The 2 structurally related peptides, neuropeptide FF (NPFF) and neuropeptide AF (NPAF), are encoded by the NPFF gene and have been identified as neuromodulators that regulate nociception and opiate-mediated analgesia via NPFF receptor (NPFFR2) in mammals. However, little is known about these 2 peptides in birds. In this study, we examined the structure, tissue expression profile, and functionality of NPAF and NPFF in chickens. Our results showed that: 1) unlike mammalian NPFF, NPFF from chicken and other avian species is predicted to produce a single bioactive NPAF peptide, whereas the putative avian NPFF peptide likely lacks activity due to the absence of functional RFamide motif at its C-terminus; 2) synthetic chicken (c-) NPAF can potently activate cNPFFR2 (and not cNPFFR1) expressed in HEK293 cells, as monitored by 3 cell-based luciferase reporter systems, indicating that cNPAF is a potent ligand for cNPFFR2, which activation could decrease intracellular cAMP levels and stimulate the MAPK/ERK signaling cascade; interestingly, gonadotropin-inhibitory hormone, a peptide sharing high structural similarity to NPAF, could specifically activate cNPFFR1 (but not cNPFFR2); 3) Quantitative real-time PCR revealed that cNPFF mRNA is widely expressed in chicken tissues with the highest level detected in the hypothalamus, whereas cNPFFR2 is expressed in all tissues examined with the highest level noted in the hypothalamus and anterior pituitary. Taken together, our data reveal that avian NPFF encodes a single bioactive NPAF peptide, which preferentially activates NPFFR2, and provides insights into potential structural and functional changes of NPFF-derived peptides during vertebrate evolution.

两种结构相关的肽，神经肽FF（NPFF）和神经肽AF（NPAF），由NPFF基因编码，并已被确定为通过NPFF受体（NPFFR2）调节伤害感受和鸦片介导的镇痛作用的神经调节剂。然而，对于鸟类中的这两种肽知之甚少。在这项研究中，我们检查了鸡中NPAF和NPFF的结构，组织表达谱和功能。我们的结果表明：1）与哺乳动物NPFF不同，NPFF预计来自鸡和其他禽类的蛋白会产生单一的生物活性NPAF肽，而假定的禽NPFF肽可能由于其C端缺少功能性RFamide基序而缺乏活性。2）合成鸡（c-）NPAF可以有效激活HEK293细胞中表达的cNPFFR2（而不是cNPFFR1），这是基于3种基于细胞的荧光素酶报告系统监测的，表明cNPAF是cNPFFR2的有效配体，该激活可以降低细胞内cAMP调节并刺激MAPK / ERK信号级联反应; 有趣的是，促性腺激素抑制激素是一种与NPAF具有高度结构相似性的肽，可以特异性激活cNPFFR1（但不能激活cNPFFR2）。3）实时定量PCR显示cNPFFmRNA在鸡组织中广泛表达，在下丘脑中检测到最高水平，而cNPFFR2在所有检查的组织中表达，在丘脑和垂体前叶中发现最高水平。综上所述，我们的数据表明禽类NPFF编码单个生物活性NPAF肽，该肽优先激活NPFFR2，并为脊椎动物进化过程中NPFF衍生肽的潜在结构和功能变化提供了见识。