Receptor-mediated GTPγS Binding and cAMP Assays

Methods for these assays have been previously described^{16 (link)}. In brief, for GTPγs binding, the potency of ligands to stimulate receptor-dependent elevation of the GTPγS-bound Gα_s subunit was determined using membrane preparations from low-receptor-density receptor clonal cell lines. Reactions were incubated for 30 min at room temperature in white, clear-bottom microtiter plates, and per cent of the maximal response was calculated using control wells as a reference. Relative EC₅₀ values were derived by nonlinear regression analysis using the per cent response versus the concentration of ligand and fitted to a four-parameter logistic equation using GraphPad Prism 7 software. For cAMP assays, kinetic cAMP assays were performed in low-density receptor cell clones transfected with the Glosensor 22 F vector (Promega). Cells were equilibrated for 5–20 min and then 20 μl of 10× ligand was added and a luminescence time course was collected. Ligand (10×) was titrated by manual serial dilution in DMSO followed by step-down into assay buffer.