Ligand binding and receptor activation in a G protein coupled receptor in baker's yeast Saccharomyces cerevisiae

• Main Author: Bajaj, Anshika
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2006
• Subjects: Biochemistry
• ISBN: 0542750880; 9780542750885

The STE2 receptor in yeast belongs to the extended family of G protein coupled receptors (GPCRs) that are involved in multiple signal transduction pathways. This receptor, upon binding its ligand α-factor, prepares haploid yeast cells for mating. The binding site of the Ste2p receptor is not well characterized and the mechanism by which ligand binding causes receptor activation remains to be understood. This thesis introduces a new flow cytometry-based assay to study ligand binding to the Ste2p receptor. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a high-throughput, sensitive, and reproducible assay. The assay utilizes NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled α-factor analogues. The NBD fluorophore is sensitive to the polarity of the environment and hence allows polarity changes in the environment of the binding site to be monitored. Based on time-dependent changes in intensity and shape of the emission spectrum of [K7(NBD),Nle12] α-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment followed by a conversion to a state in which the fluorophore moves to a more polar environment. To understand the significance of the two phases of binding, experiments were conducted using a variety of receptor-ligand combinations. We observed that fluorescent antagonists lie in a more polar receptor environment than a closely related agonist. It was also observed that a fluorescent agonist resides in a more polar binding pocket upon binding to loss-of-function mutant receptors than when bound to normal receptors. These experiments have led us to conclude that a correlation exists between receptor activation and hydrophobicity of the binding site. The thesis also investigates the possibility of a charge-charge interaction between the three positive charges on the α-factor and the eight negative charges on the extracellular surface of the Ste2p receptor. We conclude that negative charges on the extracellular surface of the receptor do not facilitate ligand binding, and maintenance of polarity at the extracellular receptor surface is more important than maintenance of charge for intracellular signaling.