Pharmacological characterization, cellular localization and expression profile of NPY receptor subtypes Y2 and Y7 in large yellow croaker, Larimichthys crocea

Comparative Biochemistry and Physiology, Part B 238 (2019) 110347

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Pharmacological characterization, cellular localization and expression profile of NPY receptor subtypes Y2 and Y7 in large yellow croaker, Larimichthys crocea

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Tianming Wanga, Jing Lianga, Xiaowei Xianga, Xu Chena, Bing Zhanga, Naiming Zhoub, ⁎ Wei Huangc, Jingwen Yanga, a b c

National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University, Zhoushan, Zhejiang 316022, PR China Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, PR China Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration, Hangzhou, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Fish GPCR Neuropeptide Y receptor Y2 Y7 large yellow croaker

Neuropeptide Y (NPY) receptors are suggested to mediate the multi-physiological functions of NPY family peptides, such as food intake, in teleost fish. However, the structure and signaling of fish NPY receptors are yet to be fully elucidated. In this study, we report the cloning and characterization of two neuropeptide Y receptor subtypes, Y2 (NPY2R) and Y7 (NPY7R), in yellow croaker Larimichthys crocea (L. crocea) (LcNPY2R, LcNPY7R). The gene structure, pharmacological characterization, cell location, and tissue expression of these two receptors were explored. The phylogenetic results showed that LcNPY2R and LcNPY7R had typical G protein-coupled receptor profiles, associated with the Y2 subfamily, with coding sequences that are highly conserved in vertebrates. The expression of both LcNPY2R and LcNPY7R could be activated by LcNPY in HEK293 cells. However, truncated LcNPY18–36 was only able to activate LcNPY2R at the same level as full length LcNPY. Expression analysis revealed that LcNPY2R mRNA was predominantly expressed in the intestine and liver, whereas LcNPY7R was expressed in the stomach, which indicated that both receptors were related to the digestive system. Overall, our data establishes a molecular basis to determine the actions of LcNPY2R and LcNPY7R, which could be used to elucidate the conserved roles of these receptor-ligand pairs in vertebrates.

1. Introduction Neuropeptide Y (NPY) is a widely distributed peptide among many species and has a variety physiological functions, including food intake, anxiety, memory, blood pressure, and circadian rhythms (Abrahamsson, 2000; Cleary et al., 1994; Huhman et al., 1996; MoralesMedina et al., 2010; Pedrazzini et al., 2003). NPY is comprised of 36 amino acids, mainly expressed in the neurosecretory cells in central and peripheral neurons (Hendry, 1993; Sundler et al., 1993), with the same length for the peptide YY (PYY), the tetrapod's pancreatic polypeptide (PP), and the fish pancreatic peptide (PY), all of which belong to the NPY family (Larhammar, 1996; Larhammar et al., 1993). Compared with PP and PYY, NPY is the most conserved and is widely distributed in the brain, whereas the other two peptides are predominantly expressed in the digestive system (Cerda-Reverter and Larhammar, 2000; Larhammar, 1996). NPY-family peptides have been proposed to be involved in evolutionary gene duplication. Studies have reported that

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teleost fishes underwent an independent tetraploidization event, known as 3R (third round of genome doubling), after two vertebrate tetraploidization (2R) (Sundstrom et al., 2008). This may have given rise to additional copies of NPY, PYY, and the ray finned fish PY (Sundstrom et al., 2005). Despite the conserved mature peptide sequence, NPYs perform a variety functions through the activation of their receptors, known as neuropeptide Y receptors (NPYR). Therefore, they are crucial for understanding the physiological mechanisms of this system. The NPYR family contains 6 or 7 members (Larhammar et al., 1998). So far, six subtypes of NPYRs have been found in humans, denoted Y1, Y2, Y3 (predictive), Y4, Y5, and Y6 (Cabrele and BeckSickinger, 2000; Ingenhoven and Beck-Sickinger, 1999). Y1 (zebrafish), Y2, Y4 (Ya), Y7, Y8a (Yc), and Y8b (Yb) have been found in bony fish (Salaneck et al., 2008; Sundstrom et al., 2013). According to their homology and functional characteristics, NPYRs are divided into Y1 subfamilies (including Y1, Y4 (Ya), Y6, and Y8 (Yb/c, not found in mammals)), Y2 subfamilies (including Y2 and Y7 (not found in

Corresponding author. E-mail address: [email protected] (J. Yang).

https://doi.org/10.1016/j.cbpb.2019.110347 Received 24 June 2019; Received in revised form 2 September 2019; Accepted 4 September 2019 Available online 06 September 2019 1096-4959/ © 2019 Published by Elsevier Inc.

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physiological role of NPYR Y2 subfamily during growth and feeding in L. crocea. Moreover, the results also provide important support for studying the evolution of NPY2R/7R and its functional analysis.

