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Abstract
This thesis provides evidence for the functional role of two types of proteins, odorant receptors and olfactory marker protein, in the signaling pathway of olfactory receptor neurons.
Odorant receptors were cloned several years ago, but the evidence for their physiological role in binding odorant molecules and activating the second messenger cascade was lacking due to difficulties with their expression in heterologous systems. Here, we have utilized an adenoviral vector to drive the expression of a particular rat odorant receptor in an excess number of sensory neurons within the olfactory epithelium. Successful expression of one odorant receptor, in a large number of olfactory receptor neurons, allowed us to screen a number of ligands and to identify a group of chemically related compounds that activate the overexpressed odorant receptor. In addition, we generated fusion proteins carrying tags on either the N-terminus or C-terminus of the odorant receptor and assayed their functional expression in olfactory epithelium, primary culture of olfactory receptor neurons and a heterologous cell line. Presented results demonstrate the functional expression of two fusion proteins, providing a tool for visualization of functional odorant receptors and allowing the future research in odorant receptor trafficking.
Another protein, for which the primary amino acid sequence was also known for years, is olfactory marker protein. This unique protein is highly conserved in vertebrate species, but its sequence does not share any similarity to any other known protein. Previous experiments demonstrated changed response kinetics in OMP-null mice and postulated a role for this protein in olfactory signaling. The direct evidence indicating the function of olfactory marker protein in signal transduction pathway was still lacking. We demonstrate a full recovery of odor response kinetics in adult OMP-null mice after short-term expression of OMP delivered to the olfactory epithelium via adenoviral vector. From these results we postulate a direct involvement of olfactory marker protein in olfactory signaling. Additional evidence places the modulatory role of olfactory marker protein downstream from the synthesis of cyclic adenosine monophosphate.
