Measuring Ligand-Induced Activation of D2L Receptors

Ligand-induced activation of the human D_2L receptors was studied employing inositol phosphate (IP) accumulation assays as described54 (link)55 (link). Briefly, HEK 293 cells were transiently co-transfected with cDNA encoding for D_2L and the hybrid G protein Gα_qi5 (Gα_q protein with the last five amino acids at the C terminus replaced by the corresponding sequence of Gαi; gift from the J. David Gladstone Institutes). Twenty-four hours after transfection, cells were transferred into 24-well plates. After adding myo-[³H]inositol (specific activity = 22.5 Ci mmol⁻¹, PerkinElmer) and incubation for 15 h, medium was aspirated, the cells were washed with serum-free medium supplemented with 10 mM LiCl, and test compounds 37 °C. Then, cells were lysed by adding 0.1 M NaOH. After neutralization with formic acid, the cell extract was separated by anion-exchange chromatography using an AG1-X8 resin (Bio-Rad) by washing and finally eluting total IP directly into scintillation counting vials. Radioactivity was determined by scintillation counting in a Beckman LS 6500 (Beckman). Data were analyzed by normalizing disintegrations per minute (d.p.m.) values; this was done by setting the data for non-stimulated receptor (buffer) equal to zero and the effect for quinpirole equal to 100%.