S1P Receptors obtained from the Missouri University of Science and Technology. CaCl2 2H2O, tetracycline HCl, and other general reagents were purchased from Sigma Aldrich or BDH. NPS R568 HCl, cinacalcet HCl, and NPS 2143 were synthesized in house, according to published methods. Molecular biology To generate a c myc tagged CaSR construct, a c myc epitope was embedded between residues D371 and T372 via Quikchange site directed mutagenesis using long complementary primers. The insertion of the c myc tag did not alter the pharmacology of the CaSR in its ability to stimulate signaling in comparison with the untagged receptor. The human c myc CaSR cDNA sequence was transferred from pDONR201 into the destination vector pcDNA5/FRT/TO using the LR clonase enzyme mix according to the manufacturers instructions. For the generation of stable Flp In HEK293 TREx c myc CaSR cells, Flp In HEK293 TREx cells were transfected with DPP-4 Lipofectamine 2000 in conjunction withthe pOG44 Flp recombinase expression vector and selected in 200 g/ml hygromycin B and 5 g/ml blasticidin.
Cell culture and assay preparation Flp In HEK293 TREx c myc CaSR cells caspase were maintained in DMEM supplemented with 16 mM HEPES, 100 g/ml hygromycin B,10%tetracycline free FBS, and 5g/ml blasticidin at 37 C in a humidified atmosphere of 5% CO2 and 95% O2. For all assays, cells were seeded into clear 96 well plates coated with 2.5 g/well poly D lysine to allow for sufficient cell adherence. Cells were harvested with 2 mM EDTA in PBS, centrifuged, resuspended, and seeded at a density of 100,000 cells/well for phosphorylation of ERK1/2 and Cai 2 mobilization assays and at 25,000 cells/ well for plasma membrane ruffling experiments. To arrest cell growth for pERK1/2 andPMruffling experiments, cells were washed twice with 100 l PBS and replaced with serum free DMEM containing 0.1 mM Ca2 and 16 mM HEPES. One hundred nanograms per milliliter tetracycline were added to cells 18 21 h before performing signaling assays to induce maximal CaSR expression. Cai 2 mobilization assay Bergenin Assays were performed in an isotonic buffer at pH 7.4 consisting of 150 mM NaCl, 2.6 mM KCl, 0.1 mM CaCl2, 1.18 mM MgCl2, 10 mM D glucose, 10 mM HEPES, 4 mM probenecid, and 0.5% BSA.
Cells grown overnight on 96 well plates, washed twice with 100 l assay buffer devoid of Ca2, and then loaded with 100l normal buffer containing 0.9MFluo 4 AM. After 1 h, cells were washed twice with 100 l Ca2 free buffer and then replaced with 160 l buffer containing 0.1mM Ca2 in each well. After 10 min, 20 l 10allosteric modulators or 20 l dimethylsulfoxide vehicle control was added for a further 20 min. The effect of Ca2 addition was measured on a Flexstation 1 microplate represents reader using 485 nm excitation and 525 nm emission filters. ERK1/2 phosphorylation assay Cells were serum starved for 3 18 h in assay buffer before the assay. Time course experiments were initially performed to determine the time at which 10mMCao 2 induced ERK1/2 phosphorylation peaked. Timecourse and concentration response assays were performed at 37 C and included FBS and DMEM as internal and vehicle controls, respectively. For interaction studies, cells were incubated with allosteric modulators for 20 min before exposure to various test Cao 2 concentrations.