Calcium Mobilization Assay

Intracellular calcium mobilization was measured with the BD calcium kit (BD Bioscience, San Jose, CA, United States). The recombinant cell lines expressing MC3R, MC4R, and MC5R with and without GPR139 or Gα₁₆ were seeded in culture media at a density of 50,000 cells/well into 96-well black/clear-bottom poly-D-lysine treated tissue culture plates. Cells were incubated overnight at 37°C and 5% CO₂. Next day, culture media was removed and cells were washed with HBSS prior to dye-loading for 1 h at 37°C and 5% CO₂. Agonist stimulation and intracellular calcium measurements were done on the FLIPR Tetra (Molecular Devices, Sunnyvale, CA, United States). For receptor inhibition analysis, antagonists (1 μM HS-024 or 10 μM JNJ-3792165) were added off-line 15 min prior to agonist addition on the Tetra. For G-protein inhibitor experiments, cells were treated with 1 μM YM-254890 for 5 min prior to agonist additions on the Tetra. Responses were normalized to buffer (0%) and EC₁₀₀ of JNJ-63533054 (100%) in Figures ​Figures3A3A–C, 4C,F,I. In Figures 4A,B,D,E,G,H responses were normalized to EC₁₀₀ of ACTH. Responses in Figure ​Figure66 were normalized to its respective agonist response without antagonist. EC_50s were calculated by fitting a three-parameter logistic curve using least-squares non-linear regression analysis as implemented in Graphpad Prism 7.0.

Concentration response of the MC agonists ACTH, α-MSH, β-MSH, and GPR139 agonists (L-Trp, JNJ-63533054, and TC-O9311) on calcium mobilization in three different host cells stably expressing human GPR139: (A) CHO-TREx, (B) HEK-293, and (C) SK-N-MC/CRE-TREx. Data are the mean ± SEM of at least three separate experiments performed in duplicates.

Concentration response of the MC agonists ACTH, α-MSH, β-MSH, and GPR139 agonists (L-Trp, JNJ-63533054, and TC-O9311) on calcium mobilization in HEK-293 cells transiently expressing MC3R (A), MC3R and G_α16 (B), MC3R and GPR139 (C) MC4R (D), MC4R and G_α16 (E) MC4R and GPR139 (F) and MC5R (G), MC5R and G_α16 (H) MC5R and GPR139 (I). Data are the mean ± SEM of at least three separate experiments performed in duplicates.

Inhibition of ACTH, α-MSH, β-MSH, L-Trp, JNJ-63533054, or TC-O9311 induced calcium release in HEK-293 cells transiently co-expressing GPR139 and MC3R (A–C), MC4R (D–F) or MC5R (G–I) by a GPR139 antagonist (10 μM JNJ-3792165, B, E, H) or a MCR antagonist (1 μM HS-024, C, F, I). Data are the mean ± SEM of at least three separate experiments performed in duplicates.