Quantifying PTH1R-mediated cAMP signaling

One day before transfection, ∼2.7 × 10⁶ HEK293T cells were seeded in a 10 cm dish using full DMEM. Cells were transfected with 1 µg of PTH1R-encoding plasmid with or without serine substitutions under control of a CMV promoter, 5 µg of the reporter construct pGL4.29 (humanized PpyRE9 firefly luciferase gene driven by a cAMP-responsive element and followed by a PEST-sequence (Branchini et al., 2010), 0.5 µg of plasmid pRL encoding Renilla luciferase driven by a CMV promoter and 3.5 µg of the empty pcDNA3.1 vector. Transfection was performed using PEI as described above. The next day, cells were trypsinized and transferred to poly-d-lysine (PDL) coated 96-well plates with a density of ∼70,000 cells per well. After 24 h, cells were stimulated at 37 °C for 3 h by the addition of 25 µl of PTH(1-34) dissolved in pure DMEM to final concentrations from 10⁻¹² to 10⁻⁶M in a 96-well. Each concentration was analyzed in four wells. The following steps were performed according to Seidel et al. 2017. Cells were washed with ice-cold HDB (12.5 mM HEPES pH 7.4, 140 mM NaCl, 5 mM KCl). Cell lysis was performed using 50 µl of luciferase buffer (10 mM MgSO₄, 25 mM glycylglycine, 4 mM EGTA, pH 7.8) supplemented with 1% Triton X-100 and 1 mM dithiothreitol (DTT) for 30 min on ice under constant gentle agitation. Luciferase activities were measured using a Omega luminometer (BMG LABTECH, Ortenberg, DE) equipped with two injectors. To each well, 50 µl of luciferin substrate buffer (luciferase buffer supplemented with 0.3 mM luciferin, 1 mM ATP, 1 mM DTT pH 7.8) were subsequently added by the first injector and the total luminescence was measured. Afterward, 50 µl of 5 µM colenterazine dissolved in HDB were added to each well (1.67 µM final concentration of coelenterazine in the well). The luminesence of Renilla luciferase was detected using a 475–30 nm emission filter. Firefly luminescence was normalized to the Renilla luminescence. Curves were fitted by non-linear regression using Prism 9 for Windows (Graphpad Software Inc., San Diego, CA). EC₅₀ values were obtained as means with the appropriate CI from at least three independent experiments, each performed in quadruplicate.