Figure 1.

hGRαR714Q has a reduced transactivation activity, about 2-fold reduction of affinity to dexamethasone, and reduced binding activity to GRIP1 in vitro. A, hGRαR714Q has a reduced transcriptional activity on the MMTV promoter in HCT116 cells. Top panel, Dexamethasone titration curves of the luciferase activity expressed from the MMTV promoter-driven reporter gene are shown. HCT116 cells were transfected with the plasmids expressing hGRα wild-type (WT) (open circles), hGRαR714Q mutant (closed circles), or a control gene (open squares) together with pMMTV-luc and pGL4.73[hRluc/SV40], and were treated with indicated concentrations of dexamethasone. Circles represent mean ± se values of the firefly luciferase activity normalized for the renilla luciferase activity in the indicated concentrations of dexamethasone. The experiment was repeated three times and representative data are shown. Bottom panel, Wild-type hGRα and hGRαR714Q are similarly expressed in HCT116 cells. Samples from HCT116 cells transfected with indicated GR-expressing plasmids were run on gels, and Western blots using anti-hGRα (top gel) or anti-β-actin (bottom gel) antibodies are shown. RLU, Relative light unit. B, hGRαR714Q has about 2-fold lower affinity to dexamethasone compared with the wild-type hGRα in whole-cell dexamethasone binding assay. Left panel shows saturation curves of radioactive dexamethasone to the wild-type hGRα (open circles) and hGRαR714Q (closed circles), and right panel demonstrates results of the Scatchard analysis examining affinity of these receptors to dexamethasone. HCT116 cells were transfected with the wild-type hGRα- or hGRαR714Q-expressing plasmid and were incubated with increasing concentrations of radioactive dexamethasone in the presence or absence of 500-fold excess amounts of cold dexamethasone. The experiment was performed three times with triplicate and mean ± se values of disassociation constant (Kd) are shown in right panel. Circles represent mean ± se values of the specific binding of radioactive dexamethasone to the receptors in A, and mean ratios of bound vs. free radioactive dexamethasone in B. C, hGRαR714Q has reduced binding activity to GRIP1 in vitro. Left panel, hGRαR714Q has reduced binding activity to various portions of GRIP1 in a GST pull-down assay. In vitro-translated and ³⁵S-labelled wild-type hGRα (top panel) or hGRαR714Q (bottom panel) was incubated with indicated fragments of GST-fused GRIP1 in the absence or presence of 10⁻⁸ m of dexamethasone (Dex). The experiment was performed three times and representative images are shown. Results obtained in the quantitative analysis of band intensity were shown in Supplemental Fig. 6A. Right panel, hGRαR714Q has reduced binding activity to the NRB domain of GRIP1 in a GST pull-down assay. In vitro-translated and ³⁵S-labelled wild-type hGRα or hGRαR714Q was incubated with GST-fused GRIP1-NRB in the presence of indicated concentrations of dexamethasone. The experiment was performed three times and representative images are shown. Results obtained in the quantitative analysis of band intensity were shown in Supplemental Fig. 6B.