mammals)), and the Y5 subfamily (currently only one member of Y5, not found in bony fish) (Larhammar and Salaneck, 2004). They may be activated by the pancreatic family polypeptides, which are represented by NPY. Among these receptors, Y1 and Y5 are thought to be involved in the regulation of orexins in mammals and fish (Day et al., 2005). Meanwhile, the receptors of the Y2 subfamily are mainly involved in appetite regulation and affective behavior in mammals (Fredriksson et al., 2004). However, the physiological functions of these receptors are still far from understood in fish. At present, the Y2 and Y7 subfamily receptors have been cloned and identified in chicken, tetrapods (frogs) (Fredriksson et al., 2004; Salaneck et al., 2000), and fish (zebrafish, rainbow trout, and grouper, among others) (Fredriksson et al., 2006; Larsson et al., 2006; Larsson et al., 2005; Wang et al., 2014), indicating that this subfamily has been highly conserved throughout evolution, thus playing multiple important roles in various physiological processes. As the target of neuropeptide Y of the cell membrane, NPYR is a G protein-coupled receptor (GPCR), which contains the typical and conserved 7-transmembrane domain. Its N-terminus is highly divergent and is mainly responsible for the binding of extracellular transmitters. The C-terminus mediates the activation of kinases. According to current studies in mammals, NPYR-mediated signaling is mainly performed via two pathways: (i) the Gαi/o coupling with pertussis toxin (PTX) sensitive pathway: inhibits adenylate cyclase activity and results in a decrease of the intracellular second messenger cAMP, while the G protein βγ subunit activates phosphatidylinositol-3 kinase (PI3K) and leads to intracellular calcium ion mobilization; (ii) the Gαq coupling with PTX insensitive pathway: the activation of phospholipase C (PLC) on the cell membrane produces diacylglycerol (DAG) and inositol triphosphate (IP3), followed by the mobilization of calcium ions by DAG and IP3. Then, the protein kinase C (PKC) is activated, which phosphorylates ERK1/2, thus mediating the regulation of downstream gene expression (Goldberg et al., 1998; Michel et al., 1998). In the field of ichthyology, the zebrafish Y2 receptor has been confirmed to be orthologous to mammalian Y2 and was pharmacologically most similar to chicken Y2. In Oncorhynchus mykiss, the Y2 and Y7 receptors may bind to the three zebrafish peptides, NPY, PYYa, and PYYb, as well as the porcine NPY and PYY at the nanomolar level (Larsson et al., 2006). However the Y2 subfamily in both zebrafish and rainbow trout showed a poor affinity with BIIE0246, which is the antagonist of human NPY2R (Doods et al., 1999; Fallmar et al., 2011). In Epinephelus coioides, the Y2 and Y8 have been previously cloned to analyze their phylogenies and binding properties. However, the majority of research has focused on the cloning and characterization of NPY and its receptor using binding assays based on mammalian cell lines (Wang et al., 2014). As such, the systematic GPCR signaling pathway and related physiological functions have not been thoroughly investigated. In this study, we present the cloning and pharmacological characterization of the NPY receptors, Y2 and Y7, in the large yellow croaker Larimichthys crocea, an important marine fishery species along the coast of China. However, due to the environmental pollution and overfishing, the natural fishery resources of the L. crocea are under threat. In order to improve our understanding of the feeding and growth profile of the large yellow croaker, the NPY receptors need to be elucidated. Here, L. crocea NPY was synthesized, and the full length of the Y2 and Y7 receptors were cloned to determine their cytobiological characteristics and their signaling pathways, which were respectively named LcNPY2R and LcNPY7R. They were fused with mammalian expression vectors pCMV-FLAG and pEGFP-N1 in vitro to determine their response to NPY activation, as well as their cellular location and internalization upon ligand stimulation. In addition, the transcriptional profiles of LcNPY2R and LcNPY7R were analyzed in different tissues for their functional evaluation. The results suggest that LcNPY2R and LcNPY7R are activated by NPY ligand and this NPY system is involved in digestive function of L. crocea. These findings provide a foundation for future studies on the signal transduction pathway and the

2. Materials and Methods 2.1. Animals and samples L. crocea samples were collected from the mari-culture farm on Dongji Island (Zhejiang, China). The fish used for cDNA cloning and mRNA expression were approximately one year old (immature fish) with an average weight of 103 g. They were kept and raised at 25 °C in an aerated seawater tank. Different tissues, including head kidney, spleen, stomach, gill, heart, liver, brain, muscle, ovary, and intestine tissues, were collected for gene expression analysis. All the tissues obtained were immediately frozen and stored in liquid nitrogen for subsequent RNA extraction. 2.2. Total RNA extraction, first-strand cDNA synthesis, and rapid amplification of cDNA ends (RACE) Total RNA was isolated from the tissues of L. crocea using Trizol reagent (TaKaRa) and phenol chloroform. The integrity of the total RNA was confirmed by electrophoresis and the RNA concentration and quality were determined using a Nanodrop 2000 (Thermo Fisher Scientific). Then, 10 μg and 1 μg of total intestine RNA were obtained to carry out the 5′ RLM-RACE and 3′ RLM-RACE protocols, respectively. These were performed using a FirstChoice® RLM-RACE kit (Ambion Inc., TX, USA), according to the manufacturer's instructions. The samples were then stored at −80 °C for the subsequent PCR step. Then, 1 μg total RNA from each sample was reverse transcribed into single-stranded cDNA by treating with M-MLV reverse transcriptase (Promega Inc., Shanghai, China) at 42 °C for 1 h using oligo(dT)20. An RNase inhibitor (Promega Inc., Shanghai, China) was used during the cDNA synthesis to avoid RNA degradation. The cDNA was maintained at −20 °C for real-time PCR. The gene-specific primers (Supplementary Table 1) were designed according to the partial coding sequence of LcNPY2R and LcNPY7R from the L. crocea genomic sequence. The genespecific primers were designed according to the CDS of LcNPY2R and LcNPY7R, as well as previously designed primers (Wang et al., 2019). All primers used in this study are listed in Supplementary Table 1. 2.3. Genomic structure and sequence analysis The cDNA sequences were compared with known sequences in GenBank for identity analysis using BLASTX 2.2.29+ (http://blast. ncbi.nlm.nih.gov/). The cDNA sequences of L. crocea were translated into the predicted amino acid sequences with DNAMAN 8.0. The deduced amino acid sequences were aligned using ClustalW2 (http:// www.ebi.ac.uk/Tools/msa/clustalw2/). The phylogenetic analysis of the amino acid sequences was carried out using the neighbor-joining (NJ) method with pairwise detection and amino acid p-distance correction using MEGA5.10 (Tamura et al., 2011). Bootstrap analysis was repeated 1000 times to compute a confidence interval. The prediction of N-glycosylation and phosphorylation sites was performed using the NetNGlyc1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and the NetPhos 2.0 Server (http://www.cbs.dtu.dk/services/NetPhos/), respectively. The prediction of the protein transmembrane helices was conducted using the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/ services/TMHMM/). In addition, the LcNPY2R and LcNPY7R protein domains were predicted using Inter-ProScan software (http://www.ebi. ac.uk/InterProScan/) and SMART (http://smart.embl-heidelberg.de/). Protein secondary structure analysis was carried out using PredictProtein (http://www.predictprotein.org/). All the accession numbers used in this study are listed in Supplementary Table 2. 2

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APO lambda blue 63 × 1.4 oil immersion lens.

