Structure-function studies of the saccharomyces cerevisiae pheromone receptor, ste2p, using novel α-factor analogs

Author

Loren Keith Henry

Date of Award

12-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Jeffery M. Becker

Committee Members

John W. Koontz. May Ann Handel, Cynthia B. Peterson

Abstract

G-protein coupled receptors (GPCRs) are heptahelical membrane proteins that allow cells to sense and respond to a variety of environmental stimuli. Study of GPCRS has begun to elucidate their important role in human disease and has allowed for the development of a number of clinical treatments.

The yeast Saccharomyces cerevisiae which uses peptide pheromones and GPCRs to mediate sexual reproduction between haploid cells presents a model system for the study of GPCRS. The studies presented here detail the use of novel peptide analogs to investigate the structure-function relationship between the Ste2p receptor and its ligand, the α-factor pheromone.

Characterization of a gamut of α-factor analogs resulted in the identification of several analogs with biological activity and sub-micromolar binding affinity (Kd) identifying them as good candidates for use in photoaffinity labeling studies to map the a-factor binding site in the Ste2p receptor. Photoaffinity labeling with the analog [Bpa¹ Y³ (¹²⁵1) R⁷ Nle¹²F¹³] α-factor and subsequent receptor fragmentation showed that position one of the α-factor peptide crosslinked to a fragment of the receptor from amino acid 190 to 294.

Formation of a dimeric α-factor molecule by covalent attachment of the epsilon amines of Lys⁷ via an aminohexanoic acid bridge resulted in a ligand which showed specific binding to the Ste2p receptor and the ability to activate the pheromone signal cascade. The binding affinity and biological activity of this α- factor dimer suggest that the Lys⁷ residue in α-factor is relatively exposed to solvent and not buried into the receptor. These findings support the current model of the structure of α-factor when it is bound to the Ste2p receptor.

Further characterization of a novel class of α-factor analogs termed synergist has revealed that these ligands do not enhance biological activity by modulating the rates of association or dissociation of agonists to the Ste2p receptor. Furthermore, the synergists do not increase the total amount of agonist binding to Ste2p since the total amount of [³H] α-factor associated to Ste2p does not change in the presence or absence of synergist. The new information from these studies has allowed for several models of synergist action to be proposed.

Recommended Citation

Henry, Loren Keith, "Structure-function studies of the saccharomyces cerevisiae pheromone receptor, ste2p, using novel α-factor analogs. " PhD diss., University of Tennessee, 2000.
https://trace.tennessee.edu/utk_graddiss/8293