The coding DNA sequence (cds) of the human GPR143 gene was inserted into

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1 Supplemental information METHODS Cloning The coding DNA sequence (cds) of the human GPR143 gene was inserted into puc19 plasmid using SalI and KpnI restriction enzymes. The mutations in the two GPR143 sorting signals (L223A-L224A and W329A-E330A) were obtained by sitedirected mutagenesis using polymerase chain reaction (PCR). Mutagenesis primers were designed with the corresponding mismatch flanked by nucleotides at the 3 - and 5 -end. Sequencing of single clones of wild type (wt) and mutant GPR143 was performed by GATC Biotech (Konstanz, Germany). The proper cds were then subcloned into DiscoverX plasmid whose ProLink (PL) tag was attached at the C terminal of GPR143 (PK2 vector, DiscoverX, Birmingham, UK) using NheI and BglII restriction sites. Cell culture and transfection Chinese Hamster Ovary (CHO) -arrestin cells were engineered from DiscoverX (Birmingham, UK) to contain the -galactosidase EA fragment fused to arrestin. Our goal was to introduce in those cells the plasmid containing wt or mutant GPR143 linked to the -galactosidase PL fragment to perform the -arrestin recruitment assay. CHO arrestin cells were cultured at 37 C with 5% CO2 in Dulbecco s modified Eagle medium-f12 (DMEM-F12) containing 10% FCS, 100 U/ml penicillin G and 100 g/ml streptomycin. CHO cells were transfected with jetprime transfection kit (Polyplus Transfection, Darmstadt, Germany) according to the manufacturer s recommendations. Melan-a cells (immortalized murine melanocytes) were grown in a mixture of 80% RPMI-20% DMEM medium supplemented with 10% FCS, 1% sodium

2 pyruvate, 1% glutamate, 5 U/ml penicillin, 5 g/ml streptomycin, 1% non-essential amino acids and 200 nm 12-O-tetradecanoyl phorbol 13-acetate. Immunostaining Transfected CHO cells were cultured on coverslips in a 12 well plate (Sarstedt) and fixed with a cold 1:1 methanol/aceton solution for 20 min at -20 C. Cells were then washed with phosphate-buffered saline (PBS) and blocked 1 h with 1% bovine serum albumin (BSA)/PBS solution. Cells were incubated in the dark 60 min with the first antibody and then 30 min with the secondary antibody, both diluted in 1% BSA/PBS. PBS was used to wash cells in between and after antibody incubations. At last coverslips were mounted using ProLong Gold antifade reagent (Invitrogen, Darmstadt, Germany) and stored in the dark at 4 C. Mouse monoclonal anti-pk/pl antibody (PathHunter DiscoverX, Birmingham, UK), rabbit polyclonal anti-pan Cadherin antibody (Abcam, Cambridge, UK), donkey anti-mouse-alexafluor594 (Jackson Immuno Research, Hamburg, Germany) and donkey anti-rabbit-alexa488 (Jackson Immuno Research, Hamburg, Germany) were used to stain GPR143 and the cells plasma membrane. A Nikon A1 Spectral confocal microscope (Pharmaceutical Institute, University of Bonn, Germany) and the NIS Element Advanced Research software 4.0 were used for image acquisition and analysis. Data analysis Results of -arrestin assay are presented as mean SEM from at least 3 independent experiments performed in triplicates. Data were analyzed using Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Differences between means or single values were tested for significance by unpaired t test (Prism). IC50 values were determined by fitting the data to a sigmoidal curve with variable slope (Prism).

3 Calcium mobilization assay CHO -arrestin cells expressing the wt or the mutant GPR143 receptor were seeded in one 175cm 2 flask and cultivated until they reach 80-90% of confluency. The cells were detached with trypsin, transferred to a falcon tube containing medium and placed into a 37 C incubator for 45 min allowing them to recover. Afterward, cells were centrifuged and the pellet resuspended in Hank s Balanced Salt Solution (HBSS; 20 mm HEPES, 13 mm NaCl, 5.5 mm glucose, 5.4 mm KCl, 4.2 mm NaHCO3, 1.25 mm CaCl2, 1 mm MgCl2, 0.8 mm MgSO4, 0.44 mm KH2PO4 and 0.34 mm Na2HPO4, ph 7.4). The calcium sensitive dye was added (Fluo-4 AM, 3 µm diluted in DMSO, Invitrogen) together with Pluronic F-127 (60 µm final concentration), a detergent permeabilizing the cell membrane to permit the dye-uptake from the cells. Cells were incubated 1 h at room temperature in the dark on a rotating wheel. The reagent plate was prepared with the compound dilutions, negative control (HBSS) and positive control (100 µm ATP). Then, the cells were centrifuged at lowest velocity and the pellet was washed three times with buffer (HBSS). The cells were seeded in a 96-well plate with clear flat bottom and incubated under light exclusion for 30 minutes allowing the cells to settle down in the wells. The cell and reagent plates were placed into the microplate reader which measured the fluorescence intensity at a wavelength of 520 nm following excitation at 480 nm. camp accumulation assay CHO -arrestin cells ( cells/well) were seeded in 24-well plates 24 h before performing the assay. Cells are then washed and incubated 2 h at 37 C and 5% CO2 with HBSS. Cells were then incubated 15 min with the phosphodiesterase inhibitor Ro (Hoffmann La Roche, Grenzach, Germany; final concentration 40 M) at 37 C and 5% CO2. To detect the Gi protein signaling pathway, different

