Figure 7.

Functional characterisation of the Akt signalling pathway by PRLR rare variants. (A) pAkt responses following PRL treatment in cells transfected with wild-type (WT) or the five extracellular domain (ECD) variant PRLRs. Asp187Glu had impaired pAkt responses when compared to WT-expressing cells. (B) FOXO1 luciferase reporter activity in cells transfected with WT, mutant His188Arg, or the five ECD variant PRLRs. Addition of 200 ng/mL PRL reduces FOXO1 luciferase activity in WT and four variant cell lines. However, no response was observed in His188Arg cells and Glu155Lys had reduced basal FOXO1 activity and impaired PRL-induced responses. (C) pAkt responses following PRL treatment in cells transfected with WT or the five intracellular domain (ICD) variant PRLRs. Arg327Gln-expressing cells had elevated basal pAkt activity. (D) FOXO1 luciferase activity in cells transfected with WT or the five ICD variant PRLRs. Addition of 200 ng/mL prolactin to WT and variant PRLRs reduces FOXO1 luciferase activity similarly in all cell lines. Data in all panels were expressed relative to WT cells treated with 0 ng/mL PRL. Mean from 4 to 6 independent assays for pAkt assays. Mean (panel D) or median (panel B) from seven independent assays for luciferase reporter assays. Statistical analyses performed by one-way ANOVA with Sidak’s or Dunnett’s multiple comparisons tests for panels A, C, and D. Statistical analysis performed by Kruskal–Wallis test with Dunn’s multiple comparisons tests for panel B. Comparisons show 0 vs 200 nM PRL (asterisks) or between WT and variant (#). ^****P < 0.0001, ^***P < 0.001, ^**P < 0.01, ^*P < 0.05, NS, not significant.