Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32

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Comparative Biochemistry and Physiology, Part A

journal homepage: www.elsevier.com/locate/cbpa

Characterization of LPXRFa receptor in the half-smooth tongue sole T

(Cynoglossus semilaevis): Molecular cloning, expression profiles, and
differential activation of signaling pathways by LPXRFa peptides
Bin Wang , Guokun Yang , Quan Liu , Jingkai Qin , Yongjiang Xu , Wensheng Li ,
a,b,1 c,1 a,d,1 c a,b c

Xuezhou Liu a,b,

⁎ , Bao Shi
a,b

a
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,
Qingdao 266071, China
b
Laboratory for Marine Fisheries and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China
c
State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, South China Sea Bio-
Resource Exploitation and Collaborative Innovation Center, Research Institute of Sun Yat-Sen University in Shen Zhen, School of Life Sciences, Sun Yat-Sen University,
Guangzhou 510006, China
d
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

ARTIC LE INFO ABSTRA CT

Keywords: Gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide, serves as a key player in the
GnIH regulation of reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive phy-
LPXRFa siology and behavior. However, little information is available in teleosts regarding the intracellular signal
LPXRFa receptor transduction pathway in response to GnIH. To this end, we first cloned the gene of LPXRFa (the piscine ortholog
Signaling pathway of GnIH) receptor in the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order
Cynoglossus semilaevis
Pleuronectiformes. The full-length cDNA of LPXRFa receptor was 2201 bp in size with an open reading frame
(ORF) of 1365 bp that encoded 454 amino acids. Tissue distribution showed that LPXRFa receptor transcripts
could be detected at high levels in the brain, to a lesser extent in the pituitary, and at low levels in the ovary and
other peripheral tissues. In vitro functional analysis revealed that putative tongue sole LPXRFa-1 and LPXRFa-2
peptides significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity in COS-7
cells transfected with the novel receptor, and these stimulatory effects were evidently reduced by two inhibitors
of the PLC/PKC pathway. In addition, neither LPXRFa-1 nor LPXRFa-2 altered the cAMP-responsive element (CRE)-
luc activity, but only LPXRFa-2 could markedly decrease forskolin-induced CRE-luc activity in COS-7 cells
expressing its cognate receptor. Taken together, our results encompass the first study reporting the existence of
LPXRFa receptor in the order Pleuronectiformes and provide novel evidence of differential activation of sig-
naling pathways by LPXRFa peptides in fish.

1. Introduction characteristic -LPXRFa (X = L or Q) motif at their C-terminus and are

In 2000, a novel hypothalamic dodecapeptide with RFamide at the
terminus was isolated from the brain of the Japanese quail and
termed gonadotropin-inhibitory hormone (GnIH) based on its ability to
directly inhibit gonadotropin release from cultured anterior pituitary
tissue (Tsutsui et al., 2000). Subsequently, GnIH orthologs have been
further detected in a variety of other vertebrates from fish to humans
(Munoz-Cueto et al., 2017; Ogawa and Parhar, 2014; Tsutsui et al.,
2015; Ubuka et al., 2016). Depending on species, the GnIH precursor is
possibly cleaved into two to four mature peptides, which possess a
designated as LPXRFa peptides from a structural perspective (Munoz-
C- Cueto et al., 2017; Ogawa and Parhar, 2014; Tsutsui et al., 2015; Ubuka
et al., 2016). In addition, the receptor for LPXRFa (LPXRFa-R) was
identifi ed as GPR147, a member of the G protein-coupled receptor
(GPCR) superfamily, and only one form of LPXRFa-R has been found in
mammals and birds (Munoz-Cueto et al., 2017; Ogawa and Parhar,
2014; Tsutsui et al., 2015; Ubuka et al., 2016). However, different si-
tuations have been observed concerning the number of paralogous
LPXRFa-R genes in teleosts. Three LPXRFa-R genes have been identified
in zebrafish (Zhang et al., 2010), goldfish (Qi et al., 2013), and common

