa/15). By functional expression in HeLa/Olf and HeLa/15 cells, we then compared the agonist specificities of seven OR (3, 5, 6, 8, 10-12) that were previously characterized by only a single concentration or few odorant concentrations in OSN (employing G{alpha}olf) or in heterologous cell systems (employing promiscuous G proteins {alpha}15 and {alpha}16). In HeLa/Olf cells but not in HeLa/15 cells, we verified 2-heptanone as an agonist for mOR912-93 that had been identified in HEK-293 cells and transfected with DNAs coding for G{alpha}15 and a modified G{alpha}q (Table 1) (11). For another 2 of the 7 OR and their known odorant agonists, we show that employment of non-typical G protein {alpha}15 (HeLa/15 cells) resulted in a dramatically altered EC50-ranking agonist profile, different from the odorant specificity observed in HeLa/Olf cells (Ors6 (S6) and Ors86 (S86) in Table 1) or OSN (see Table 1) (3). In single-cell Ca2+ imaging, FLIPR, and [35S]GTP{gamma}S binding experiments with HeLa/Olf cells expressing Ors6 and in FLIPR experiments with Ors86, we observed an odorant selectivity for nonanedioic acid and nonanoic acid, respectively, but not for octanoic acid (Figs. 4 and 5), similar to what was observed in OSN (3). In contrast, in HeLa/15 cells, octanoic acid outperformed nonanedioic and was as good an agonist as nonanoic acid for Ors6 and Ors86, respectively (Fig. 4 and Table 1). Because octanoic acid worked as an agonist for both Ors6 and Ors86 in HeLa/15 cells but did not induce signaling in Ors6- or Ors86-expressing HeLa/Olf cells, we consequently investigated in these cells a possible antagonistic function of octanoic acid on Ors6 and Ors86. Here, octanoic acid acted as a competitive antagonist by shifting the EC50 values of nonanedioic acid and nonanoic acid from 0.5 ± 0.03 µM (n = 3) and 3.3 ± 0.5 µM (n = 3) to 1.6 ± 0.3 µM (n = 2) and >100 µM (n = 2), respectively (Fig. 5, a and b). Similarly, when using cAMP as a readout in Ors6- or Ors86-expressing HeLa/Olf cells, octanoic acid shifted the concentration-response relations of nonanedioic and nonanoic acid to the right (Fig. 5, c and d). Octanoic acid at 30 µM did not inhibit the db-cAMP-stimulated CNGA2 channel in HeLa/Olf cells (data not shown).