Structural models were generated for L. crocea NPY2R and NPY7R homology modeling (Fig. 3). Suitable structural templates (PDB: 5zbh.1.A) were identified comparing the BLAST search against the Protein Data Bank proteins database (pdb), as implemented in the SWISS-MODEL Protein Modelling Server (Arnold et al., 2006). Homology modeling was performed with SWISS-MODEL in automated mode. The model quality was assessed using ModFOLD Model Quality Assessment Server version 4.0 (http://www.reading.ac.uk/bioinf/ ModFOLD/ModFOLD_form_4_0.html) in the Swiss Model structure assessment tool.

2.7. Ligand competition binding assay HEK293 cells stably expressing Flag-LcNPY2R and Flag-LcNPY7R were plated in 96-well plates coated with poly-L-lysine one day before the experiment. The cells were washed twice with cold PBS. Then, 50 μl of PBS containing 0.2% BSA, 25 μl rhodamine-labeled LcNPY (40 nM), and 25 μl unlabeled LcNPY (control, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM) were added to each well at a total volume of 100 μl for the displacement binding analyses. After incubation at 4 °C for 60 min, the cells were transferred onto ice. The fluorescence-labelled LcNPY, in the absence of unlabeled ligand, was used to determine total binding. Then, the cells were washed three times with 100 ml of ice-cold PBS with 0.1% BSA and the fluorescence intensity was measured using a fluorometer (Infinite F200Pro, Tecan). Binding is presented as the percentage of total binding. The binding displacement curves were analyzed and the Ki values were determined using GraphPad Prism.

2.4. Construction of the mammalian expression vectors To construct the LcNPY2R and LcNPY7R plasmids, reverse transcription PCR (RT-PCR) was performed as previously described(Wang et al., 2017). To amplify the full-length sequence encoding LcNPY2R and LcNPY7R, forward primers (LcNPY2R-vec-F or LcNPY7R-vec-F) and reverse primers (LcNPY2R-vec-R-EGFP, LcNPY7R-vec-R-EGFP, LcNPY2R-vec-R-FLAG, and LcNPY7R-vec-R-FLAG) were designed based on the full-length cDNA sequence to allow for subcloning into the pEGFP-N1 and pCMV-Flag plasmids, respectively (Supplementary Table 1). The pEGFP-N1 and pCMV-Flag vectors were purchased from Clontech Laboratories, Inc. (Palo Alto, CA), and Sigma (St. Louis, MO), respectively. The PCR products were inserted into the final pEGFP-N1 and pCMV-Flag expression vectors using the Hind III and BamH I restriction enzymes (Beyotime, Haimen, China) and a Rapid DNA Ligation Kit (Beyotime, Haimen, China). The resulting vectors were named LcNPY2R-EGFP, LcNPY7R-EGFP, Flag-LcNPY2R, and Flag-LcNPY7R, respectively, and sequenced to verify sequence fidelity, orientation, and reading frame.

2.8. cAMP accumulation measurement After seeding in a 96-well plate overnight, HEK293 cells were stably co-transfected with the Flag-LcNPY2R, Flag-LcNPY7R, and pCRE-Luc vectors and grown to 80–85% confluence. The cells were then treated with the indicated concentrations of LcNPY (1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM) in DMEM without FBS and incubated for 4 h at 37 °C; the experiments were repeated a total of three times. Luciferase activity was detected using a firefly luciferase assay kit (Kenreal, Shanghai, China).

2.5. Peptide synthesis

2.9. Real-time quantitative PCR (qRT-PCR)

The sequences of L. crocea mature neuropeptides were obtained from the genomic data. Their validity was verified by degenerate primer PCR and sequencing. Then, the NPY were synthesized by GL Biochem Shanghai (China) and purified by preparative reverse-phase high-performance liquid chromatography using a Dynamax-300 Å C18 25 cm × 21.4 mm ID column with a flow rate of 9 ml/min and two solvent systems of 0.1% TFA/H2O and 0.1% TFA/acetonitrile. Fractions containing the appropriate peptide were pooled together and lyophilized. The purity of the final product was assessed by analytical reversephase high-performance liquid chromatography, capillary electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of solid-phase synthesis and chemical modified peptide to activate its receptor has been verified by previous studies (Fredriksson et al., 2006; Yang et al., 2013).

Total RNA was extracted from the head kidney, spleen, stomach, gill, heart, liver, brain, muscle, ovary, and intestine tissues of L. crocea. Reverse transcription was conducted using a M-MLV reverse transcriptase cDNA Synthesis Kit (TaKaRa). Specific qRT-PCR primers for LcNPY2R, LcNPY7R, and Lcβ-actin were designed based on CDS (Supplementary Table 1). The primers were tested to ensure the amplification of single discrete bands with no primer-dimers. qRT-PCR assays were carried out using the SYBR PrimeScript™ RT reagent kit (TaKaRa, Kusatsu, Japan) according to the manufacturer's instructions, using ABI 7500 Software v2.0.6 (Applied Biosystems, UK). qRT-PCR was performed for 35 cycles with the following conditions: 95 °C/5 s, 60 °C/30 s. The relative levels of gene expression were calculated using the 2-ΔΔ Ct method and the data were normalized by the internal control gene. Differences among tissues for a given gene were tested using oneway analysis of variance (ANOVA) followed by Tukey's post hoc test, using PASW Statistics 18.00 (SPSS Inc., Chicago, IL, USA). Significance was set at p < .05, and an extremely significant difference was set at p < .01.