⁎
Corresponding author at: Yellow(X.
E-mail address: liuxz@ysfri.ac.cn SeaLiu).
Fisheries Research Institute, Chinese Academy of Fishery Sciences, No.106 Nanjing Road, Qingdao 266071, China.
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.cbpa.2018.05.008
Received 28 January 2018; Received in revised form 19 April 2018; Accepted 3 May 2018
Available online 07 May 2018
1095-6433/ © 2018 Elsevier Inc. All rights reserved.
B. Wang et al.
Table 1
Information of primers used in this study.
Primer name Primer sequence(5′-3′)
LPXRFa-R F1 ACCTCCTGGTGGGCATCT
LPXRFa-R R1 CTTCTGTTGTTATTAGCCGTGT
LPXRFa-R F2 CTGCAGTTCTGTTCGCTCACATCTACCTGG
LPXRFa-R R3 ACTGCTAACACCCAGATAAA
LPXRFa-R R4 ACCAAGGTGAAGACCGAGGC
UPM (long) CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
UPM (short) CTAATACGACTCACTATAGGGC
LPXRFa-R-orf-F GCTCTAGAATGGAGTTACTGGATGGAAT
R plasmid LPXRFa-R-orf-R CCCAAGCTTTCAGTTTTCCCACGCCTGAT
LPXRFa-R F5 GCTTTTCATGTTGTCCTGGTTG
LPXRFa-R R5 GGGTTGATGCTTGAGTTGGAG
18S-F GGTCTGTGATGCCCTTAGATGTC
18S-F AGTGGGGTTCAGCGGGTTAC
carp (Peng et al., 2016), while only one type of LPXRFa-R gene was
obtained from grass puffer (Shahjahan et al., 2011), Nile tilapia (Biran
et al., 2014), orange-spotted grouper (Wang et al., 2015), and cin-
namon clownfish (Choi et al., 2016).
The involvement of the LPXRFa system in the regulation of fish
reproduction was evaluated, but the exact physiological role of LPXRFa
is still uncertain in teleosts (Munoz-Cueto et al., 2017; Ubuka and
Parhar, 2018). A recent study performed in Senegalese sole showed that
intramuscular injection of LPXRFa-3 provoked a significant reduction in
brain gonadotropin-releasing hormone 3 (gnrh3) expression (Aliaga-
Guerrero et al., 2017), and zebrafish LPXRFa-3 reduced gnrh3 transcript
levels in brain slices of zebrafish (Spicer et al., 2017). Similarly, in-
traperitoneal (ip) injection of goldfish LPXRFa-2 and LPXRFa-3 down-
regulated hypothalamic gnrh3 mRNA levels in the same species (Qi
et al., 2013), but stimulatory effects of grouper LPXRFa-3 on gnrh3
expression have been reported in ip-injected orange-spotted grouper
(Wang et al., 2015). Interestingly, the effects of LPXRFa-2 on gnrh2
expression in the brain of male sea bass appear dependent of the route
of administration because intracerebroventricular (icv) injection sea
bass LPXRFa-2 reduced gnrh2 mRNA levels, whereas intramuscular in-
jection of LPXRFa-2 increased gnrh2 expression (Paullada-Salmeron
et al., 2016a; Paullada-Salmeron et al., 2016b). Notably, the effects of
LPXRFa on fish pituitary hormone synthesis and secretion also seem to
differ between species. For example, goldfish LPXRFa-1, −2, and −3
peptides stimulated the release of luteinizing hormone (LH), follicle-
stimulating hormone (FSH) and growth hormone (GH) from cultured
pituitary cells of sockeye salmon (Amano et al., 2006). Similarly, both
in vivo and in vitro studies revealed that tilapia LPXRFa-2 induced a
signifcant increase in LH and FSH secretion (Biran et al., 2014), and
Cichlasoma dimerus LPXRF-2 could act as an FSH-releasing factor in
intact pituitary cultures of this cichlid fish (Di Yorio et al., 2016).
Likewise, goldfish LPXRFa-1 enhanced lhβ, fshβ, gh and prolactin (prl)
gene expression in grass puffer pituitary in vitro (Shahjahan et al., 2016;
Shahjahan et al., 2011). On the other hand, ip injection of zebrafish
LPXRFa-3 reduced the plasma LH levels in goldfish (Zhang et al., 2010),
and Cichlasoma dimerus LPXRFa-1 also inhibited LH and FSH release in
intact pituitary cultures (Di Yorio et al., 2016). Ip injection of goldfish
LPXRFa-2 and LPXRFa-3 suppressed fshβ mRNA levels in the pituitary
gland of goldfish, while only LPXRFa-2 was able to decrease lhβ ex-
pression significantly (Qi et al., 2013). Moreover, orange-spotted
grouper LPXRFa-2 decreased lhβ mRNA levels in the pituitary gland of
grouper in vivo (Wang et al., 2015), and zebrafish LPXRFa-3 down-
regulated lhβ and common α subunit expression in vitro (Spicer et al.,
2017). It should be noted that goldfish LPXRFa-3 exerted both stimu-
latory and inhibitory effects on pituitary LH release and gonadotropin
subunit mRNA levels depending on maturational status and adminis-
tration route (Moussavi et al., 2012, 2013). Altogether, this diversity in
actions could be related to the species, the sex of the animals, the
physiological status, the route, and the time elapsed after
24
Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32
Purpose
Partial CDS
3′RACE
5′RACE, 1st PCR
5′RACE, 2nd PCR
Universal primers
Construction of pcDNA3.1-LPXRFa-
qPCR
administration of the LPXRFa peptide.
Despite its functional significance, the molecular mechanism of
LPXRFa action in the target cells has not been fully elucidated and is
just emerging in fish (Munoz-Cueto et al., 2017; Ubuka et al., 2016). In
mammals, LPXRFa reduced forskolin-induced cAMP production in CHO
cells expressing its cognate receptor, suggesting that LPXRFa-R may
couple to G protein and inhibit adenylyl cyclase (AC) activity
αi
(Fukusumi et al., 2001; Hinuma et al., 2000). Similarly, activation of
chicken and orange-spotted grouper LPXRFa-Rs also inhibited the for-
skolin-evoked cAMP response (Shimizu and Bedecarrats, 2010; Wang
et al., 2015). However, Nile tilapia LPXRFa-R was most likely coupled
to G protein (Biran et al., 2014). On the other hand, tilapia LPXRFa-2
αs
increased SRE-luc activity in COS-7 cells expressing its cognate receptor
(Biran et al., 2014). In contrast, grouper LPXRFa-1 suppressed SRE-luc
activity in COS-7 cells transfected with grouper LPXRFa-R (Wang et al.,
2015). Most recently, all three zebrafish LPXRFa peptides activated
LPXRFa-R2 and LPXRFa-R3 via the PKA/cAMP pathway (Spicer et al.,
2017). However, no dose response of the SRE pathway was detected by
any of the three LPXRFa peptides with any of the three LPXRFa-Rs in
this species (Spicer et al., 2017).
Half-smooth tongue sole is an economically important marine flat-
fish that is widely cultured in China. In nature, the body length of
mature females is twice larger and the body weight is over six times
greater than those of mature males, exhibiting a sexual dimorphism of
growth (Wang et al., 2017a). In our preliminary study, the full-length
cDNA sequence of LPXRFa encoding two putative LPXRFa peptides was
obtained from the brain of tongue sole and the variations of LPXRFa
mRNA during ovarian maturation were examined (Wang et al., 2018).
Identification of LPXRFa receptor is essential to understand the mode of
action of LPXRFa and further studies on LPXRFa and its receptor will
give insight into the physiological significance of LPXRFa in teleosts.
Accordingly, the aims of this study were to (1) obtain the full-length
cDNA sequence of LPXRFa-R, (2) study the expression profiles of
LPXRFa-R in various tissues, and (3) confirm the interaction of LPXRFa
peptides with LPXRFa-R, and analyze the intracellular signal trans-
duction pathways via LPXRFa-R.
2. Materials and methods
2.1. Animals
All of the animal experiments were approved by the Animal Care
and Use Committee of the Chinese Academy of Fishery Sciences.
Approximately 2-year-old female tongue sole were purchased from a
local fishery in Qingdao, China. The fish were reared in an indoor
concrete tank with recirculating seawater at room temperature under a
cyclical light–dark photoperiod (12 h:12 h). The fish were fed to satia-
tion twice daily with a commercially available dry diet (Shengsuo
Aquafeed Co., Ltd., Yantai, China).
B. Wang et al. Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32
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25 Fig. 1. Nucleotide sequence and deduced amino acid sequence of the cDNA
encoding LPXRFa-R in the half-smooth tongue sole. The ORF is indicated by
capital letters, and the 5′- and 3′-UTRs are indicated by lower-case letters. The
start codon is boxed, and the stop codon is indicated by an asterisk. Putative
seven hydrophobic TM domains are underlined. Putative N-linked glycosylation
and protein phosphorylation sites are indicated by diamonds (a CBS prediction
server score > 0.50) and circles (score > 0.90), respectively.

