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.
Krieger
J
J
University Stuttgart-Hohenheim, Institute of Zoophysiology, Germany.
Schleicher
S
S
Strotmann
J
J
Wanner
I
I
Boekhoff
I
I
Raming
K
K
De Geus
P
P
Breer
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002923:Cilia / Q000737:chemistry; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D007150:Immunohistochemistry; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009812:Odorants; D009831:Olfactory Mucosa / Q000737:chemistry*; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D018035:Receptors, Odorant / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015290:Second Messenger Systems
D000906:Antibodies; D007295:Inositol Phosphates; D018035:Receptors, Odorant; D000242:Cyclic AMP