Homologous and heterologous binding
For the competition binding experiments, COS-7 cells were transfected
with receptor plasmid or pcDNA3.1(+) and seeded with 150 000 cells per
well in CulturPlate-24 (PerkinElmer; Waltham, MA) one day after
transfection. The number of cells seeded per well was selected on the
basis to obtaining 5-10% specific binding of the radioligands. Day 2
after transfection, the cells were washed twice in prechilled binding
buffer (50 mM HEPES buffer, supplemented with 1 mM
CaCl2, 5 mM MgCl2 and 0,5% (w/v) BSA)
at pH 8 and incubated for 15 min at 4C°. Increasing concentration of
unlabeled ligand followed by a stable concentration of the radioligand
(20 000cpm/well) were added to the cells, which were then incubated for
additional 3 hours at 4C°. After incubation, the cells were washed twice
in prechilled binding buffer, lysed, and counted using a Wizard gamma
counter (PerkinElmer; Waltham, MA).