We have put HeLa/Olf cells to the test in a functional genomics approach to identify OR further with high sensitivity and specificity for (-)citronellal. In 96-well FLIPR experiments, we first screened (-)citronellal at a concentration (1 µM) below its EC50 value for Olfr49 against 93 Rho-tag(20)-M4 chimeric mouse OR (10) separately expressed in HeLa/Olf cells. The Rho-tag(20)-M4 chimera of Olfr49 was always included as a positive control (96-well coordinate A5) and consistently responded to (-)citronellal (Fig. 6a). We identified two new OR, Olfr43 (A1) and MOR267-1 (F2), that responded to 1 µM (-)citronellal (Fig. 6a). Full-length Rho-tag(39)-Olfr43, Rho-tag(39)-Olfr49, and Rho-tag(39)-MOR267-1 were expressed at the plasma membrane level in HeLa/Olf cells (Fig. 6b). The expression of these rhodopsin-tagged OR at the plasma membrane level of HeLa/Olf cells amounted to 25-40% of that of rhodopsin as a reference GPCR (Fig. 6c). The identification of OR-specific chaperones might improve this further (17). Rho-tag(39)-Olfr43, Rho-tag(39)-Olfr49, and Rho-tag(39)-MOR267-1 activated a (-)citronellal-induced Ca2+ influx in a receptor-dependent (Fig. 6d) and odorant concentration-dependent manner (Fig. 6e). Olfr43 emerged as the most sensitive OR for (-)citronellal (Fig. 6e and Table 1). To further characterize and establish an odorant-response pattern of Olf43, we then screened HeLa/15 cells and HeLa/Olf cells expressing full-length Rho-tag(39)-Olfr43 versus 94 odorants (listed in Supplemental Table 2) at a concentration of 30 µM (Fig. 7, a and b). 14 or 10 odorants elicited Ca2+ responses of varying amplitudes in full-length Rho-tag(39)-Olfr43-transfected HeLa/15 or HeLa/Olf cells, respectively, but not in mock-transfected cells (Fig. 7, a and b). Interestingly, the pattern of Olfr43-activating odorants differed depending on the G protein employed but overlapped in a set of seven odorants that were agonists in both OR/G protein systems. These odorants turned out to be the most potent agonists of Rho-tag(39)-Olfr43 in HeLa/Olf cells. We obtained an EC50-based rank order of potencies: (-)citronellal > helional > E-4-decenal, octanal > heptanal, {beta}-citronellol (Fig. 7, c and d, and Table 1) with no effect of citronellic acid.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 6.
De-orphaning of OR for (-)citronellal by FLIPR screening of an OR expression library in HeLa/Olf cells. a, HeLa/Olf cells transfected with DNAs of 93 Rho-tag(20)-M4 chimeric mouse OR and screened versus 1 µM (-)citronellal in 96-well FLIPR experiments. Asterisks, responders to odorants. Coordinates A10-A12 contained mock-transfected cells. Time scale, 3 min. b, confocal fluorescence images of anti-Rho-tag/Alexa-488-labeled non-permeabilized HeLa/Olf cells, demonstrating expression of full-length Rho-tag(39)-Olfr43, Rho-tag-(39)-Olfr49, and Rho-tag(39)-MOR267-1. Control, mock-transfected cells. Scale bar, 8 µm. c, densitometric analysis (Tina software, raytest) of fluorescence signals from confocal images of HeLa/Olf cells expressing rhodopsin or full-length Rho-tag(39)-OR. Fluorescence signals from entire single cells were normalized to cell area and background-substracted. Data are fluorescence from non-permeabilized cells (filled bars, representing plasma membrane expression of receptors) in percent of fluorescence from permeabilized cells (open bars) given as means ± S.D. from at least 25 cells. d, single-cell Ca2+-imaging experiments with HeLa/Olf cells that were transiently transfected with DNA coding for full-length Rho-tag(39)-OR and stimulated with 1 µM (-)citronellal. Shown are averaged traces from all of the responsive cells within the camera field: Olfr43 (13 responders/33 cells); Olfr49 (7/22); MOR267-1 (12/43); and all of the cells in mock). Experiments were done under thapsigargin. Similar results were observed in two independent experiments. e, concentration-response relations of (-)citronellal versus Olfr43 (), EC50 2.1 ± 0.2; Olfr49 ({blacksquare}), EC 3.3 ± 0.4; and MOR267-1 ({blacktriangleup}), EC50 8.2 ± 0.3. Data are mean of ± ± S.D. two independent FLIPR experiments.

 



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 7.
Odorant specificity patterns and EC50-ranking odorant profile of Olfr43. a and b, HeLa/15 cells (a) and HeLa/Olf cells (b) transfected with DNA for Rho-tag(39)-Olfr43 and tested with 30 µM of 94 different odorants. All of the odorants and their coordinates in the 96-well format are listed in Supplemental Table 2. Asterisks, responders to the same odorants in both cell lines: C9, (-)citronellal; C10, (+)citronellal; D6, geraniol; E4, E-4-decenal; G3, helional; G6, bourgeonal; and H6, octanal. c, concentration-response relations and EC50 values of (-)citronellal () 2.1 ± 0.2 µM (same data as in Fig. 6d); helional ({blacktriangleup}) 3.6 ± 0.5 µM; octanal ({diamondsuit}) 22.5 ± 3.9 µM; and E-4-decenal ({blacksquare}) 30.4 ± 1.4 µM on Ca2+ influx into HeLa/Olf cells transiently transfected with DNA for full-length Rho-tag(39)-Olfr43. EC50 values are means ± S.D. from two independent experiments. d, set of odorants that activated Olfr43 in both HeLa/Olf and HeLa/15 cells. Increasing distance from (-)citronellal as depicted by the lengths of the solid arrows reflect increasing EC50 values of these odorants in HeLa/Olf cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have designed HeLa/Olf cells to function as sensors for odorant/OR-induced cAMP signaling. To stably reconstitute OR signal transduction, we employed the homomeric wild-type olfactory CNGA2 channel, which has a rather high EC50 for cAMP (65 µM) (24). However, we refrained from using a mutant homomeric CNGA2 channel that had been described to have a similar low EC50 (3 µM) for cAMP as does the heteromeric wild-type channel (25). An increased sensitivity for cyclic nucleotides may result in an increased resting conductance of CNG channels (26), which in the long run might lead to noxious Ca2+ concentrations, hampering the establishment of stable cell lines. The lower sensitivity for cAMP of the HeLa/CNGA2 cell system was compensated by the overexpression of OR-coupling G protein, G{alpha}olf or G{alpha}s.