2.2. Molecular cloning of tongue sole LPXRFa receptor cDNA

The fish were anesthetized with 0.05% MS222 (Sigma, USA) and
decapitated. Total RNA was extracted from the brain of tongue sole
using the RNAiso Plus reagent (Takara, Dalian, China) according to the
manufacturer's protocol. The first strand cDNA was synthesized using
the PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian,
China) as follows: one microgram of total RNA was incubated with
gDNA Eraser at 42 °C for 2 min to eliminate contaminating genomic
DNA, and then reverse transcription was performed in a volume of
20 μL of the reaction mixture containing 1 μL of PrimeScript RT Enzyme
Mix I, 1 μL of RT Primer Mix, 4 μL of 5 × PrimeScript Buffer, and 4 μL of
RNase-free water. The reaction condition of reverse transcription was
37 °C for 15 min, followed by 85 °C for 5 s. A partial cDNA fragment was
obtained by PCR using primers (Table 1) designed based on the con-
served regions of LPXRFa-R sequences from other teleost species al-
ready published. PCR amplification was performed with the Re-
combinant Taq DNA Polymerase mix (Takara, Dalian, China) and PCR
conditions were as follows: denaturation at 95 °C for 5 min, followed by
35 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 70 s, and a final
incubation for 10 min at 72 °C. PCR products of expected sizes were
isolated, purified and subcloned into the pEASY-T1 vector (TransGene
Biotech, Beijing, China) for DNA sequencing. Based on the nucleotide
sequence obtained, new primers were synthesized and 3′/5′ RACE PCRs
were conducted to obtain the full-length cDNA sequence using the
SMARTer® RACE Kit according to the manufacturer's instructions
(Clontech, USA).
Sequence alignment of the LPXRFa-R was performed using Clustal
Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and was modified
manually. The NetPhos 2.0 Server (http://www.cbs.dtu.dk/services/
NetPhos/) was used to analyze the phosphorylation sites and the
NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) was
used to examine the N-linked glycosylation sites. Hydrophobic trans-
membrane (TM) domains were predicted with TMHMM 2.0 Server
(http://www.cbs.dtu.dk/services/TMHMM/). Phylogenetic analysis of
the LPXRFa-R sequences was performed using Mega6 software (Tamura
et al., 2013). Sequences were aligned by ClustalW, and neighbor-joining
trees were constructed using the Poisson model with 1000 bootstrap
replicates.

2.3. Tissue distribution of tongue sole LPXRFa receptor mRNA by real-time

PCR

To detect the tissue distribution of LPXRFa receptor mRNA in

tongue sole, three females (body weight = 730.9 ± 96.3 g and gona-
dosomatic index, the percentage of gonad weight to body weight,
=1.08 ± 0.10%) were anesthetized and decapitated. Tissue samples of
the brain, pituitary, gill, heart, liver, spleen, kidney, stomach, intestine,
muscle, and ovary were quickly collected and snap-frozen in liquid
nitrogen. RNA extraction and reverse-transcription were performed as
mentioned above. Gene expression levels were determined with quan-
titative real-time PCR according to the standard procedures established
in our lab (Wang et al., 2016; Xu et al., 2017). In brief, a total of 20 μL
of PCR reaction volume contained 10 μL of 2 × SYBR® Premix Ex Taq II
(Takara, Dalian, China), 0.8 μL of forward and reverse primers (10 μM
each), 2 μL of 10-fold diluted cDNA templates and 7.2 μL of water. The
amplification of samples was carried out with the Mastercycler® ep
B. Wang et al. Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32

