FIG 7.

Regulation of GCM1 posttranslational modification and activity by hCG. (A) Stimulation of Ser269 and Ser275 phosphorylation in GCM1 by hCG. (Left) 293T cells were transfected with the empty vector or pHA-LH/CGR for 24 h. Cells were then treated without or with 50 or 100 IU/ml of hCG for an additional 24 h before they were harvested for cAMP assays. Mean values and standard deviations obtained from three independent experiments are presented. **, P < 0.01. (Right) On the other hand, 293T cells were transfected with pGCM1-FLAG and pHA-LH/CGR and treated without or with 100 IU/ml of hCG from different commercial suppliers (#1 to #3). Cells were subjected to immunoprecipitation (IP) with p-Ser269275-GCM1 or Ac-K Ab followed by immunoblotting (IB) with FLAG Ab. (B) Stimulation of GCM1 phosphorylation and acetylation in placental cells by hCG. BeWo31 cells were mock treated or treated with the indicated amounts of hCG for 24 h before being harvested for immunoprecipitation analysis with p-Ser269275-GCM1 or Ac-K Ab followed by immunoblotting with HA Ab. (C) Stimulation of GCM1 transcriptional activity by hCG. BeWo31 cells were transfected with the GCM1 reporter plasmid p(GBS)₄E1bLuc for 24 h. Cells were then treated with hCG or hCG plus 5 μM H89 for an additional 24 h before being harvested for luciferase assays. Mean values and standard deviations obtained from three independent experiments are presented. There was a significant correlation (by the Spearman rank correlation test) between GCM1-mediated transcriptional activation and hCG treatment. **, P < 0.01. (D) hCG stimulates Ser269 and Ser275 phosphorylation of GCM1 in primary trophoblast cells. Human primary trophoblast cells were mock treated or treated with 200 IU/ml hCG and then harvested for immunoprecipitation with GCM1 Ab, followed by immunoblotting with p-Ser269275-GCM1 Ab.