Mechanistic Investigations of Pheromone and Phytohormone G-Protein Coupled Receptors

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G-protein coupled receptors (GPCRs) form a super-family of cell surface receptors with 1000's of representative genes identified across taxa. They comprise the vast majority of current drug targets in pharmaceutical industry today. Ste2p and Ste3p are the opposite mating type GPCRs responsible for pheromone based signalling in the mating of yeast. Ste2p in particular has served as a model par excellence in basic studies of structure-function relationships in GPCRs. Previously we developed a high-yield recombinant expression system for these receptors and characterized homo-oligomerization of Ste2p in vitro. We further demonstrated novel putative roles for Ste2p in plasmogamy (cytoplasmic fusion), including mediating cell wall degradation and possibly membrane fusion. We linked these functionalities to residues of the N-terminal domain and transmembrane 6 of the receptor. We now propose to elucidate the mechanisms of Ste2p action associated with these plasmogamy events. At the same time, we are initiating a long-term mechanistic investigation of a recently characterized plant GPCR. Arabipdopsis thaliana GTG-1 (GPCR Type G-Protein 1) is a receptor for the agriculturally and environmentally pertinent plant hormone abscisic acid (ABA) and is to date the only plant GPCR of known ligand. We have developed a recombinant overexpression/purification system and demonstrated binding of ABA to this reconstituted recombinant GTG-1. We will continue to develop tools to study this GPCR, including a yeast based bio-assay to enable screening and carry out mutagenic structure-function analyses. In the much longer term we are interested in attempting to crystallize these receptors, based on advances that have led to the publication of 6 mammalian GPCR structures in the last 5 years. Overall, this work will increase our knowledge of the structure and function of GPCRs while yielding novel prospects for pharmaceutical and agbiotech development in the future.