Cloning and expression of odorant receptors

Abstract

Myriads of odorous molecules that vary widely in structure are nevertheless readily detected and discriminated by the sense of smell, but how this is achieved by the olfactory system has been a long-standing puzzle. Several different models have been proposed, and previous observations indicate that the recognition sites for odorous molecules could be G-protein-coupled receptor proteins, an idea supported by the discovery of a new gene family that probably encodes a diversity of odorant receptors. Here we report the identification of new members of the gene family encoding putative odorant receptors and demonstrate that they are indeed transcribed in olfactory receptor neurons. Furthermore, the receptor-encoding complementary DNA is expressed in non-neuronal surrogate cells, which generate second messenger responses upon stimulation with appropriate odorants, indicating that the receptors recognize odorants and couple to G proteins of the host cells.

Olfactory Receptor Proteins

“…Four peptides from different segments of the olp4 sequence were synthesized: the N-terminal peptides NT1 (positions 1-12) and NT2 (positions [16][17][18][19][20][21][22][23][24][25][26][27][28][29], the cytoplasmic loop-3 peptide CL3.2 (positions 220-238) and the C-termihus peptide CT2 (positions 292-308), except for the N-terminal serine, which was changed to cysteine. The peptides were coupled to keyhole limpet hemocyanin and used to immunize three-month-old New Zealand White rabbits with 1 mg coupled peptide in complete Freund's adjuvant as described [33].…”

Multiple genetic control of acetate-induced olfactory responses in Drosophila melanogaster larvae

“…Support for this hypothesis -or at least for the involvement of a very large number of genes in olfaction -has come from Buck & Axel (1991) who cloned and characterized in rats several dozen members of a multigene family of proteins, apparently transmembrane receptors, the expression of which was limited to olfactory epithelium. One of these proteins has been shown to act as an olfactory receptor when expressed in Xenopus eggs (Raming et at., 1993), and *Correspondence 444 the rat sequences have been used to find similar expression patterns in the cat-fish (Ngai et at., 1993).…”

Probing olfactory receptors with sequence‐specific antibodies

“…The peptide E464, with 14 amino acid residues (DMSALLKLACSDTR) corresponding to the second extracellular loop between hydrophobic domains 4 and 5 of the odorant receptor OR5 [8], was synthesized by the Fmoc (9-fluorenylmethoxycarbony1)-polyamide method using an automated solid-phase peptide synthesizer (Applied Biosysterns). The synthetic peptides were purified by reverse-phase HPLC and freeze dried.…”

“…It has recently been demonstrated by in situ hybridisation that a defined receptor subtype is only expressed in a distinct subpopulation of olfactory neurons [5 -71. Meanwhile, several receptor-encoding cDNAs have been expressed in surrogate cells; for one of the receptor subtypes, it has been demonstrated that upon stimulation with certain odorants second-messenger signals are elicited in the host cells [8].The deciphering of odorant receptor primary structure permits investigation of the structure and function of native receptor proteins in olfactory neurons using immunological approaches. Antibodies generated against chemically synthesized peptides based on sequence information derived from molecular cloning, have been found to react specifically with native molecules [9].…”

“…It has recently been demonstrated by in situ hybridisation that a defined receptor subtype is only expressed in a distinct subpopulation of olfactory neurons [5 -71. Meanwhile, several receptor-encoding cDNAs have been expressed in surrogate cells; for one of the receptor subtypes, it has been demonstrated that upon stimulation with certain odorants second-messenger signals are elicited in the host cells [8].…”