Abstract
Mammalian odorant receptors (ORs) are Class I G-protein coupled receptors (GPCRs) located within the nasal epithelium. Odorant receptors interact with Galpha olfactory, a Galpha S type G-protein. Activated Galpha olfactory stimulates adenylate cyclase and the resulting increase in cAMP concentration opens cyclic nucleotide gated channels allowing Ca2+ to enter the cell. The increased Ca2+ then activates a Ca2+ activated Cl- channel which further depolarizes the cell. This depolarization initiates an action potential that reaches the axon of the olfactory sensory neuron located in the main olfactory bulb. Information from the main olfactory bulb is then transmitted to higher regions of the brain. Olfactory information is initially coded through the interaction of odorant molecules with hundreds of distinct ORs, but difficulty in exogenous expression of odorant receptors has delayed the identification of ligands for individual ORs. However, expression of mouse odorant receptors in Xenopus laevis oocytes allows for a systematic screening for potential ligands, as well as for efficient study of the structure-function relationship of the receptors and their ligands. My screening of odorant receptors using Xenopus oocytes included the coexpression of a signal transduction system and the use of robotic two-electrode voltage clamp electrophysiology. In this study, I investigated the structural basis for ligand recognition in mouse odorant receptors. First, I expanded the molecular receptor ranges of seven Class I odorant receptors. By use of a high throughput assay, I was able to expand upon current knowledge in the field for the mouse odorant receptors 23-1, 31-4, 32-11, 40-4, 42-1, 42-2 and 42-3. I then examined one receptor (MOR23-1) in more detail. I used the substituted cysteine accessibility method to identify residues within transmembrane domain five of this receptor that are accessible from the extracellular space. These residues may line the ligand binding site or the ligand access pathway. Conventional mutations of A205 caused little alteration in the molecular receptive range of the receptor, suggesting that this residue may not play a significant role in ligand interaction within the binding pocket. Mutagenesis of G111, a residue within transmembrane domain three caused significant shifts in the molecular receptive range of the receptor, but the location of this residue within the binding pocket could not be confirmed by the substituted cysteine method. Previous reports had suggested significant similarity between the molecular receptive ranges of the seven mouse odorant receptors that I used in my research. By expanding upon the known aliphatic ligands for each receptor identified new ligands for each receptor, I was able to show that the molecular receptive ranges of these receptors are in fact distinct. The experimental identification of residues located within the binding pocket on transmembrane five of mouse odorant receptor 23-1 provides an improved understanding of ligand recognition by this receptor class and will aid in better computer modeling of these receptors. This increased accuracy of the computer models of these basic Class I GPCRs may aid in future drug discoveries. Since GPCRs constitute a significant fraction of current drug targets, understanding the mechanism of ligand interactions with mouse odorant receptors may aid in the development of more efficacious compounds in the treatment of many common ailments.