Spectrophotometric Determination of Affinities of α‐Factors for Their G Protein‐Coupled Receptors in Saccharomyces cerevisiae

Abstract

A new and relatively simple spectrophotometric technique has been developed for the accurate determination of α‐factor pheromone affinities for Ste2p whole cell receptor in yeast a‐cells. We designed and tested nine detector peptides containing mono‐ (ε412 = 14 500) or tri‐cysteine residues (ε412 = 43 660). The free unbound detector was detected using Ellman's reagent at 412 nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 ( MATa ) at a concentration of 2.5 × 1011 cells/mL with the highest affinity detector 1, [Orn6]α‐factor‐[Cys]3, resulted in a dissociation constant ( K D ) of 1.67 × 10−7 and total binding sites per cell (B cell = 29 500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic α‐factor analogs allowed for the determination of each K D value. [Orn6,d‐Ala9]α‐factor showed the highest receptor affinity ( K D = 1.03 × 10−7 M), which was threefold higher than that of native α‐factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic α‐factors and eliminates the need for radioactive waste disposal.

Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p

“…The procedure followed the assay protocol previously described by Hong [13]. Relative activities of the different analogs were determined by comparing the amount of peptide causing formation of a 30 mm halo from the regression line (Fig.…”

“…All target analogs investigated in this paper were efficiently synthesized on trityl resin using a solid phase approach [13]. In general, the crude products of TFA cleavage obtained from the resin contained approximately 85% to 95% of the major components, as indicated by analytical reversed-phase HPLC.…”

“…Transformed strain LM102 containing a plasmid encoding STE2 gene was grown in liquid media containing 2% galactose, which is an inducer of STE2 gene expression. After cell growth, Ste2p receptor expression was determined by the steady-state saturation binding technique [13]. Since a given number of cells is expected to possess a finite number of receptors, saturation was assessed by examining binding as a function of increasing detector concentration using 2.5 × 10 cells (Fig.…”

“…In our previous study, [13] we reported ligand-binding assays for GPCRs from yeast whole cells based on spectrophotometric measurement using a tri-cysteine detector. The method was demonstrated to be rapid and convenient for measuring the receptor affinities of α-factors, but its major drawback was a low sensitivity requiring high concentrations of both cells and detector.…”

“…]α-Factor-[Cys] 3 (Fig. S9) The procedure followed the assay protocol previously described by Hong [13]. Steady-state saturation binding for Scatchard analysis is shown in Fig.…”