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

Twenty analogs of [Orn ⁶ ,D-Ala ⁹ ]α-factor were synthesized and assayed for their biological activities: seven analogs of [Orn ⁶ ,X ⁹ ]α-factor, seven analogs of [X ⁶ ,D-Ala ⁹ ]α-factor, five analogs of [X ⁵ ,X ⁶ ,D-Ala ⁹ ]α-factor, and native α-factor (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native α-factor (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU- STE2 vector. [Dap ⁶ ,D-Ala ⁹ ]α-factor with weak halo activity (10%) showed the highest receptor affinity (＞ 230%) and the highest gene induction activity (167%). [Arg ⁶ ,D-Ala ⁹ ]α-factor showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, [Arg ⁶ ,D-Ala ⁹ ]α-factor-Edan, with high sensitivity (12,500-fold higher than the absorption-based detector [Orn ⁶ ]α-factor-[Cys] ₃ ).