Fig. 1.

High-throughput screen and counter-screen for activators of compromised tethered-peptide-agonist GPR56. (A) Schematic of the tethered agonist region of GPR56 7TM in comparison with the tethered-peptide-agonist-compromised GPR56-A386M 7TM receptor and alignment of the GPR56 activating peptide (P7). Artificial initiator methionine placements are shown in red. (B) Optimization of the high-throughput screen was performed in a 384-well plate with increasing concentrations of TYFAVLM (P7) peptide. The data are the mean ± S.D. of 16 replicates. Z′ assay quality scores were calculated for each concentration of TYFAVLM (P7) peptide tested. HEK293T cells transiently transfected with the SRE-luciferase reporter and the (C) GPR56-A386M 7TM (high-throughput screen) plasmid, or (D) empty vector (high-throughput counter-screen) were seeded into wells containing ∼3–5 μM DMSO-solubilized compounds from the Spectrum Collection. Luminescence was measured after an 18-hour incubation period. Each point represents one individual compound tested.