In the course of establishing HeLa/Olf cells for the de-orphaning of OR, our experiments revealed coupling of Olfr49 or {beta}-AR to both G{alpha}olf and G{alpha}s, in line with and extending previous findings (9, 27). Moreover, the coupling of Olfr49 to G{alpha}olf, as well as coupling of {beta}-AR to G{alpha}s, appeared to be preferential. The preference of {beta}-AR signaling via G{alpha}s seemed to be more pronounced than the preference of Olfr49 signaling via G{alpha}olf. However, a more rapid deactivation of G{alpha}olf-GTP by GTP hydrolysis and GTP dissociation relative to G{alpha}s-GTP (27) may account for these differences. Although the differences in signaling efficacy of an OR coupling to either one of the rather homologous G proteins {alpha}olf or {alpha}s appear subtle in HeLa cells, they may have a physiological relevance during development when both G proteins are differentially expressed in the olfactory epithelium (28). However, drastic differences in odorant profiles of a given OR appeared when employing a non-typical G protein such as G{alpha}15 in HeLa cells. 2 (Ors6 and Ors86) of the 8 OR in our study displayed a dramatically altered odorant profile in HeLa/15 cells as compared with HeLa/Olf or OSN. For another OR (mOR912-93), the identified agonist in HEK-293 cells transfected with G{alpha}15, together with modified G{alpha}q (11), could be confirmed in HeLa/Olf but not in HeLa/15 cells. The EC50 values of the known agonists of the other five OR did not change when tested in both G protein systems. However, increasing the number of odorants tested on Olfr43 revealed the discrepancy of its odorant recognition patterns in HeLa/15 versus HeLa/Olf cells. Interestingly, only the most potent agonists of Olfr43 in HeLa/Olf cells appeared as agonists also in HeLa/15 cells.

The phenomenon that agonists on the same receptor differ in their signaling efficacy and the stimulus pattern they produce in different physiological systems (or cell lines) was observed for a variety of GPCR (29). It was proposed that a ligand-receptor interaction may stabilize a certain G protein-activating receptor conformation or a G protein may stabilize a ligand-receptor interaction (30). As a result, different G protein pathways (31) may be stimulated with different efficacies ("ligand-selective agonism" (29)).

Coupling of OR to non-typical G proteins {alpha}15 or {alpha}16 is unlikely to occur in the OSN and hence is not relevant for odorant coding in the olfactory epithelium. However, because of the excessive numbers of OR and their combinatorial odorant coding, EC50-ranking agonist and antagonist profiles are the only reliable pharmacological parameters of OR that will have to be determined in heterologous cell systems. We conclude from this study that it is crucial to couple OR via G{alpha}olf in heterologous cell systems to avoid false odorant profiles in a functional genomics approach.

Our experiments clearly show that odorants structurally related to OR agonists can function as competitive antagonists at the receptor level, depending on the G protein employed. Acknowledging several recent reports on odorant antagonism at the level of OR (32-34), the functional characterization of OR will have to be modeled on GPCR pharmacology. Hence, the basic concept of establishing odorant recognition patterns of OR needs to be extended to OR-specific EC50-ranking agonist and antagonist profiles in a cellular system, such as HeLa/Olf, that provides for OR-specific signal transduction.

OR may have a broadly tuned odorant specificity because of combinatorial odorant coding and depending on odorant concentration. An advantage of nonspecific over specific tuning of OSN and thus OR for quality and intensity coding by means of a mixture of receptive fields of greatest possible diversity has been proposed recently in a mathematical model (35). However, another study has suggested (4) that principal odor qualities are encoded by the most sensitive receptors for a given odorant. Are Olfr43, Olfr49, and MOR267-1 such receptors for (-)citronellal? HeLa/Olf cells proved suitable to answer this question by screening (-)citronellal versus an OR library and screening the best responding OR versus an odorant library.

