Differential Activation of Their Cognate Receptors by NPFF and GnIH Peptides in the Half-Smooth Tongue Sole (Cynoglossus semilaevis)

Neuropeptide FF (NPFF) belongs to the RF-amide peptide family and is homologous to gonadotropin-inhibitory hormone (GnIH). The NPFF precursor encodes two mature peptides, NPFF and NPAF (neuropeptide AF). Both peptides share the conserved C-terminal PQRFa motif. However, there is very limited information available on receptor cross-reactivity for NPFF and GnIH peptides in teleosts. As a first step, we cloned two cognate receptor genes for NPFF, designated as NPFFR2-1 and NPFFR2-2, in the flatfish species half-smooth tongue sole. Tissue distribution analysis revealed that npffr2-1 and npffr2-2 transcripts were present at high levels in the brain and pituitary gland, and at lower levels in some peripheral tissues. In vitro functional analysis indicated that NPFF significantly stimulated CRE-luc and SRE-luc activity in COS-7 cells expressing either NPFFR2-1 or NPFFR2-2. However, NPAF increased CRE-luc and SRE-luc activity only via NPFFR2-1. Moreover, NPFF exerted an inhibitory effect on NFAT-RE-luc activity in COS-7 cells transfected with NPFFR2-1, whereas NPAF elicited an evident stimulatory effect via NPFFR2-2. Neither GnIH1 nor GnIH2 altered CRE-luc activity in COS-7 cells transfected with NPFFR2-1 or NPFFR2-2; however, forskolin-induced CRE-luc activity was significantly reduced by these two peptides. Furthermore, neither basal nor forskolin-stimulated CRE-luc activity was modified by NPFF or NPAF in COS-7 cells expressing the GnIH receptor (GnIHR). Both GnIH1 and GnIH2 significantly increased SRE-luc activity in COS-7 cells expressing NPFFR2-1 or NPFFR2-2, and vice versa. Taken together, our findings provide novel evidence that both NPFF and GnIH peptides could exert their functions via three different receptors, and that PKA, PKC, and Ca2+ signaling pathways are potential mediators.