2.6. Cell culture, transfection, and confocal microscopy The human embryonic kidney cell line (HEK293) was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, China) and 4 mM L-glutamine (Invitrogen, CA). The LcNPY2R-EGFP and LcNPY7R-EGFP plasmid constructs were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen, CA) and X-tremeGene HP (Roche, Switzerland) according to the manufacturers' instructions. The selection for stable expression was initiated by the addition of G418 (800 μg/ml) 1–2 days after transfection. For receptor surface expression analysis, HEK293 cells expressing LcNPY2R-EGFP LcNPY7R-EGFP were seeded onto glass coverslips coated with 0.1 mg/ml of poly-L-lysine and allowed to attach overnight under normal growth conditions. After 24 h, the cells were stained with the membrane probe DiI (Beyotime, China) at 37 °C for 5–10 min, fixed with 4% paraformaldehyde for 15 min, and finally incubated with DAPI for 10 min. The cells were visualized by fluorescence microscopy using a Leica TCS SP5II laser scanning confocal microscope using an HCX PL

3. Results 3.1. Isolation, characterization, and phylogenetic analysis of L. crocea NPY2R and NPY7R The full-length 1462 bp LcNPY2R cDNA sequence cloned from L. crocea contains an open reading frame (ORF) that is 1224 bp in length and encodes 407 amino acids, a 5′ untranslated region (UTR) that is 102 bp in length, and 3′ UTR that is 136 bp in length (Fig. 1). The cDNA sequence was submitted to the NCBI GenBank (accession no. MH106713). One potential polyadenylation signal was identified within the 3′ UTR. The putative protein was predicted to have a molecular mass of 45.06 kDa and an isoelectric point (pI) of 9.11. The amino acid sequence of LcNPY2R contains the following potential sites: 3

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Fig. 1. LcNPY2R cDNA sequence and deduced amino acid sequence. The seven transmembrane domains (TM1-TM7) are indicated in black underline. The Nglycosylated sites are highlighted in gray. The phosphorylation sites are labeled in box with full lines. The initiation codon (ATG) and the termination codon (TGA) are shown in bold. The potential polyadenylation signal (AATAAA) is indicated by the double underscore. The numbers on the left refer to the position of the nucleotides and the amino acids.

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Fig. 2. LcNPY7R cDNA sequence and deduced amino acid sequence. The seven transmembrane domains (TM1-TM7) are indicated in black underline. The Nglycosylated sites are highlighted in gray. The phosphorylation sites are labeled in box with full lines. The initiation codon (ATG) and the termination codon (TGA) are shown in bold. The potential polyadenylation signal (AATAAA) is noted by the double underscore. The numbers on the left refer to the position of the nucleotides and the amino acids.

NPYRs from various other species, a phylogenetic tree was constructed with Mega 6.0 using ClustalW multiple alignment and the protein sequences of LcNPY2R and LcNPY7R with 28 alternate NPYRs obtained from the GeneBank (Fig. 5). This revealed that the NPYRs can be divided into three main groups: NPY2R/NPY7R, NPY1R/NPY4R/NPY6R, and NPY5R receptors. Initially, both LcNPY2R and LcNPY7R clustered with other fish NPY2Rs. Then, both the deduced LcNPY2R and LcNPY7R protein sequences were positioned alongside the Y2/Y7 subfamily receptors in the phylogenetic tree and clustered with other vertebrate NPYRs subfamilies.

two typical N-glycosylation sites within N-terminal (N8) and C-terminal (N403), two conserved cysteine residues (C116 and C216) within TM2 and EC2, and 33 phosphorylation sites at 20 serine (S26, S61, S82, S133, S150, S194, S204, S224, S231, S244, S255, S262, S270, S271, S274, S334, S368, S373, and S399), 11 threonine (T10, T93, T96, T134, T171, T217, T228, T283, T284, T308, and T394), and 2 tyrosine (Y230 and Y250) residues (Fig. 1). The predication of the 7tm_1 domain of this putative amino acid sequence indicated that LcNPY2R was a member of the rhodopsin-type (class A) GPCR family. The deduced amino acid sequence of L. crocea LcNPY2R was compared with four other homologous NPY2R sequences, which vary in length from 379 to 405 amino acid residues. Pairwise ClustalW analysis of these amino acid sequences was carried out to evaluate their homologous relationships. The full-length LcNPY7R cDNA sequence cloned from L. crocea was 2236 bp long and contained an 1116 bp open reading frame encoding 371 amino acid proteins, 104 bp of 5′ untranslated regions (UTR), and 1016 bp of 3’ UTR (Fig. 2). The full-length cDNA sequence was submitted to the NCBI GenBank (accession no. MN031135). The putative protein had a theoretical molecular weight of 42.10 KDa and a pI of 9.22. The LcNPY7R amino acid sequence contained several potential sites for modification: two N-linked glycosylation sites (N8 and N25) in N-terminal domain, two conserved cysteine (C99, C147) residues within the TM2 and IC2 domains, and 28 phosphorylation sites including 18 eight serine (S2, S7, S54, S129, S133, S160, S201, S209, S216, S218, S247, S249, S250, S254, S311, S342, S348, and S353), 7 threonine (T6, T138, T157, T208, T261, T262, and T358), and 3 tyrosine (Y235, Y301, and Y349) sites (Fig. 2). According to the predicted 7tm_1 domain of this putative amino acid sequence, LcNPY7R was classified as belonging to the class A GPCR family. The predicted L. crocea NPY2R amino acid sequence showed similarities with the Epinephelus coioides putative NPY2R sequence (89.0% identity), as well as other reported NPY2R amino acid sequences, albeit with lower levels of similarity (from 86.5% to 58.5% identity). The predicted L. crocea NPY7R amino acid sequence showed similarities with the Takifugu rubripes putative NPY7R sequence (89.2% identity), whereas a relatively low similarity was found with other species (from 81.2% to 74.5% identity). The multiple sequence alignment analysis reveals that the amino acid sequences of both LcNPY2R and LcNPY7R with other species' are very conserved in the 7 transmembrane domains but not in the N/C terminal region (Fig. 3). The LcNPY2R protein binding regions were analyzed, with two regions (2, 54–55 aa) predicted in the extra cellular N-terminal domain and one region (293 aa) predicted in the EC3 domain, which may be involved in ligand binding. Furthermore, protein binding regions (175 aa; 247, 257–259 aa) were also predicted in the IC2 and IC3 domains, respectively, which are thought to play key roles in the G protein coupling (Fig. 4B). As for LcNPY7R, nine sites were predicted for ligand binding (1–2, 4, 37, 40–41 aa in N-terminal; 110 aa in EC1, 187–188, 190, 201–204 aa in EC2; 292–296 aa in EC3). Six sites were predicted for intracellular protein binding and G protein coupling (155, 158 aa in IC2; 248–249, 256 aa in IC3; 337–342, 345–347 aa in C-terminal). The determined three-dimensional (3D) structure of the LcNPY2R and LcNPY7R proteins are shown in Fig. 4 and Supplementary Data 3. The protein structure of LcNPY2R and LcNPY7R was predicted using SWISSMODEL. Homology modelling revealed that this protein was similar to 5zbh.1.A in the Protein Data Bank (Fig. 4A, B). While the secondary structure was predicted by PredictProtein (Fig. 4C, D), the protein binding region sites were predicted and marked on the constructed model. To determine the relationship of LcNPY2R and LcNPY7R with

3.2. Genomic analysis of LcNPY2R and LcNPY7R Five exons and four introns were identified by comparing the LcNPY2R cDNA sequence to the corresponding genomic DNA fragments. The lengths of exons 1, 2, 3, 4, and 5 were 189, 133, 166, 154, and 582 bp (Palczewski et al., 2000), whereas introns 1, 2, 3, and 4 were 215, 7325, 1238, and 1141 bp, respectively (Fig. 6). As for LcNPY7R, only one exon and one intron were detected, which were 1116 bp and 3356 bp, respectively (Fig. 6). The comparison between the nucleotide sequences of LcNPY2R and NPY2R sequences from other teleosts revealed a high degree of similarity regarding the exon-intron organizations. As shown in Fig. 6A and B, each NPY2R precursor spanned five exons and four introns, and the location of exon/intron junction sites were conserved. Although slight variations existed among the ORF-encoding regions, the main differences were caused by the sizes of introns. Further analysis revealed that all of the ORF-encoding regions 2, 3, and 4 were the same size, that is 133, 166, and 154 bp, respectively. The intron of LcNPY7R was found at the 3’ UTR of ORF-encoding region, compared to the 5’ UTR intron of T. rubripes and D. rerio NPY7R. In addition, the length of these introns in different species varied greatly. 3.3. A rhodamine red-labeled LcNPY directly binds to and activates LcNPY2R and LcNPY7R To confirm the direct interaction of LcNPY peptides with the LcNPY2R and LcNPY7R receptors, we designed and synthesized an Nterminal rhodamine red-tagged LcNPY (Rho-LcNPY) peptide. The subsequent functional assays demonstrated that Rho-LcNPY could activate LcNPY2R and LcNPY7R to inhibit the forskolin-induced luciferase activity in HEK293 cells, with the respective IC50 values of 49.1 ± 0.27 nM and 13.2 ± 0.13 nM (Fig. 7A, C). Using a displacement analysis method, Rho-LcNPY was found to compete with unlabeled LcNPY, with a Ki value of 6.39 ± 0.21 nM (LcNPY2R) and 7.25 ± 0.19 nM (LcNPY7R) (Fig. 7 B, D), suggesting that LcNPY directly binds and activates LcNPY2R and LcNPY7R. 3.4. cAMP inhibition in LcNPY2R/LcNPY7R expressing cells stimulated by NPY Previous studies have shown that NPY2R and NPY7R are activated by NPY, resulting in cAMP inhibition in zebrafish (Fredriksson et al., 2004); however, the detailed signaling pathways remain to be elucidated. To examine LcNPY2R and LcNPY7R mediated G protein coupling and signaling, HEK293 cells were co-transfected with pFLAG-LcNPY2R/ pFLAG-LcNPY7R and a reporter gene system (pCRE-Luc vector) consisting of the firefly luciferase coding region under the control of cAMPresponse elements. As shown in Fig. 8B and C, upon stimulation with LcNPY (NPY) and the truncated LcNPY (NPY18–36) (Fig. 8A), LcNPY2R 6

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Fig. 3. Amino acid alignment of the Y2 and Y7 receptors from Larimichthys crocea together with the other NPYRs. LcNPY2R receptor compared with Epinephelus coioides NPY2R, Xiphophorus maculatus NPY2R, and Danio rerio NPY2R. LcNPY7R receptor compared with Takifugu rubripes NPY7R, Oncorhynchus mykiss NPY7R, and D. rerio NPY7R. The relative sequences were obtained from GenBank, along with a list of accession numbers (Supplementary Table 2). Alignment was generated using CLUSTAL W. The seven transmembrane domains (TM1–TM7) are indicated with a black horizontal line above the sequence alignment. The three extracellular (EC) and three intracellular (IC) domains are noted above the sequence alignment. Black triangles indicate conserved cysteine residues. A rectangular frame indicates potential glycosylation sites.

protein (EGFP) fused to the C-terminus. HEK293 cells were transiently transfected with the LcNPY2R-EGFP/LcNPY7R-EGFP vector and the resulting GFP signals were analyzed by confocal microscopy. Significant cell surface expression of LcNPY2R was detected under fluorescence microscopy (Fig. 9A), suggesting that the C-terminal GFP tag did not affect LcNPY2R expression. However, as for LcNPY7R, GFP signal was detected both on the membrane and in cytoplasm, which may be caused by overexpression. However, the internalization upon the activation of 1 μM LcNPY and a dramatic redistribution of the receptor in the cytoplasm with distinct perinuclear accumulation was observed for both LcNPY2R and LcNPY7R (Fig. 9B, C).

was activated to induce significant inhibition of intracellular cAMP, with IC50 values of 0.22 ± 0.17 and 1.67 ± 0.10 nM, respectively. LcNPY7R was activated, with an IC50 value of 0.655 ± 0.08 nM, by full length LcNPY, whereas a low affinity of IC50 87.85 ± 0.07 nM resulted from truncated LcNPY activation. (Fig. 8B, C). 3.5. Cellular localization and internalization of L. crocea neuropeptide Y receptor To analyze the possible sub-cellular localization of LcNPY2R and LcNPY7R, LcNPY2R-EGFP and LcNPY7R-EGFP vectors were constructed to express the respective receptors with enhanced green fluorescent 7

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Fig. 4. Predicted LcNPY2R and LcNPY7R protein structure and respective domains. Predicted 3D structure of the LcNPY2R (A) and LcNPY7R (B) protein. Proteinbinding regions are represented as black dots. Seven transmembrane domains (TM1–TM7), three extracellular (EC), and three intracellular (IC) domains are marked. The predicted 3D structure of the LcNPY7R protein was generated using SWISS-MODEL. LcNPY2R (C) and LcNPY7R (D) protein binding domain and transmembrane region. The dashboard overview was generated using PredictProtein. Red diamonds indicate protein binding sites. A purple rectangular frame indicates the transmembrane helix.

receptor family (Y3 has not been identified) (Larhammar et al., 2001) and lost the Y7 and Yb/Yc which belong to fish, Atlantic cod and rainbow trout (Arvidsson et al., 1998; Larson et al., 2003). Previous research has reported that Y1 and Y5 are also absent in zebrafish, T. rubripes, and Tetraodon nigroviridis (Larsson et al., 2006). Studies have proposed that Y7 originates from the duplication of Y2, whereby the two receptors would originate from a common ancestral gene through a chromosome duplication event (Larsson et al., 2006) (Fredriksson et al., 2004). Our results for the cloning and characterization of the L. crocea Y2 and Y7 receptors will help to further our understanding of the differences between fish and mammals. As for the phylogenetic tree, our results showed that LcNPY2R and LcNPY7R were first grouped with E. coioides and T. rubripes, respectively, then together with other fish, with the Y2 and Y7 groups individually and then combined with the Y2 subfamily. Afterwards, they were found to cluster with the Y1/Y4 and Y5 subfamilies. The Y2 and Y7 in the large yellow croaker share a sequence similarity of 47.8%. Moreover, the alignments clearly showed that LcNPY2R and LcNPY7R belong to the same subfamily and are separate from other neuropeptide Y receptor subtypes, in zebrafish and O. mykiss (Fredriksson et al.,

3.6. Expressional quantification of LcNPY2R and LcNPY7R mRNA qRT-PCR for LcNPY2R and LcNPY7R transcripts was performed in multiple tissues of an adult female large yellow croaker. The LcNPY2R gene was found to be highly expressed in the brain, but most notably in the digestive system, including the stomach, liver, and intestine. No expression or low levels were detected in the head kidney, spleen, gill, heart, muscle, and ovary (Fig. 10). 4. Discussion In the present study, L. crocea neuropeptide Y2 and Y7 receptors, both of which belong to the Y2 subfamily, were characterized. Neither Y1 nor Y5 are currently found in the published genomic data of the large yellow croaker (Wu et al., 2014). Previously, NPY receptors from several different mammalian species have been cloned and characterized pharmacologically, including chicken, teleost, and Chondrichthyes (Larhammar et al., 2004; Larhammar and Salaneck, 2004; Salaneck et al., 2003). Although the neuropeptide Y receptors are clustered in the Y1, Y2, and Y5 subfamilies, mammals have only five members of the Y 8

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intron organization of NPY2R and NPY7R indicated that the distribution of five exons of L. crocea Y2 are relatively similar with those of four other bony fish, while the L. crocea Y7 only has one exon, with its intron at the 3’ UTR, which is in contrast to the 5’ UTR intron of T. rubripes and zebrafish. Studies on the chromosomal organization of pufferfish, zebrafish, and chicken have provided further evidence of the duplication of Y2 and Y7 from a common ancestral gene through a chromosome duplication event (Larhammar and Salaneck, 2004; Larsson et al., 2008). Taken together, these findings suggest that Y2 is more conserved than Y7. The two large yellow croaker neuropeptide receptors both displayed typical GPCR features. For instance, both contain conserved seven transmembrane helix and loops, an N-linked carbohydrate in the Nterminal, and a conserved Cys anchoring the tail to the membrane in the C-terminal. The Y2 receptor has been previously identified in frogs, zebrafish, trout, chicken, and mammals, including human and rat (Gerald et al., 1995; He et al., 2016; Pjetri et al., 2012). The Y7 receptor was first found in the chicken (Bromee et al., 2006; He et al., 2016), and was then characterized in zebrafish, frog, T. rubripes, O. mykiss, and pufferfish (Larsson et al., 2005; Larsson et al., 2008; Sundstrom et al., 2013; Sundstrom et al., 2012). Bioinformatics was conducted to analyze the LcNPY2R/LcNPY7R 3D structure and predict its molecular structure. These sites are mainly involved in protein localization, function, activity, diversity, signal transduction, and gene expression (Parker and Balasubramaniam, 2008; Shi and Javitch, 2004; Sjodin et al., 2006). The L. crocea Y2 and Y7 receptors were functionally expressed in HEK293 cells and characterized in ligand binding studies. In 1986, the subtype of the Y-receptor family was described pharmacologically as a predominantly presynaptic NPY/PYY receptor (Wahlestedt et al., 1986). Here, the N-terminal rhodamine-labeled LcNPY was synthesized to compete with the wild type LcNPY, as fluorescence-labeled peptide binding assays proved to be valid in previous studies (Breen et al., 2016; Lewis and Daunert, 1999; Yang et al., 2013). Our results showed that both receptors could bind L. crocea NPY with affinities in the nanomolar range (FLAG-vector as the negative control), which is consistent with the results obtained for chicken, zebrafish and rainbow trout (Fallmar et al., 2011; Fredriksson et al., 2004; Fredriksson et al., 2006; He et al., 2016; Larsson et al., 2006). The most prominent pharmacological feature of the Y2 receptor is that it can bind several truncated peptide fragments. NPY or PYY lacking 13, 18, or even 22 amino acids at the amino terminal is still able to bind human, rat, and mouse Y2 with high

Fig. 5. Phylogenetic tree of LcNPY2R and LcNPY7R with 28 other NPYRs superfamily receptors amino acid sequences. The tree was generated based NJ algorithms using MEGA 5.0. The topological stability of the NJ tree was achieved by running 1000 bootstrapping replications. Bootstrap values (%) are indicated by the numbers at the nodes. The accession numbers are listed in Supplementary Table 2.

2006; Larsson et al., 2006). In addition, previous studies have revealed that an ancestral repertoire of Y receptors seems to have differentiated between mammals and teleost fishes primarily due to differential losses of receptor genes (Palczewski et al., 2000). A comparison of the exon-

Fig. 6. Comparison of the exon-intron organization of teleost NPY2R and NPY7R genes. The boxes and bars represent the exons and introns, respectively. The numbers above each line refer to the sizes (bp) of the corresponding exons or introns. The accession numbers for the genomic sequences are listed in Supplementary Table 2. 9

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Fig. 7. Direct interaction of LcNPY2R and LcNPY7R with LcNPY. N-terminal labeled rhodamine LcNPY activity was assayed using the CRE-driven luciferase system. LcNPY and rhodamine-labeled LcNPY (Rho-LcNPY) both induced the inhibition of the forskolin-stimulated cAMP increase in a ligand concentration-dependent pattern in HEK293 cells stably co-transfected with FLAG-LcNPY2R (A) and FLAG-LcNPY7R (C). Binding of Rho-LcNPY to LcNPY2R (B) and LcNPY7R (D) expressed in HEK293 cells were measured in the presence of different concentrations of unlabeled peptides or in the absence of unlabeled peptides (total binding). The extent of binding was determined by fluorescence intensity and is presented as a percentage of the total binding. The results represent the mean ± standard deviation (S.D.) (n = 3). All images and data are representative of at least three independent experiments.

confirmed that the sensitivity to N-terminal truncation of peptide ligands makes Y7 distinct from the mammalian and chicken Y2 receptors (Fredriksson et al., 2004). Thus, we proved that LcNPY2R and LcNPY7R expressed in HEK293 cells are functionally coupled to Gαi protein, and can lower forskolin-induced cAMP levels while regulating the cAMP/ PKA signaling pathway, which is consistent with the findings that all NPY receptors except Y6 are coupled to Gαi/cAMP pathway in mammals and chicken (He et al., 2016; Michel et al., 1998; Persaud and

affinity (Michel et al., 1998). However, Y7 could only bind to intact NPY, and not the N-terminal truncated peptides of zebrafish and rainbow trout. In the cAMP assay, both LcNPY2R and LcNPY7R could be activated by LcNPY, which decreased the intracellular cAMP, with an IC50 of 0.1–1 nM (red spots and lines). Meanwhile, LcNPY2R was able to respond to fragment LcNPY18–36 with IC50 of 1–10 nM, whereas the fragment LcNPY18–36 had an IC50 of 10–100 nM, which is much higher than that of full length LcNPY for LcNPY7R. These results

Fig. 8. LcNPY-induced cAMP inhibitation. (A) Amino acid sequence alignment of the full length of LcNPY (1–36 amino acids) and truncated LcNPY (18–36 amino acids: NPY18–36). HEK293 cells stably co-transfected with FLAG-LcNPY2R/FLAG-LcNPY7R and pCRE-Luc were stimulated with various ligands (final concentration 1 μM). (B)LcNPY and LcNPY18–36 inhibited the forskolin-stimulated increase in cAMP in a ligand concentration-dependent pattern in HEK293 cells stably cotransfected with FLAG-LcNPY2R. (C)LcNPY inhibited the forskolin-stimulated increase in cAMP in a ligand concentration-dependent pattern in HEK293 cells stably co-transfected with FLAG-LcNPY7R, whereas LcNPY18–36 had a relatively high IC50 value. All data shown are representative of at least three independent experiments and were analyzed using the Student's t-test (*, p < .05, **, p < .01, ***, p < .001). 10

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Fig. 9. Confocal microscopy of HEK293 cells expressing the LcNPY2R- and LcNPY7R-EGFP fusion protein. (A)LcNPY2R distribution in HEK293 cells. Cells were stained with a membrane plasma probe (DiI) and a nuclei probe (DAPI). Cells stably expressing LcNPY2R-EGFP/LcNPY7R-EGFP were seeded onto glass-bottom 6-well plates overnight, incubated with DiI (10 μM) and DAPI, and examined by confocal microscopy, as previously described. (B) Internalization of LcNPY2R-expressing cells. (C) Internalization of LcNPY7R-expressing cells. HEK293 cells transfected with LcNPY2R-EGFP/LcNPY7R-EGFP were activated by treatment with 1 μM LcNPY during 30 min and detected by confocal microscopy. “DMEM” denotes the control without LcNPY stimulation. All images represent at least three independent experiments.

