Date of Award

12-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey M. Becker

Committee Members

Cynthia B. Peterson, Todd B. Reynolds, Timothy E. Sparer, Elias J. Fernandez

Abstract

The Saccharomyces cerevisiae alpha-factor receptor, Ste2p, belongs to the G proteincoupled receptors (GPCRs), a class of integral membrane proteins that are characterized by seven-transmembrane (TM) domains. Ste2p-alpha-factor pair has been used extensively as a paradigm for investigating GPCRs structure and function. Upon binding of alpha-factor to Ste2p, a signal is transduced via an associated guanine-nucleotide binding protein, Gpa1p, initiating a cascade of events similar to those for mammalian GPCRs signal transduction. GPCRs are essential in many physiological processes associated with human diseases. Many aspects of structure and function are highly conserved across GPCRs, irrespective of primary amino acid sequence. This dissertation investigated the interactions of Ste2p with alpha-factor and Gpa1p. An overview of GPCRs in general with specific emphasis on Ste2p interactions with alpha-factor and Gpa1p are discussed in part I. Cross-linking studies of alpha-factor analogs containing 3,4-dihydroxylphenylalanine (DOPA) at positions 1 and 13 indicated that Trp1 and Tyr13 of α-factor are in close proximity to Lys269 and Cys59 of Ste2p, respectively when alphafactor is bound to Ste2p. An alpha-factor synergist lacking the last two amino acids required for binding could only inhibit the cross-linking of DOPA at position 1 suggesting that the alphafactor synergist interacts with the N-terminus of alpha-factor (described in part II). Part III describes the first report of an unnatural amino acid, p-benzoyl-L-phenylalanine (Bpa), replacement in a GPCR expressed in its native environment, and the use these receptors to photocapture a peptide ligand. Many of the Bpa-substituted Ste2p receptors exhibited biological activity and two of them, Phe55-Bpa and Tyr193-Bpa, were able to selectively capture alphafactor in the ligand binding site after photoactivation; indicating that these residues may be in direct contact or in close proximity to alpha-factor when bound to Ste2p. Part IV reports for the first time the involvement of the third intracellular loop (IL3) in Ste2p homo-dimer formation, and also conformational changes at the cytoplasmic ends of TM5-TM6 of Ste2p induced by alpha-factor binding. Conformational changes in the C-terminus of Gpa1p occurring during Ste2p and Gpa1p activation are also discussed for the first time in part V. Variants of Ste2p- Gpa1p fusion proteins that displayed activities similar to Ste2p and Gpa1p are described in part VI. The final part of this dissertation discusses the overall conclusions and contains suggestions for future studies. The results obtained during this study should provide very important information about the mechanisms underlying the activation of GPCRs and G proteins.

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