In summary, Olfr43 emerged as the most sensitive from 93 OR for citronellal, which in turn was the best agonist for Olfr43 from a collection of 94 odorants. Olfr43 appeared to have a prevalence for aldehydic odorants. A more detailed characterization of Olfr43 and its human homologs is in process.

Here, we have described the functional reconstitution of OR together with their stably reconstituted olfactory signal transduction molecules in the human cell line HeLa/Olf to investigate odorant coding at the receptor level. We demonstrated the central role of G{alpha}olf in defining OR-specific odorant profiles as well as defining the function of some odorants as either agonists or antagonists. Our approach points the way to solve the riddle of odorant coding and structure-function relations of OR by functionally screening an entire OR proteome versus odorant libraries using high-throughput screening methods and establishing EC50-ranking odorant profiles.


   FOOTNOTES
 
* This work was supported in part by Grants Kr-1548 (to D. K.) and Wi 270/24-1/2 (to K. W.) from the Deutsche Forschungsgemeinschaft, grants from the International Foundation for the Promotion of Nutrition Research and Nutrition Education (to E. S.), and Funds of the Chemical Industry (to K. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains supplemental material and Supplemental Fig. 1 and Table 2. Back

§ A former Gottfried-Daimler/Karl-Benz fellow and is supported by a fellowship from the German Federal Ministry of Education and Research (BMBF). Back

|| Recipient of a stipend from the Graduierten Kolleg: Funktionelle Proteindomaenen. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. Tel.: 49(0)33200-88568; Fax: 49(0)33200-88384; E-mail: dietmark{at}mail.dife.de.

1 The abbreviations used are: OR, olfactory receptor(s); AC, adenylyl cyclase; RT, reverse transcriptase; hG{alpha}olf, human guanine nucleotide-binding G protein {alpha}; {beta}-AR, {beta}-adrenergic receptor; CCD, charged coupled device; CNGA2, cyclic nucleotide-gated channel subunit A2; Cx-43, connexin-43; db-cAMP, dibutyryl cyclic AMP; GTP{gamma}S, guanosine 5'-O- (thiotriphosphate); DMEM, Dulbecco's modified Eagle's medium; ECL, enhanced chemiluminescence; HEK, human embryonic kidney; FLIPR, fluorescence-imaging plate reader; GPCR, G protein-coupled receptor(s); HBS, HEPES-buffered saline; IBMX, 3-isobutyl-1-methylxanthine; OSN, olfactory sensory neuron(s). Back

