Figure 3.
125I-MLT binding to MT1 and MT2 in the presence of GPR50. (A, B) HEK 293 cells stably expressing GPR50-YFP (HEK-GPR50) transiently expressed 5–10 fmol of MT1-Rluc per mg of protein (A) or 10–15 fmol of MT2-Rluc per mg of protein (B). Expression was monitored with the luciferase assay and by 125I-MLT binding (500 pM). (C) HEK 293 cells stably expressing 70–80 fmol of Flag-MT1 per mg of protein were transfected with untagged GPR50 or C-terminal YFP fusion constructs of GPR50, MT2 or MT2C113A. Expression of YFP fusion proteins was monitored by measuring YFP fluorescence, relative expression of GPR50 and GPR50-YFP was detected by Western blot using anti-GRP50-specific antibodies (not shown). Expression of Flag-MT1 was monitored by flow cytometry using anti-Flag antibodies (not shown) and by 125I-MLT binding. (D) Increasing concentrations of GPR50-YFP were expressed in HEK 293 cells stably expressing Flag-MT1, and YFP fluorescence, 125I-MLT binding and MT1 surface expression were determined. (E) HEK 293 cells transiently expressing MT1-Rluc in the absence (▪) or presence of GPR50-YFP (▵) were incubated with increasing concentrations of 125I-MLT. Data are means±s.e.m. of at least three independent experiments each performed in duplicate (A–C) or are representative of three further experiments (D, E) (***P<0.001; NS, P>0.05).