Probing olfactory receptors with sequence-specific antibodies.

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 (M(r) 50,000), and the predicted protein size based on the deduced primary structure from cloned receptor genes (M(r) 30,000-35,000). 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.