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J. Biol. Chem., Vol. 280, Issue 12, 11807-11815, March 25, 2005

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Identification of Specific Ligands for Orphan Olfactory Receptors

G PROTEIN-DEPENDENT AGONISM AND ANTAGONISM OF ODORANTS^*

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

From the Departments of 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

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 olf and adenylyl cyclase type-III to the Ca²⁺ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Golf in odorant-specific signaling out of OR. The employment of the non-typical G protein 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 G15 or Golf. 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.

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

^* 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.

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

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

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

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.

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