TM1 TM2 TM3

zebrafish 1 ------------------MDTM------------TLNHSAANLSWQNCTLLPYYIHSAGMAVSYILSYLLVLLLCVVGNGLVCLVVIRNRNMRSVTNLFILNLAVSDLLVGIFCVPTTLIDSLISGWPFSQITCTMSNLVQGMSVSASVFTLVAIAVDRFTGIVYPFHHRLRPVTALFAI
zebrafish 2 -------MAGEPGEMETSQELTTLDMN--ISLSNAYTNNSNATNHSSITYYPYYQHSLPVAAALTLAYLFIFLLCMVGNGLVCLIVLENRRMRTVTNLFILNLAVSDLLVGVFCIPTTLVDNLITGWPFTNTVCKMSGLVQGMSVSASVFTLVAIAVDRFRCIVYPFQPKLTLLVAKVTI
zebrafish 3 -------MEG--SETVGLWADISICVF-SNIHCTNQTNVTNSSTMAGIILSPYYQHSLPTAALFSLAYLFIFLLCLMGNALVCVIVLRNRHMRTVTNIFILNLAVSDLLVGIFCIPTTLVDNLITGWPFSNTVCKLSGLVQGMSVSASVFTLVAIAVDRFRCIVYPFKPKLTLFIAKVSI
goldfish 1 ------------------MDLNFF--YSENIS---INHSMLFNDSRNLTFLPFYQHSLAVASVIILAYVLIFSLCMLGNILVCFIVLKNRQMRTVTNIFILNLAISDLLVGILCLPITLVDNLITGWPFDVVICKLSGLVQGASVSASVFTLVAIAVERFRCIVYPFQQKLTRRQAIITI
goldfish 2 -------MAEEPAEIEMSQELSTLYLN--SSLQNDYINNSNVTNHTGITYYPYYQHSLPVAAALTMAYLFIFLLCMVGNGLVCLIVLENRRMRTVTNLFILNLAVSDLLVGVFCIPTTLVDNLITGWPFTNTVCKMSGLVQGMSVSASVFTLVAIAVDRFRCIVYPFQPKLSLLVAKVTI
goldfish 3 -------MEA--S--EESWDTSSVYIISDLVSCTNQTNSTNSSTAGGDVLFPYYQHSLPTAALFTLAYLFIFLLCLMGNALVCVIVLRNRHMRTVTNIFILNLAVSDLLVGIFCIPTTLVDNLITGWPFSNTICKLSGLVQGTSVCASVFTLVAIAVDRFRCIVYPFKPKLTLFVAKVSI
grass puffer MEGVEASEGLEGLELEGSTTTGTVN-----------F--STDAGAINITFYPYYQHSVYVAASFVLAYFSIFLLCMVGNILVCLIVVENRHMRTVINLFILHLAVSDLLVGIFCIPTTLVDNLITGWPFSKIVCKMSGFVQGVSVSASVFTLVAIAVERFGCIVYPPHPKPTICVAQAAI
tiger puffer MEGVEASEGLEGLELEGSTTTGTVN-----------I--STDAGAINITFYPYYQHSVYVAASFVLAYFSIFLLCMVGNILVCLIVVENRHMRTVINLFILNLAVSDLLVGIFCIPTTLVDNLITGWPFSKIVCKMSGFVQGVSVSASVFTLVAIAVERFRCIVYPLHPKPTICVAQAAI
Nile tilapia -MDILDVVDEDGMAFEGSIITSV-LL---------NTSLLGVSNVTNITFFPYYQHSLYVAAIYTLAYSFIFLLCMVGNILVCLIVLENRRMRTVTNLFILNLAISDLLVGIFCIPTTLVDNLITGWPFSNTVCKMSGFVQGVSVSASVFTLVAIAVERFRCIVYPLQPKLTVFVAKLAI
grouper -MEILDSMGEEGMELEGSAATAA-TV---------NTSLDGVTNITNITFSPYYRHSLYVAATYILAYFFIFLLCMVGNILVCLIVLENRHMRTVTNLFILNLAISDLLVGIFCIPTTLVDNLITGWPFSNTVCKMSGFVQGMSVSASVFTLVAIAVERFRCIVYPLQPKTTILVANAAI
tongue sole -MELLDGMMGQEMEFEGSATAAAHKK---------NISTDIVTNVTNVTFSPYYQHSLYVAASYILAYFFIFLLCMVGNILVCLIVLENHRMRTVTNLFILNLAISDLLVGIFCIPTTLVDNLITGWSFSNTVCKMSGFVQGVSVSASVFTLVAIAVERFRSIMYPLQPKATVLVAKLAI
*:* ** * ::* :: **::** ***.:*:.*:.**:* *:***.**:******::*:* **:*.**:** * *.:* :*** **.***********:** *:** : : * :*
