Date of Award

12-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey M. Becker

Committee Members

Ana A. Kitazono, Elizabeth E. Howell, Timothy E. Sparer, Todd B. Reynolds

Abstract

G protein-coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by seven-transmembrane (7TM) domains connected by intracellular and extracellular loops, an extracellular N-terminus, and an intracellular Cterminus. GPCRs recognize neurotransmitters, sensory molecules and chemotactic agents and are involved in the control of many aspects of metabolism. Since GPCRs play important roles in diverse processes such as pain perception, growth and blood pressure regulation, and viral pathogenesis, GPCRs became important target for therapeutic agents. The tridecapeptide α-factor pheromone (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) of Saccharomyces cerevisiae and Ste2p, its cognate GPCR, have been used extensively as a model for peptide ligand-GPCR structure and function. The power of yeast genetics has been used to examine the structure and function of Ste2p. Recently, GPCR homodimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. In this dissertation, Ste2p has been used to investigate GPCR dimerization.

Part I of this dissertation is an overview of the GPCR structure and its ligandinduced conformational change with specific emphasis on the peptide pheromone α- factor and its receptor Ste2p. Part II of this dissertation is a study originally designed to probe inter-helical interaction between TM1 and TM7 of Ste2p. Site-directed mutagenesis and cysteine cross-linking with targeted residues of Ste2p were carried out. Although the anticipated inter-helical interactions were not identified from this study, the results provided strong evidence for Ste2p dimerization. Part III of this dissertation describes dimer interfaces including TM1 and TM7 of Ste2p. By using the disulfide cross-linking methodology, we studied the participation of specific residues at the intracellular boundary between TM1 and intracellular loop one and the entire TM7 in Ste2p dimerization. The final part of this dissertation contains a study of the participation of the Ste2p N-terminus in homo-dimer formation and the effect of ligand binding on this interaction. This part also includes overall conclusions and suggestions for future experiments that could contribute to an understanding of the dimer interfaces in Ste2p and the role of dimerization in the function of this receptor.

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