2 K. Schmiedeberg and D. Krautwurst, unpublished observation. Back

3 P. Bedner and K. Willecke, unpublished observation. Back

4 Randall Reed (Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD), personal communication. Back


   ACKNOWLEDGMENTS
 
We thank R. Reed (rat G{alpha}olf), S. Offermanns (G{alpha}15), B. Bufe (rat G{alpha}s), and U.-B. Kaupp (bovine CNGA2) for plasmid DNA. We thank P. Hargrave for B6-30 anti-rhodopsin antibody and H. Falk for the anti-G{alpha}s antibody. We are grateful to M. Sieber, S. Bandholtz, N. Brune, K. Klass, and F. Neuschaefer-Rube for technical assistance, M. Pyrski for helpful comments on the manuscript, and C. Barth and H.-G. Joost for continuous support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zhang, X., Rodriguez, I., Mombaerts, P., and Firestein, S. (2004) Genomics 83, 802-811[CrossRef][Medline] [Order article via Infotrieve]
  2. Malnic, B., Godfrey, P. A., and Buck, L. B. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2584-2589[Abstract/Free Full Text]
  3. Malnic, B., Hirono, J., Sato, T., and Buck, L. B. (1999) Cell 96, 713-723[CrossRef][Medline] [Order article via Infotrieve]
  4. Hamana, H., Hirono, J., Kizumi, M., and Sato, T. (2003) Chem. Senses 28, 87-104[Abstract/Free Full Text]
  5. Mombaerts, P. (2004) Nat. Rev. Neurosci. 5, 263-278[CrossRef][Medline] [Order article via Infotrieve]
  6. Zhao, H., Ivic, L., Otaki, J. M., Hashimoto, M., Mikoshiba, K., and Firestein, S. (1998) Science 279, 237-242[Abstract/Free Full Text]
  7. Touhara, K., Sengoku, S., Inaki, K., Tsuboi, A., Hirono, J., Sato, T., Sakano, H., and Haga, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4040-4045[Abstract/Free Full Text]
  8. Bozza, T., Feinstein, P., Zheng, C., and Mombaerts, P. (2002) J. Neurosci. 22, 3033-3043[Abstract/Free Full Text]
  9. Kajiya, K., Inaki, K., Tanaka, M., Haga, T., Kataoka, H., and Touhara, K. (2001) J. Neurosci. 21, 6018-6025[Abstract/Free Full Text]
  10. Krautwurst, D., Yau, K. W., and Reed, R. R. (1998) Cell 95, 917-926[CrossRef][Medline] [Order article via Infotrieve]
  11. Gaillard, I., Rouquier, S., Pin, J. P., Mollard, P., Richard, S., Barnabe, C., Demaille, J., and Giorgi, D. (2002) Eur. J. Neurosci. 15, 409-418[CrossRef][Medline] [Order article via Infotrieve]
  12. Wetzel, C. H., Oles, M., Wellerdieck, C., Kuczkowiak, M., Gisselmann, G., and Hatt, H. (1999) J. Neurosci. 19, 7426-7433[Abstract/Free Full Text]
  13. Kenakin, T. (2002) Trends Pharmacol. Sci. 23, 403-404[CrossRef][Medline] [Order article via Infotrieve]
  14. Murrell, J. R., and Hunter, D. D. (1999) J. Neurosci. 19, 8260-8270[Abstract/Free Full Text]
  15. Illing, N., Boolay, S., Siwoski, J. S., Casper, D., Lucero, M. T., and Roskams, A. J. (2002) Mol. Cell Neurosci. 20, 225-243[CrossRef][Medline] [Order article via Infotrieve]
  16. Barber, R. D., Jaworsky, D. E., Yau, K. W., and Ronnett, G. V. (2000) J. Neurosci. 20, 3695-3704[Abstract/Free Full Text]
  17. Saito, H., Kubota, M., Roberts, R. W., Chi, Q., and Matsunami, H. (2004) Cell 119, 679-691[CrossRef][Medline] [Order article via Infotrieve]
  18. Reed, R. R. (1992) Neuron 8, 205-209[CrossRef][Medline] [Order article via Infotrieve]
  19. Offermanns, S., and Simon, M. I. (1995) J. Biol. Chem. 270, 15175-15180[Abstract/Free Full Text]
  20. Elfgang, C., Eckert, R., Lichtenberg-Frate, H., Butterweck, A., Traub, O., Klein, R. A., Hulser, D. F., and Willecke, K. (1995) J. Cell Biol. 129,
  21. Ludwig, J., Margalit, T., Eismann, E., Lancet, D., and Kaupp, U. B. (1990) FEBS Lett. 270, 24-29[CrossRef][Medline] [Order article via Infotrieve]
  22. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drobak, B. K., Bjerrum, P. J., Christensen, S. B., and Hanley, M. R. (1994) Agents Actions 43, 187-193[CrossRef][Medline] [Order article via Infotrieve]
  23. Bidlack, J. M., and Parkhill, A. L. (2004) Methods Mol. Biol. 237, 135-143[Medline] [Order article via Infotrieve]
  24. Finn, J. T., Krautwurst, D., Schroeder, J. E., Chen, T. Y., Reed, R. R., and Yau, K. W. (1998) Biophys. J. 74, 1333-1345[Medline] [Order article via Infotrieve]
  25. Fagan, K. A., Schaack, J., Zweifach, A., and Cooper, D. M. (2001) FEBS Lett. 500, 85-90[CrossRef][Medline] [Order article via Infotrieve]
  26. Pun, R. Y., and Kleene, S. J. (2003) Biophys. J. 84, 3425-3435[Medline] [Order article via Infotrieve]
  27. Liu, H. Y., Wenzel-Seifert, K., and Seifert, R. (2001) J. Neurochem. 78, 325-338[CrossRef][Medline] [Order article via Infotrieve]
  28. Menco, B. P., Tekula, F. D., Farbman, A. I., and Danho, W. (1994) J. Neurocytol. 23, 708-727[CrossRef][Medline] [Order article via Infotrieve]
  29. Kenakin, T. (2003) Trends Pharmacol. Sci. 24, 346-354[CrossRef][Medline] [Order article via Infotrieve]
  30. Gudermann, T., Kalkbrenner, F., and Schultz, G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 429-459[Medline] [Order article via Infotrieve]
  31. Offermanns, S. (2003) Prog. Biophys. Mol. Biol. 83, 101-130[CrossRef][Medline] [Order article via Infotrieve]
  32. Spehr, M., Gisselmann, G., Poplawski, A., Riffell, J. A., Wetzel, C. H., Zimmer, R. K., and Hatt, H. (2003) Science 299, 2054-2058[Abstract/Free Full Text]
  33. Oka, Y., Omura, M., Kataoka, H., and Touhara, K. (2004) EMBO J. 23, 120-126[CrossRef][Medline] [Order article via Infotrieve]
  34. Araneda, R. C., Kini, A. D., and Firestein, S. (2000) Nat. Neurosci. 3, 1248-1255[CrossRef][Medline] [Order article via Infotrieve]
  35. Sanchez-Montanes, M. A., and Pearce, T. C. (2002) Biosystems 67, 229-238[CrossRef][Medline] [Order article via Infotrieve]
  36. Araneda, R. C., Peterlin, Z., Zhang, X., Chesler, A., and Firestein, S. (2004) J. Physiol. (Lond.) 555, 743-756[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Sci SignalHome page
H. Saito, Q. Chi, H. Zhuang, H. Matsunami, and J. D. Mainland
Odor Coding by a Mammalian Receptor Repertoire
Sci. Signal., March 3, 2009; 2(60): ra9 - ra9.
[Abstract] [Full Text] [PDF]