TM4 TM5 TM6 TM7
zebrafish 1 VFIWLLAFAIIFPSAATLTVIHLDDMYMVQND---QIYPLFVCFEDWPRADMRRVYTTVIFVHVYLAPLGLISIMYGCIAAKLSSNLQEN------------------------
RLRSRRRMKVIKMLIMVAVLFMVSWLPLWTLMLLTDYQDLDRQQIDFLSSYLFPVAHWLAFFNSGV zebrafish 2
VMIWVLAVVIMCPSAVTLTVERVEHHYMVRGEDYNHTYPLFSCFENWANPQMRKVYTTVLFAHIYLIPLTLITLMYGRIGIKLYTTSVISGNDQHDGGQPHTSPQAPGGQQEGRPLISQKKIKVIKMLSIVALLFTISWLPLWTLMLLTDYGGLNEDELELLSGYVFPFAHWLAFSNSSV zebrafish 3
GTIWLLALTIMFPSVLMLTVEQERAHFMIYNDDYNNTYPLYSCYETWPDPEMRKIYTTVLFIHIYVIPLALIMLMYGRIGAKLYSAAVSEHAN----A------------------QGKRKIRVVKMLIMVALLFMLSWLPLWTLMMLTDYGHPDNDSLEILTSYIFPLSHWLAFSNSSV goldfish 1
AFIWALAVSIMCPSAVTLTVSQDVLHFTVDGD--NETHPLYTCWEAWPDQTMRKIYTTVLFSHIYLAPVTLIFIMYSRIAVRLVKPP-----------------------------VFRRKLRVVNMLLMVALLFAVSWLPLWILMMLTDYGNLSAAQLDLVAVYVFPFAHWLAFFNSSV goldfish 2
VMIWVLAVVIMCPSAVTLTVERVEHHYMVHNEDYNHTYPLFSCFENWANPEMRKVYTTVLFAHIYLIPLTLITLMYGRIGIKLYTTSVISGNDQHESRQPHVSPPAPGVQQEVRPLISQKKIKVIKMLSVVALLFTLSWLPLWTLMLLTDYGGLNEEELDLLSGYVFPFAHWLAFSNSSV goldfish 3
GMIWVLALTIMFPSVLMLTVQQEKGHVMVHGD--NSTYPLYSCYETWPDPEMRKVYTTVLFLHVYVIPLALIMLMYGRIGAKLYATAVLTRAEQPDVP------------------ASHRKIRVIKMLMVVAVLFMLSWLPLWTLMLLTDYARPDEDSLELLTSYVFPLSHWLAFSNSSV grass puffer
LLIWVLAVAIMCPAALALTVEQIPNHYIIYNDDLNHMLPIYSCYENFANPRMTKVYTVVLFVHIYLVPLTVITIMYVSIGVKLCSSVLANREPPVA-----DGTVEVRERRGGQPMISRKKLMVIKMLILVALLFMLSWLPLWTLMMMADYAGLDSDQVDLLTSYIFPFAHWLAFANSSI tiger puffer
LLIWVLAVAIMCPAALALTVEQIPNHYIIYNDDLNHTLPIYSCYENFANPRMTKVYTVVLFVHIYLVPLTVITIMYVSIGVKLCSSVLANREPPVA-----DGTVEVRVRRGGQPMISRKKLMVIKMLILVALLFMLSWLPLWTLMMMADYAGLDSDQVDLLTSYIFPFAHWLAFANSSI Nile tilapia
ISIWVLAVAFMCPAAVALTVEKVPYHYMIYNNNFNHTLPWYTCYENFANPRMRKVYTVVLFAHIYLLPLTVISLMYISIGVKLCSSVFANKEPHLA-----NATVQVGSRRHGHPRVSRKKIKVIKMLIVVALLFMLSWLPLWTLMMITDYADLDGNQLDLLTSYIFPFAHWLAFSNSSV grouper
VLIWVLAVVMMCPAAVALTVEEVPYHYILYNDDFNHTLPLYTCYENFANPQMRKVYTTVLFAHIYVVPLAIITLMYGSIGGKLCFSVVANREPQLA-----NATVQAGGRRGGHPTISQKKIRVIKMLIVVALLFMLSWLPLWTLMMMTDYAGLDRDQLDLLANYIFPFAHWLAFSNSSV tongue sole
FFIWVLAVAMMCPAVMALTVKKFPSHYIVYDDDFNHTLPVYMCYENFDKPQMRKIYTAVLFAHIYLVPLTLITVMYGCIGIKLCTSVVPNREPQLD-----NATVAVG-RRAPQPMISQKKIKAIKMLTLVALLFMLSWLPLWTLMMITDYAGWDMDQLDLLTSYMFPFAHWLAFSNSSI
** **. :: *:. *** . : : * : *:* : * ::**.*:* *:*: *: :* :** *. :* :::: .::** :**:** :****** **:::** . .::::: *:**.:***** **.:

zebrafish 1 NPIIYGFFNENFRRGFQAAVACGFCSSVAS-EM----------------------------RHTHFVLPPPN-KVSDGSRGVTSGRK-----------ERCFPVIPHGAAHGIQG----ILL------EDMNG-
TVASHRVLGAWME zebrafish 2 NPIIYGYYNENFKRGFQAVCRAHSCCFEGM-TA----------RRGMRRKARG-ETRDPAVNSNPLNFGARN-RVYTDGD-
LKNSGTCLELEHRRTGRLCNNSVCSNETGSSVGSGIKGVTNQKAFQMEEAEKKSPIRGSKNQAWDQ zebrafish 3 NPIIYGYFNENFKRGFQAVFQRQACFRRRR-KM----------RFRVKRPRQG-CSPL-----
NTGVLGSKTNRIFTESD-L-TGCVRLELEHRRASTIENKA----------EGGNSGSSGRREIHFEEIEKVSSVGGKIYNAWEH goldfish 1 NPIVYGYFNENFRRGFQEAFKLGLCVVDEP-TQ----------
PRRN--------------------AAWKHNRVFADRD-ETNAKSD------GR-----REFCSGEQR-------------DELVLEDLD--------------- goldfish 2 NPIIYGYYNENFKRGFQAVCRTHSCCCGGM-
RV----------RGGMRQKARG-DTRDPVANSNPLNFGARN-RVYTDGV-TKNSRMCLELEHRRTGRLCNNSLCSNDTGSSAGSGIKGVTNQKVFQMEELEKISPVRVGKNQAWDQ goldfish 3
NPIIYGYFNENFKRGFQAACQHQVCCWGRE-KT----------RFRIKRPRAG-SQLNRAASGHPLSLGSRTNRIFTESD-L-TGCVCLEMDTKGS-----SA----------EGGNSSTSI-----TREIGKVSSTGGKIYNAWER grass puffer
NPIIYGYYNENFKRGFQAVCKSRPLCCLLQCHHCERMTRWGKRDRSMEEPHQM----DGTSARNHIVLRVRN-RVHNSAR-LKDAEE-----VKRHARA---AVRPQRENFD-QTIEMAILHNKDHHSQDSNRVSSLEASVYKAWEK tiger puffer
NPIIYGYYNENFKRGFQAVCKSRPLCCLLQCHHCERMTRWGKRDRSMEEPHQM----DGTSARNHIDLRVRN-RVHNSAR-LKDAEE-----VKRHARA---AVRPQRENFD-QTIEMAILHNKDHHSQDSNRVSSLEASVYKAWEK Nile tilapia
NPIIYGYYNENFKRGFEAICKSRPFCCLVQSQLWSRIVGWRKKRRSVQAPCEGTVSRE-VVNHNHFVLGLRN-RVHNDNK-LNDTAE-----VNRSARVECAVVHSGRALSD-QELEMTAVNKKSSNEEESEKVSSLAVSPYQVWDN grouper
NPIIYGYYNEKFKRGFQAVCKSRPFCCLVQCQLWKRMVRWDRKDRTVQALCGGADFRDATNNHNHLVLGLRN-RVHHDNK-VTETAE-----VNRSVKVGCVVVHSEGRLSD-RGLEMTAVHKKGSNGEESQRLSSLAASVYQAWDN tongue sole
NPIIYGYYNENFRRGFKAVCKSQQLCCSLG---------------------------ETTVHHHPVVVRLTN-RVHNDNE-LKDTAN-----NNRSAPVGNPMVLTQRCPSN-QVMEMASLPKNSS--AESERVGSLAVSVHQAWEN
*** *::**:*: *: :: :

