Probing olfactory receptors with sequence‐specific antibodies

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Abstract
Molecular cloning has revealed the structure of several putative odorant receptors. Chemically synthesized peptides, that correspond to a predicted extracellular domain of the encoded proteins, were employed to generate receptor-specific antibodies. Immunohistological approaches as well as Western-blot analysis confirmed the specificity of the antipeptide sera. Furthermore, deglycosylation experiments explained the observed discrepancy between the molecular mass of odorant receptors, as determined by SDS/PAGE and Western-blot analysis of ciliary proteins (Mr 50000), and the predicted protein size based on the deduced primary structure from cloned receptor genes (Mr 30000–35000). Receptor proteins become phosphorylated upon odorant stimulation of olfactory cilia preparations; this was demonstrated by immunoprecipitation experiments employing the sequence-directed, receptor-specific antibodies. Functional assays revealed that the receptor-specific antibodies significantly attenuate second messenger signalling elicited by inositol 1,4,5-trisphosphate-inducing odorants, whereas activation of the cAMP cascade by appropriate odorants was not affected. These observation indicate that the sequence-specific antibodies not only recognize odorant receptors, but also discriminate between receptor subtypes coupling to different second-messenger pathways.