The rat olfactory system is used as a model in which to study chemosensory transduction. Although the mechanism whereby odorants are encoded has been elusive, an odorant binding protein (OBP) has been purified which demonstrates specific, saturable and physiologically relevant binding to several classes of odorants. Rat olfactory explants in culture and isolated rat olfactory cilia (the chemoreceptive neuronal surface) are used to elucidate the role and regulation of OBP. Conditions for the use of explants of olfactory epithelium to study OBP turnover shall be established. Pulse-chase experiments using (35S) methionine are used to determine synthetic and degradative rates of OBP under different conditions. Quantitation of labelled OBP is done by immunoprecipitation with rabbit anti-rat OBP and gel electrophoresis. Initially, OBP turnover shall be investigated in control or isoproteronol-treated rats and rats with olfactory bulb lesions. A cDNA to OBP shall be identified from a rat olfactory epithelium library using a synthetic oligonucleotide probe to the N-terminal sequence of OBP. This cDNA is then used for in situ hybridization to determine the site of biosynthesis of OBP. The protein sequence for OBP as predicted by the cDNA can be compared to other putative odorant binding proteins. The cDNA is also used to quantitate the mRNA to OBP under conditions previously described. Finally, isolated rat olfactory cilia are used to study the initial events of olfactory signal transduction. It is known that these cilia contain adenylate cyclase whose activity is increased by certain odorants in a GTP-dependent manner. The effects of OBP with or without odorants on cyclase activity shall be studied. In addition, the consequences of cyclase activation with respect to targets for a protein kinase shall be studied.
NIH ExPorter
NIH ExPorter