Figure 6.

Functional characterisation of the JAK2-STAT5 signalling pathway by PRLR ICD variants. (A) pSTAT5 responses following prolactin (PRL) treatment in cells expressing wild-type (WT), mutant His188Arg, or ICD variants Phe255Ser, Gly263Asp, Asp320Tyr, Arg327Gln, Val535Met. PRL-induced pSTAT5 production was abolished in His188Arg expressing cells, significantly reduced in Phe255Ser and significantly increased in Arg327Gln expressing cells when compared to WT cells. Additionally, pSTAT5 was reduced in Gly263Asp- and Val535Met-expressing cells when treated with high (1000 ng/mL) PRL. (B) CISH luciferase reporter activity in cells transfected with WT, mutant His188Arg, or the five ICD variant PRLRs. CISH reporter activity was abolished in His188Arg-expressing cells, significantly reduced in Phe255Ser and significantly increased in Arg327Gln-expressing cells when compared to WT cells. Mean from four to five independent assays for all panels. Statistical analyses show comparisons between WT and the five ICD PRLR variants (black) and WT and the His188Arg mutant (grey) by two-way ANOVA with Dunnett’s or Tukey’s multiple comparisons tests. ^****P < 0.0001, ^***P < 0.001, ^**P < 0.01, ^*P < 0.05.