4 concentrations of compounds in 5% DMSO/HBSS buffer (final DMSO concentration: 1%) were added and, after a 5 min incubation at 37 C, forskolin (10 µm final concentration) was added in every well. The supernatant was removed after an incubation of 15 mins at 37 C. On the other hand, to detect the Gs protein signaling pathway, different dilutions of compounds in 5% DMSO/HBSS buffer were added to the cells and incubated 15 min at the same conditions described above. The supernatant was then removed and 500 l of hot lysis buffer (90 C; 4 mm EDTA and 0.01% Triton X-100, ph 7.4) were added. After 1 h of mixing on ice, the camp amount in the lysates was determined by competitive radioligand binding experiments l of lysate was incubated with 30 l of [ 3 H]cAMP solution in lysis buffer (final radioligand concentration 3 nm) and 40 l of camp binding protein (50 g/vial). For the camp standard curve, 50 l of different camp concentrations were measured instead of the cell lysate. Total binding was obtained mixing radioligand solution and camp binding protein to lysis buffer, and the background was defined in the absence of binding protein. The mixture was incubated 60 min on ice and then filtered through GF/B glass fiber filters using a Brandel harvester. The filters were then washed with ice-cold Tris buffer (50 mm, ph 7.4), transferred into vials and incubated for 6 h with 2.5 ml of scintillation cocktail (LUMASAFE, Perkin Elmer, Rodgau, Germany). The vials were then counted in liquid scintillation counter (Tricarb 2900TR, Perkin Elmer, Rodgau, Germany). Independent experiments were performed in duplicates. Amounts of camp were calculated by linear regression from a standard curve and normalized to the maximal effect induced by 100 M forskolin (set as 100%). MTT assay Melan-a cells (5000 cells/well) were seeded in a 96-well plate and cultivated

5 overnight in the incubator. The compounds dissolved in DMSO and ethanol were diluted in the medium (80% RPMI-20% DMEM medium), added to the cells and incubated 72 h at 37 C with 10% CO2. The reagent is composed by a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2h-tetrazolium] which needs to be mixed with an electron coupling reagent (phenazine methosulfate). The mixed reagents were applied on the cells (100 l) after the supernatant disposal and the cells were incubated for 1 h at the same conditions described above. The absorbance was then read at 490 nm. The cells treated with the vehicles were used as control and set as 100% cell viability. Independent experiments were performed in quadruplicates. Western Blot Cell lysates (30 µg total protein/well) were separated in 10% SDS-PAGE gel and then transfered onto a nitrocellulose membrane (PROTRAN Nitrocellulose Transfer Membrane Whatman, Sigma, Taufkirchen, Germany). The Novex Sharp Prestained Protein Standard (Thermo Fisher Scientific) was used as marker. The membrane was blocked 1 hour in a 5% powdered milk/pbs-tween solution. Afterward, the membrane was incubated with the primary antibody overnight at 4 C or 1 hour at RT, washed 1 h with PBS-Tween, incubated with secondary antibody and washed again. The detection was performed with ECL kit (GE Healthcare, Amersham, Arlington, IL Dassel, Germany) according to the manufacturer's instructions. The following antibody combinations were used: mouse monoclonal anti- PK/PL (1:1000 dilution, PathHunter DiscoverX, Birmingham, UK) and anti-mouse- HRP (1:5000 dilution, Jackson Immuno Research, Hamburg, Germany) or rabbit antiserum PEP-7 3 and anti-rabbit-hrp (Sigma), respectively.

6 References 1. Nordstedt C, Fredholm BB. A modification of a protein-binding method for rapid quantification of camp in cell-culture supernatants and body fluid. Anal Biochem 1990;189: Pomerantz SH. L-tyrosine-3,5-3H assay for tyrosinase development in skin of newborn hamsters. Science 1969;164: Jimenez M, Tsukamoto K, Hearing V. Tyrosinases from two different loci are expressed by normal and by transformed melanocytes. J Biol Chem 1991;266: