Cloning, expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper, Epinephelus coioides

Abstract

As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp(6.59) might be a common NPY-binding site, while Asp(2.68) of the Y8b receptor and Glu(5.24) of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.

Recent advances in neuropeptide-related omics and gene editing: Spotlight on NPY and somatostatin and their roles in growth and food intake of fish

“…And transgenic mice in which the vesicular glutamate transporter 2 gene was disrupted selectively in orexin-producing neurons were used to study the roles of orexin neurons in mediating methamphetamine-induced changes in body temperature and heart rate ( 119 ). It is well known that in the neuropeptide and receptor family, there are usually several receptors corresponding to one ligand, neuropeptides, such as NPY and somatostatin ( 182 , 183 ). Therefore, the Cre-loxP conditional knockout system is very useful and efficient for identifying specific receptor types that mediate ligand function in specific tissues.…”

“…The y1 gene is only found in the zebrafish genome ( 189 ), speculating that most teleost lost Y1. Studies have revealed that Y4, Y8a, Y8b, Y2 and Y7 are all highly expressed in the brains ( 190 , 218 ), eyes, heart, kidney, liver, intestine and gonads ( 182 , 220 ). Research on the distribution of teleost NPY family receptors in brain regions, especially in various sub-regions other than the hypothalamus, remains to be studied.…”

Neuropeptide Y and melanocortin receptors in fish: regulators of energy homeostasis

“…Receptors of Y1 (zebrafish), Y4 (Ya), Y8a (Yc) and Y8b (Yb) are identified as the Y1 receptor subfamily; receptors of Y2 and Y7 belong to Y2 receptor subfamily (Larsson et al 2005). The Y1 subtypes are identified widely in teleost species whereas the Y2 subtypes are found only in limited teleost species, such as zebrafish, rainbow trout (Oncochynchus mykiss), grouper and large yellow croaker (Larimichthys crocea) (Fredriksson et al 2004;Larsson et al 2006;Wang et al 2014Wang et al , 2019. With zebrafish, in addition to Y2 and Y7 receptors, the Y2 subfamily contains Y2-2 receptor, which is obtained by the local duplication of the Y2 receptor (Fällmar et al 2011;Sundström et al 2013).…”

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“…Here we investigated the expression of appetite regulating genes in the brains of cichlids adapted to herbivorous and carnivorous trophic niches and identified two appetite-regulating genes, cart (or cartpt1) and npy2r, to have higher expression in the carnivore brains prior to the initiation of feeding across all three lakes. Both genes are indicated to have anorexigenic function in different groups of teleost fishes (Matsuda et al, 2012;Babichuk & Volkoff, 2013;Wang et al, 2014;He et al, 2015;Volkoff, 2016;Porter, Roberts & Maruska, 2017). The first gene, cart, or cocaineand amphetamine-regulated transcript, encodes a pre-proprotein which proteolyzes to multiple active peptides and participates in biological processes related to regulation of appetite, energy balance, stress response, and reward and addiction responses (Volkoff, 2006(Volkoff, , 2016Koylu, Balkan & Pogun, 2006;Vicentic et al, 2007;Rogge et al, 2008).…”

“…Orexigenic (Grone et al, 2012;Matsuda et al, 2012;Babichuk & Volkoff, 2013;Volkoff, 2016;Das et al, 2019) npy2r Neuropeptide Y receptor type 2 Perciformes Cypriniformes Anorexigenic Diet transition (Matsuda et al, 2012;Wang et al, 2014;He et al, 2015) pacap Pituitary adenylate cyclase activating polypeptide Cichliformes Cypriniformes Anorexigenic (Matsuda et al, 2005;Zhou et al, 2013;Costa et al, 2016) pomc Pro-opiomelanocortin preproprotein Cichliformes Cypriniformes Anorexigenic (Volkoff, 2016;Porter, Roberts & Maruska, 2017) pyy Prepro-peptide YY Perciformes Cypriniformes Orexigenic Anorexigenic (Murashita et al, 2006;Volkoff, 2016) trh, trhra Thyrotropin-releasing hormone and its receptor Cypriniformes Orexigenic Diet transition (He et al, 2015;Volkoff, 2016)…”