Characterization of the primary interaction between the mating pheromone, alpha-factor, and its receptor in Saccharomyces cerevisiae

Alpha-factor is a peptide of thirteen amino acids which is required for mating between the haploid mating types, a and α, in Saccharomyces cerevisiae. An analogue of alpha-factor, DHP⁸ DHP¹¹ Nle¹² tridecapeptide, was catalytically reduced in the presence of ³H gas for production of a radiolabelled pheromone suitable for use in binding studies. Incorporation of tritium resulted in ³H-alpha-factor with high specific activity, purity, biological activity and long shelf-life. Binding studies revealed that alpha-factor interacts with its receptor via a simple, reversible process which obeys the law of mass action. Association and dissociation kinetics indicate values of 2.92 X 10⁶ M^-1 min^-1 for k₁ and between 4 and 7 X 10^-2 min^-1 for k_-1. Saturation binding studies reveal an equilibrium dissociation constant equal to 2.32 X 10^-8 M which approximates the kinetically-derived K_D of 2.12 X 10^-8 M. Scatchard and Hill analyses as well as dissociation behavior in the presence of excess unlabelled ligand indicate alpha-factor interacts with a homogeneous population of binding sites (13000 sites/cell) which do not interact and exhibit one affinity for the alpha-factor pheromone. Studies using unlabelled competitors confirm the specificity expected for a receptor-mediated process, i.e., binding affinities of alpha-factor analogues parallel their efficiency at inducing biological activities. Preliminary evidence obtained from the thermodynamic analysis of the temperature dependence of binding of alpha-factor (an agonist) and desTrp¹ Ala³ dodecapeptide (an antagonist) suggests that agonist binding is entropy-driven and antagonist binding is enthalpy-driven. This may reflect differences observed when binding of the ligand involves interaction with, or traversing of, the cell wall.