Role of ECL2 in GPR21 Activation

To investigate the role of ECL2 in the activation of GPR21, three GPR21 mutants were generated. The residues 169–178 (immersed region of ECL2), residues 179–188 (cap region of ECL2), and residues 169–187 (the whole ECL2) were replaced by a flexible GS linker (GGSGGS), respectively. Wild‐type GPR21 and GPR21 mutants were cloned into pCDNA3.1 vector and expressed in HEK293 cells (Invitrogen). Plasmids were transfected in HEK293 cells using lipofectamine 3000 (Invitrogen). cAMP accumulation was performed using HTRF cAMP kit (Cisbio Bioassays) following the manufacturer's instructions. In brief, HEK293 cells were seeded into 384‐well plates. Then, 5 μl of cAMP‐d2 reagent and 5 μl of cAMP Eu‐cryptate antibody were added to each well. After incubation at room temperature for 1 h, fluorescence was measured using a microplate reader (Envision 2105, PerkinElmer) with excitation at 330 nm and emission at 620 and 665 nm. cAMP accumulation was calculated from a standard dose–response curve using GraphPad Prism 8.0 (GraphPad Software). The cAMP levels of GPR21 mutants were normalized to that of wild‐type GPR21 (100% level).