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. 2005 Jun 20;169(6):885–896. doi: 10.1083/jcb.200409150

Figure 2.

Figure 2.

Gβ1, Gβ2, and Rack1 suppress the transcriptional activity of GR on the MMTV promoter, and endogenous Gβ and Gγ are associated with GR in vivo. (A) Gβ1, Gβ2, and Rack1 dose dependently suppress the transcriptional activity of GR on the MMTV promoter in HCT116 cells. HCT116 cells were transfected with indicated amounts of Gβ1, Gβ2, or Rack1-expressing plasmids together with pRShGRα, pMMTV-Luc, and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10−6 M of dexamethasone. (B) GR(Δ262-404) has stronger transcriptional activity than the wild-type GR and Gβ2 loses its suppressive effect on GR(Δ262-404)-induced transactivation in HCT116 cells. HCT116 cells were transfected with Gβ2-expressing plasmids and pRShGRα or pRShGRα(Δ262-404), together with pMMTV-Luc and pSV40-β-Gal. Bars represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence or presence of 10−6 M of dexamethasone. (*), P < 0.01; n.s., not significant, compared with the baseline. (C) Expression of Gβ2 shifts the dexamethasone titration curve of the luciferase activity from the pMMTV-Luc in HCT116 cells. HCT116 cells were transfected with pRShGRα, pMMTV-Luc, and pSV40-β-Gal in the absence or presence of Gβ2-expressing plasmid. Cells were then stimulated with increasing concentrations of dexamethasone. Open and closed circles represent mean ± SEM values of the luciferase activity normalized for β-galactosidase activity in the absence (open circle) or presence (closed circle) of the Gβ2 expression. (D–F) Abrogation of Gβ1 and Gβ2 enhanced dexamethasone-activated TAT activity (D) and suppressed mRNA (E) and protein (F) levels of Gβ1 and Gβ2 in HTC cells. HTC cells were transfected with control or Gβ1 and Gβ2 siRNAs and the cells were treated with 10−6 M of dexamethasone for 24 h. Cell lysate and total RNA were harvested and the TAT activity (D), Gβ1 and Gβ2 mRNA abundance (E) and protein levels of Gβ1 and Gβ2 in Western blots using their specific antibodies in the absence of dexamethasone (F), were determined. Bars represent mean ± SEM values of TAT activity (D) or fold induction of Gβ1 or Gβ2 mRNAs (E) in the absence or presence of 10−6 M of dexamethasone. (*), P < 0.01; n.s., not significant, compared with the baseline. (G) Endogenous Gβ and Gγ, but not Gαi, are associated with GR in vivo. HeLa cells were stimulated with 10−6 M of dexamethasone and coimmunoprecipitation was performed with control or anti-GRα antibody. After blotting the precipitated proteins on nitrocellulose membranes, the associated Gβ, Gγ, or Gαi was detected with their specific antibodies. Expression of Gβ, Gγ, Gαi, and GR was also examined in 10% whole homogenates in Western blots.