Neuropeptide S (NPS) and its receptor (NPSR1) in chickens: cloning, tissue expression, and functional analysis

Neuropeptide S (NPS) and its receptor neuropeptide S receptor 1 (NPSR1) have been suggested to regulate many physiological processes in the central nervous system (CNS), such as arousal, anxiety, and food intake in mammals and birds, however, the functionality and tissue expression of this NPS-NPSR1 system remain unknown in birds. Here, we cloned NPS and NPSR1 cDNAs from the chicken brain and reported their functionality and tissue expression. The cloned chicken NPS is predicted to encode a mature NPS peptide of 20 amino acids, which shows a remarkable sequence identity (∼94%) among tetrapod species examined, while NPSR1 encodes a receptor of 373 amino acids conserved across vertebrates. Using cell-based luciferase reporter systems, we demonstrated that chicken NPS could potently activate NPSR1 expressed in vitro and thus stimulates multiple signaling pathways, including calcium mobilization, cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways, indicating that NPS actions could be mediated by NPSR1 in birds. Quantitative real-time PCR revealed that NPS and NPSR1 are widely expressed in chicken tissues, including the hypothalamus, and NPSR1 expression is likely controlled by a promoter upstream exon 1, which shows strong promoter activities in cultured DF-1 cells. Taken together, our data provide the first proof that the avian NPS-NPSR1 system is functional and helps to explore the conserved role of NPS and NPSR1 signaling in tetrapods.