Home page
 Chem SensesHome page
G. Sanz, T. Thomas-Danguin, E. H. Hamdani, C. Le Poupon, L. Briand, J.-C. Pernollet, E. Guichard, and A. Tromelin
Relationships Between Molecular Structure and Perceived Odor Quality of Ligands for a Human Olfactory Receptor
Chem Senses, September 1, 2008; 33(7): 639 - 653.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
E. Shirokova, J. D. Raguse, W. Meyerhof, and D. Krautwurst
The human vomeronasal type-1 receptor family-- detection of volatiles and cAMP signaling in HeLa/Olf cells
FASEB J, May 1, 2008; 22(5): 1416 - 1425.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Schmuker and G. Schneider
Processing and classification of chemical data inspired by insect olfaction
PNAS, December 18, 2007; 104(51): 20285 - 20289.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Zhuang and H. Matsunami
Synergism of Accessory Factors in Functional Expression of Mammalian Odorant Receptors
J. Biol. Chem., May 18, 2007; 282(20): 15284 - 15293.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Abaffy, A. Malhotra, and C. W. Luetje
The Molecular Basis for Ligand Specificity in a Mouse Olfactory Receptor: A NETWORK OF FUNCTIONALLY IMPORTANT RESIDUES
J. Biol. Chem., January 12, 2007; 282(2): 1216 - 1224.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
T. B. Huber, B. Schermer, R. U. Muller, M. Hohne, M. Bartram, A. Calixto, H. Hagmann, C. Reinhardt, F. Koos, K. Kunzelmann, et al.
Inaugural Article: Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels
PNAS, November 14, 2006; 103(46): 17079 - 17086.
[Abstract] [Full Text] [PDF]

Home page
 Chem SensesHome page
Y. Nie, J. R. Hobbs, S. Vigues, W. J. Olson, G. L. Conn, and S. D. Munger
Expression and Purification of Functional Ligand-Binding Domains of T1R3 Taste Receptors
Chem Senses, July 1, 2006; 31(6): 505 - 513.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. E. C. Von Dannecker, A. F. Mercadante, and B. Malnic
Ric-8B promotes functional expression of odorant receptors
PNAS, June 13, 2006; 103(24): 9310 - 9314.
[Abstract] [Full Text] [PDF]

Home page
 Chem SensesHome page
P. C. Lai, M. S. Singer, and C. J. Crasto
Structural Activation Pathways from Dynamic Olfactory Receptor-Odorant Interactions
Chem Senses, November 1, 2005; 30(9): 781 - 792.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/12/11807    most recent
M411508200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shirokova, E.
Right arrow Articles by Krautwurst, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shirokova, E.
Right arrow Articles by Krautwurst, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement