The chicken leptin receptor (chLEPR) activates Janus kinase (JAK) -signal transducers and activator of transcription (STAT) signaling pathway after leptin stimulation. We have previously developed a bioassay using leptin inducible reporter gene in cultured cells. However, we failed to detect leptin-like activity in chicken blood. In the present study, we expressed green fluorescent protein (GFP) fused chSTAT3 (GFP-chSTAT3) in cells expressing chLEPR and analyzed leptin dependent activation of the chSTAT3. Leptin phosphorylated GFP-chSTAT3 and by which fluorescent signal translocated into nuclei in COS-7 cells transiently expressing GFP-chSTAT3 with chLEPR. Furthermore, we established CHO-K1 cells stably expressing chLEPR and chSTAT3 (CHO-chLEPR/chSTAT3), and in which detected time-and dose-dependent activation of chSTAT3 by leptin. Therefore, the CHO-chLEPR/STAT3 cells would be an excellent tool to detect and monitor leptin-like activity in avian tissues.

Avian prolactin receptor (PRLR) possesses two homologous repeated units in its extracellular domain and of which the membrane-proximal extracellular unit specifically binds with prolactin (PRL). However, whether another membrane-distal extracellular unit of avian PRLR is capable to bind specifically with PRL is still unknown. Therefore we analyzed whether the each two putative ligand-binding domain of chicken PRLR (chPRLR) is able to interact specifically with PRL by monitoring activation of Janus kinase (JAK)-Signal transducer and activator of transcription (STAT) signaling pathway in vitro. We primarily investigated that ovine PRL activates JAK-STAT signaling cascade in CHO-K1 cells transiently expressing chPRLR, which was not demonstrated in avian PRLR. Thereafter we revealed that PRL increased luciferase activity via STAT responsive reporter gene and phosphorylated STAT3 in LMH cells transiently expressing mutant chPRLR possessing a membrane-distal or a membrane-proximal ligand binding domain. Occurrence of signal transduction by PRL in cells expressing the mutant chPRLRs indicates that each putative ligand-binding domain in authentic chPRLR may bind specifically with PRL.

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