Fig. 2. Alignment of the amino acid sequences of LPXRFa-R in different fish species. Gaps introduced in some sequences to maximize the alignment are indicated by
hyphens. Identical sequences are indicated by asterisks. Commas denote conserved amino acids and colons indicate highly conserved amino acids. The putative TM
domains are boxed. GenBank accession numbers of the sequences used for alignment are as follows: tiger puffer RFRPR (BAF34887), grass puffer LPXRFa-R
(BAJ41196), goldfish LPXRFa-R1 (AFY63167), LPXRFa-R2 (AFY63168), LPXRFa-R3 (AFY63169), zebrafish LPXRFa-R1 (ADB43133), LPXRFa-R2 (ADB43134),
LPXRFa-R3 (ADB43135), Nile tilapia LPXRFa-R (AIE89789), and half-smooth tongue sole LPXRFa-R (APR72387). The sequence of orange-spotted grouper LPXRFa-R
was obtained from the literature (Wang et al., 2015).
26

realplex Real-time PCR System (Eppendorf, Hamburg, Germany) using

the following thermal cycling profiles: 95 °C for 30 s, and 40 cycles of
95 °C for 5 s and 60 °C for 20 s. Each sample was analyzed in triplicate.
At the end of the amplification, a melting curve analysis was generated
to confirm the presence of a single PCR product. No amplification was
observed in non-template reactions (water instead of cDNA) that served
as a negative control. The 18S gene was used as the internal reference,
and the 18S levels remained stable across various tissues. The ampli-
fication efficiencies of the qPCR primers used to quantify LPXRFa-R and
18S mRNA levels were determined with ten-fold serial dilutions of
cDNA, and the amplification efficiency values of LPXRFa-R and 18S
were 96% and 100%, respectively (Wang et al., 2017b). Relative ex-
pression levels of LPXRFa-R mRNA were normalized to the 18S levels
and were quantified with the comparative Ct method.

