FIGURE 5.
Specificity of p10-mediated activation of GPHRs. A, COS-7 cells transfected with LHR and TSHR and their inactive mutants E354K and E409K, respectively, were incubated with their respective glycoprotein hormone (LHR: 500 nm hCG; TSHR: 100 milliunits/ml bTSH) and p10 and cAMP levels were determined. Basal cAMP level for pcDps was 1.8 ± 0.8 nm/well. B, COS-7 cells transfected with TSHR, LHR, and FSHR were incubated with their GPH (100 milliunits/ml bTSH, 500 nm hCG, 5 milliunits/ml FSH) and p10 E5K as indicated. Basal cAMP level for pcDps (empty vector) was 1.7 ± 0.5 nm/well. cAMP levels were determined (see “Experimental Procedures”). C and D, Nthy-ori 3-1 cells (40) were incubated with TSH, p10, and p10 E5K. In C and D, cAMP levels (basal cAMP levels: 1.1 ± 0.3 nm/well) (C) and mRNA expression levels (D) of the NIS and TG prior to and after stimulation were determined (see “Experimental Procedures”). Expression of NIS and TG was normalized to β2-microglobulin (Ct 15.7 ± 0.2). Gene regulation values are given as means of 2−ΔΔCt ± S.E. Data are means ± S.E. of three independent experiments performed in triplicate. *, p < 0.05, **, p < 0.01, ***, p < 0.001 (paired Student's t test).