A study of interactions between saccharomyces cerevisiae α-factor and its G protein-coupled receptor, Ste2p

Author

Byung-Kwon Lee

Date of Award

12-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey Becker

Committee Members

Robert N. Moore, David Brian, John W. Koontz, Elizabeth E. Howell

Abstract

During the life cycle of the yeast Saccharomyces cerevisiae, haploid cells of opposite mating type can fuse during sexual conjugation to form a diploid cell. In preparation for conjugation, haploid cells secrete small diffusible peptide molecules [α-factor, a tridecapeptide pheromone and a-factor, a modified dodecapeptide pheromone] that specifically bind to cell surface receptors found on the opposite mating type cell. The basic structure of the receptors (Ste2p for α-factor and Ste3p for a-factor) is evolutionarily conserved and places them among the 7-transmembrane, G protein-coupled receptors (GPCRs). Part 1 of this dissertation is an overview of the structure and the molecular mechanisms involved in ligand recognition and activation these receptor families with specific emphasis on peptide hormones and α-factor receptors. Part 2 of this dissertation is a study of a-factor analogs in which Tyr¹³ was replaced with a number of side chains for the design of an iodinatable ligand for affinity labeling studies as a direct iodination at Tyr¹³ abolished function of α-factor. The result of binding and biological activity assays of these analogs showed the lack of strict requirement for Tyr¹³ and allowed the design of several multiple replacement analogs in which Phe or p-F-Phe were substituted at position 13 and Tyr was placed in other positions of peptide. One potential receptor ligand [Tyr(¹²⁵I)¹, Nle¹², Phe¹³]α-factor exhibited saturable binding with a Kdof 81 nM and was competed by α-factor for binding. In Part 3, an analysis of the α-factor receptor was carried out using random and site-directed mutagenesis to try to understand pheromone binding and receptor activation mechanisms. Three receptors containing mutations F55V, S219P, and S259P were screened for their altered ligand specificity and analyzed for their biological responses to various α-factor analogs and for their ligand binding profiles. The S259P mutation demonstrated ligand dependent biological response to all peptides tested (α-factor, antagonists and a synergist). The S219P mutation responded to α-factor, some antagonist peptides and the synergist, but not to other antagonists. The F55V mutant receptor responded only to α-factor and the synergist peptide and not to any antagonist analogs. These results confirmed previous findings that the fifth and sixth transmembrane domain of the receptor are important for receptor activation. In addition, changes in binding affinity of α-factor and its analogs indicate that residue 55 of α-factor receptor is involved with ligand binding. Part 4 of this dissertation is a study of identification of the α-factor binding region of Ste2p using site-directed mutagenesis and ligand modification. Affinities and activities of mutant receptors at serine 47 and threonine 48 residues were determined with analogs in which Gln¹⁰ of α-factor was replaced with various functional groups. All mutant receptors showed a similar number of binding sites and efficacy but different Kd and EC₅₀ values for a-factor compared to those of wild type receptor. A mutant receptor (S47K, T48K) had dramatically reduced affinity and activity for K¹⁰ and Orn¹⁰-α-factors while the affinity of S. kluyveri α-factor (E¹⁰ with additional four variant residues) was increased over 40-foid compared to that of wild type receptor. In contrast to KK substitution, the affinity of K¹⁰- and Orn¹⁰-α-factor was greatly increased in a S47E, T48E mutant receptor while the binding of S. kluyveri α-factor was decreased over 100-fold. E¹⁰-α-factor showed about two fold higher affinity in this mutant receptor than KK mutant receptor. The affinity of K¹⁰- and Orn¹⁰-α-factors for the EE mutant, however, dropped 4-6 fold in the presence of 1M NaCI while affinity of α-factor was not affected by this treatment. The results indicate that 10^th Gin residue of S. cerevisiae α-factor when bound to the receptor is adjacent to Ser47 and Thr48 residues in the receptor.

Recommended Citation

Lee, Byung-Kwon, "A study of interactions between saccharomyces cerevisiae α-factor and its G protein-coupled receptor, Ste2p. " PhD diss., University of Tennessee, 2000.
https://trace.tennessee.edu/utk_graddiss/8333