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INVESTIGATOR: Leary, G. P.

PERFORMING INSTITUTION:
UNIVERSITY OF MONTANA
COLLEGE OF FORESTRY AND CONSERVATION
MISSOULA, MONTANA 59812

STRUCTURE AND FUNCTION OF THE EUROPEAN CORN BORER SEX PHEROMONE RECEPTORS

NON-TECHNICAL SUMMARY: The insect order Lepidoptera (moths and butterflies) includes over 180,000 species and is one of the largest lineages of plant eating organisms (Grimaldi & Engel, 2006). Reproduction in moths almost exclusively depends on a unique pheromone blend released from the female's pheromone gland which is detected by highly specific odorant receptors of the male antenna over distances of 10 to 1000 meters. This ability to locate mates at long distances has allowed for the extensive spread of moth species, and it also provides an ideal target for the management of species, specific, pest populations. The potential for adopting bio-rationale approaches to developing semiochemicals for insects has surged with the identification of a large number of olfactory receptors genes (Clyne et al, 1999, Vosshall et al, 1999, Gao and Chess,1999). In this proposal, we use the male specific European Corn Borer (ECB) sex pheromone receptors (ECBOR 1 and 3-7 expressed with the co-receptor ECBOR2) as a model system to distinguish these homologous receptors (Wanner et al, 2010). With an eventual goal to rationally design molecules for the Lepidoptera sex pheromone receptors and insect ORs in general, we utilize methods pioneered in pharmaceutical drug design that is rooted in heterologous expression systems, a basic understanding of receptor-ligand interaction, and a fundamental knowledge of the protein signal transduction mechanism.

OBJECTIVES: In Objective 1, we will develop a large data set defining the molecular pharmacology for each ECBOR (1 & 3-7). In the Lepidoptera, most mating pheromones are long hydrocarbon chains that have variations of carbon chain length, type of terminal functional group, double bond location, number of double bonds, and/or stereochemistry around the double bond. From these five characteristics, we have created a library of European corn borer pheromone related molecules to test on ECBOR1 & 3-7 expressed in Xenopus laevis oocytes with the necessary co-receptor ECBOR2. This effort will provide a framework to identify the ligand structural determinants that confer receptor selectivity and agonist/antagonist activity in this receptor family. In Objective 2, we will identify the structural determinants in two closely related receptors, ECBOR3 and ECBOR6, that confer selectivity for ECBOR6 selectively binding of the primary mating pheromone, Z11-tetradecenyl acetate. Sequence alignment of the predicted transmembrane and extracellular domains reveals that ECBOR6 and ECBOR3 are 72.4% identical and 94% similar. The most obvious divergence in sequence is between the beginning of transmembrane domain (TMD) 3 and the end of TMD4, which includes the largest extracellular domain of the protein. We hypothesize that residues within these transmembrane and extracellular domains form direct amino acid contacts that distinguish carbon chain length and stereochemistry of the pheromones. We will test this by engineering chimeras and using site directed mutagenesis to define changes that convert the highly specific response profile of ECBOR6 to the broad response profile of ECBOR3. In Objective 3, we will characterize the the novel pheromone-gated ion channel in this seven transmembrane domain pheromone receptor family. My preliminary data show that application of the thiol reactive probe, methane thiosulfonate (MTSEA), selectively blocks a conductance in oocytes expressing ECBOR3 and the mandatory co-receptor ECBOR2. Two separate research groups reported a cation channel within the insect odorant receptors that is independent of Gprotein activation; however, the activating mechanism and location of the channel remains unresolved (Sato et al, 2008 and Wicher et al, 2008). We hypothesize that MTSEA modifies an endogenous cysteine residue in the receptor that either blocks the permeation pathway for cations or directly interferes with gating of the channel. Through site directed mutagenesis, we will identify which of seven extracellular cysteine residues is modified and accounts for the block. We will distinguish the effects in ion permeation vs ion gating by studying how MTSEA affects the single channel currents recorded in outside-out patches pulled from ECBOR3&ECBOR2 expressing oocytes. Finally, we will define the channel structure using cysteine-scanning mutagenesis.