Figure 7.

Activation of G proteins and adenylyl cyclase and coimmunoprecipitation of GPR30 with G proteins in croaker GPR30-transfected HEK293 cell plasma membranes. A, Effects of 100 nm E2 treatment (20 min) on specific [³⁵S]GTPγS binding to G proteins in membranes of transfected HEK293 cell plasma membranes; top, results obtained using original assay procedure (18); bottom, results obtained using modified protocol described in Materials and Methods. Boiled indicates that membrane samples were boiled for 15 min before assay. *, P < 0.05; n = 4. B, Immunoprecipitation of [³⁵S]GTPγS bound to G proteins activated by 100 nm E2 with specific anti-Gs and anti-Gi antibodies or control IgG. CTL, Control nonimmunized IgG. *, P < 0.05 compared with respective vehicle. C, Coimmunoprecipitation of 100 nm E2-activated G protein with specific G protein antibodies (Gs, Gi) and croaker GPR30 antibody (left) and detection of GPR30 (G30), Gs, and Gi protein in GPR30-transfected HEK 293 cell plasma membranes (right). M, Size marker; V, vehicle; CTL, control IgG. D, Effects of treatment with E2, Tmx, and ICI 182,780 (ICI) on cAMP level changes in the plasma membranes of HEK 293 cells transfected with croaker GPR30 cDNA (upper panel) and the nontransfected control HEK cell membranes (lower panel). *, P < 0.05 compared with respective 0 nm control (Tukey’s). Each value represents the mean ± sem of three to four determinations. All assays were repeated three times with ovarian tissue from different donors, and similar results were obtained in each assay.