Identification and functional study of the GPR50 variant. (A) Sanger sequence of codons 37-39 in exon 1 of GPR50 in a wild-type (WT) subject and proband B confirms the presence of the T38I variation. The variant nucleotide residue is indicated by a red rectangular. (B) The wild-type and variant GPR50 protein both located on the membrane of HEK293 cells. GFP-tagged GPR50 wild-type and variant plasmids were transfected into HEK293 cells. The red plasma membranes were labeled by Concanavalin A (Con A) and the nuclei were stained blue with DAPI. (C) The variant GPR50 protein did not alter the effect on melatonin-induced MT1 signaling. GPR50 wild-type and variant plasmids were transfected into MT1 stable transfection HEK293 cells, no difference of cAMP levels was deteced when treated with forskolin and melatonin, 48 h after trnasfection (ns, P > 0.05).