cell surface expression was observed by fluorescence microscopy, suggesting that the C-terminal EGFP tag did not affect L. crocea Y2 and Y7 expression of their orientation in the cell membrane of HEK293 cells. These results also coincide with the subcellular distribution found in the orange-spotted grouper, where two neuropeptide receptors were found to be localized on the cell membrane (Wang et al., 2014). Internalization is one of the predominant mechanisms that control GPCR signaling

Bewick, 2014; Salaneck et al., 2000). Green fluorescent protein (GFP) has been widely used to study the localization, distribution, and function of proteins via fusion expression in different systems. In this study, we constructed a chimeric protein in which EGFP was fused to the C terminus of LcNPY2R and LcNPY7R (LcNPY2R-EGFP and LcNPY7R-EGFP, respectively), which was stably expressed in HEK293 cells and exhibited functional activity. Significant

Fig. 10. Relative expression of LcNPY2R/LcNPY7R mRNA in different tissues (head kidney, spleen, stomach, gill, heart, liver, brain, muscle, ovary, and intestine). The expression value was normalized against the expression of the internal control gene (β-actin). Each vertical bar represents the mean ± S.D. (n = 6). Different lowercase letters above the bars indicate significant differences (p < .05) between different tissues. 11

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Acknowledgments

and which ensures appropriate cellular responses to stimulation. Observation using confocal microscopy revealed that the fluorescence of LcNPY2R-EGFP and LcNPY7R-EGFP was dramatically and rapidly internalized into the cytoplasm in HEK293 cells in response to treatment with 1 μM of LcNPY of 30 min. These results revealed an interaction between LcNPY and LcNPY2R/LcNPY7R. Our study clearly showed that both the LcNPY2R and LcNPY7R mRNA transcripts are ubiquitously expressed in the digestive system and brain. Y2 was highly detected in the liver, intestine, stomach, and brain, while Y7 was also present in the gills and showed a relatively wide distribution. The Y2 subfamily receptors are one of the most abundant receptors in the central nervous system (CNS) and are widely expressed in hypothalamus, hippocampus, brainstem, amygdala, and lateral septum (Parker and Balasubramaniam, 2008). In the central nervous system, the interaction between stress and food intake is regulated by neuroanatomical pathways in the hypothalamus, in which NPY plays a key role, which may explain why Y2 and Y7 are expressed at relatively high levels in theL. crocea brain and intestine, known to be involved with food intake regulation and body weight (Reichmann and Holzer, 2016). These findings are consistent with previous findings in mammals where Y2 was reported to be widely expressed in various regions of the CNS and was implicated with the inhibition of food intake. A previous study inactivated the Y2 receptor subtype in mice and found that the body weight, food intake, and fat deposition increased in the mice (Naveilhan et al., 1999). Other studies have also demonstrated that the NPY/NPYR system is involved in the regulation of food intake in teleosts (de Pedro et al., 2000; Li et al., 2017). Our findings are consistent with the predominant expression of Y2 in brain and liver in rainbow trout and orange-spotted grouper (Larsson et al., 2006; Wang et al., 2014). Y7 is also mainly present in the brain and intestine of zebrafish and rainbow trout (Fredriksson et al., 2004). However, in chicken, the Y2 receptor was detected in all organs except the liver and gizzard, and the Y7 receptor was exclusively observed in the adrenal gland (Bromee et al., 2006). These differences are most likely due to species differences, and suggest that the Y2 subfamily may mediate a broad spectrum of actions, including feeding, growth, and homeostasis. In conclusion, this study investigated the molecular, bioinformatic, and cellular biology characteristics of the Y2 and Y7 receptors in L. crocea. Our results demonstrated that the two receptors belong to the Y2 subfamily and have the typical G protein-coupled receptor 7 transmembrane structure. The TM domains of both receptors are conserved in other species. While the N-terminal and C-terminal showed the most sequence variation, both LcNPY2R and LcNPY7R were expressed on the cell membrane of the HEK293 cells. Moreover, both were internalized in the cytoplasm upon LcNPY stimulation. The binding properties showed that both receptors bind to LcNPY with affinities in the nanomolar range. LcNPY was able to activate both Y2 and Y7, leading to the intracellular cAMP inhibition in a dose-dependent manner in HEK293 cells. However, truncated LcNPY18–36 could only active LcNPY2R with an IC50 comparable to that of full length mature NPY, which could not be detected in LcNPY7R. Hopefully, the cloned receptor and constructed expression vector described here can be used to develop subtype-selective ligands, agonists, and antagonists for functional studies in vivo. On one hand, these results improve our understanding of the fish NPYR signaling pathway, while, on the other hand, enriching the exploration of the physiological roles of NPY/NPYR system for the aquaculture and farming of the large yellow croaker. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpb.2019.110347.

The authors of this paper would like to thank Prof. Jiayan Xie for their technical assistance and equipment. Funding This work was supported by the National Nature Science Foundation of China [grant number 41606150, 41876154], the Technical Applied Research Project of Zhoushan [grant number 2017C41010] and the Fundamental Research Funds for Zhejiang Provincial Universities and Research Institutes [grant number 2019JZ00007]. References Abrahamsson, C., 2000. Neuropeptide Y1- and Y2-receptor-mediated cardiovascular effects in the anesthetized guinea pig, rat, and rabbit. J. Cardiovasc. Pharmacol. 36, 451–458. Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 22, 195–201. Arvidsson, A.K., Wraith, A., Jonsson-Rylander, A.C., Larhammar, D., 1998. Cloning of a neuropeptide Y/peptide YY receptor from the Atlantic cod: the Yb receptor. Regul. Pept. 75-76, 39–43. 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Declaration of Competing Interests The authors declare no conflict of interests.

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