2.4. Reagents

Putative peptides (GenBank accession no. KU612223) corre-

sponding to tongue sole LPXRFa-1 (SLDLERLNMRVTPTASKSSLPTIIK-
LYPPTVNPHIHANMPMRF-NH2) and LPXRFa-2 (EVEP.
EDDQSHNTPNMPQRF-NH2) were predicted from the precursor
cDNA sequence using the ProP 1.0 Server (http://www.cbs.dtu.dk/
services/ProP/) and provided by ChinaPeptides Co., Ltd. (Shanghai,
China) with a purity of at least 95%, as determined by HPLC. LPXRFa
peptides were prepared with distilled water and the aliquots of con-
centrated stock solutions were stored at −80 °C. These stock peptides
were previously used for pharmacological analysis and showed biolo-
gical potency under in vitro conditions (Liu et al., 2017; Wang et al.,
2017c). U73122 was obtained from Sigma. GF109203X and forskolin
were purchased from Calbiochem. All of the chemicals were prepared
 R, transfection efficiency)
an containing the Renilla luciferase reporter gene were co-transfected into
as described in detail elsewhere (Wang et al., 2014).
d the cells in 500 μL serum-free medium using Lipofectamine 3000 re-
50 agent (Invitrogen, USA). Six hours post-transfection, culture medium
2.5. Cell culture, transfection, and signal transduction analysis ng was replaced and (1) cells were treated with vehicle or LPXRFa peptides
pR at doses ranging from 0.1 to 1000 nM for 24 h; (2) cells were exposed to
The ORF of tongue sole LPXRFa-R cDNA was subcloned into the
L- a 24-h challenge with 10 μM forskolin (FSK) alone or in combination
XbaI and HindIII sites of pcDNA3.1 expression vector (Invitrogen, USA)
TK with various concentrations (1–1000 nM) of LPXRFa peptides; (3) cells
and all constructs were verified by sequencing. Cell culture and trans-
(fo were incubated for 24 h with 1 μM LPXRFa peptides alone or in the
fection experiments were generally performed as previously described
r presence of 5 μM U73122 (a phospholipase C (PLC) inhibitor) and
(Wang et al., 2015) with slight modifications. In brief, the COS-7 cells
no 10 μM GF109203X (a PKC inhibitor). The concentrations of these in-
(ATCC, USA) were seeded in 24-well tissue culture plates at a density of
rm hibitors were chosen based on previous reports (Wang et al., 2014;
1 × 105 cells/well in 1 mL of DMEM containing 10% FBS and 1% pe-
ali Wang et al., 2017c). At the end of each incubation period, cells were
nicillin/streptomycin (Gibco, USA) at 37 °C under an atmosphere of 5%
zat harvested and luminescence was measured by Dual Luciferase Kit
CO2. Twenty hours later, 500 ng CRE-Luc (a PKA-specific reporter) or SRE-
ion (Promega, USA). Each experiment was repeated twice, and all treat-
Luc (a PKC-specific reporter) plasmid, 300 ng pcDNA3.1-LPXRFa-
of ments included three or four replicate wells in each experiment. A
B. Wang et al. Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32

Fig. 3. Phylogenetic analysis of LPXRFa-R in vertebrates.

The phylogenetic tree was constructed by MEGA 6.06
using the neighbor-joining method with 1000 bootstrap
replicates. The number shown at each branch indicates
the bootstrap value (%). GenBank accession numbers of
the sequences used in this analysis for LPXRFa-R are as
Mammals follows: human NPVFR (AAK94199), dog NPFFR1
(XP_851935), horse NPFFR1 (XP_001917713), pig
NPFFR1 (NP_001193386), cattle NPFFR1
(NP_001179676), rat NPFFR1 (NP_071627), mouse
NPFFR1 (NP_001170982), rabbit NPFFR1 (AEZ01938),
opossum NPFFR1 (XP_001374817), Japanese quail
GnIHR (BAD86818), and chicken GnIHR (BAE17050).
See the legend of Fig. 2 for GenBank accession numbers
Birds of the LPXRFa-R sequences in teleosts.

Fish

Fig. 4. Relative expression of LPXRFa-R

2 mRNA in various tissues of half-smooth
tongue sole. Data were normalized to the
abundance of 18S RNA expressed in the
1.5 same tissue and presented as the
mean ± SEM (n = 3). Groups with dif-
d ferent letters are significantly different
from each other (p < 0.05; ANOVA fol-
1 lowed by Duncan's multiple range test).
c

0.5
b
ab ab ab
ab ab
a a a
0

brain pituitary gill heart liver spleen kidney stomach intestine muscle ovary
Th
full-
leng
parallel control transfection experiment was performed with empty th
pcDNA3.1, CRE-luc or SRE-luc and the internal control pRL-TK. DN
A
LPX
2.6. Statistical analysis RFa-
R
Data are presented as the mean ± SEM and were analyzed by one- a
way ANOVA followed by Duncan's multiple range test using SPSS17.0 olate
d
software (Chicago, IL, USA). Normality and homoscedasticity assump-
o
tions were tested prior to the analysis. Differences between groups with h
p < 0.05 were considered statistically significant. rai
of
3. Results

3.1. cDNA cloning, identification, and phylogenetic analysis of LPXRFa

receptor
 e has been deposited in the
GenBank database under accession no. KX839491. As shown in Fig. 1,

27 ton the tongue sole LPXRFa-R cDNA was composed of 2201 base pairs (bp)
gu which contained a 5′-untranslated region (UTR) of 60 bp, an ORF of
e 1365 bp encoding LPXRFa-R of 454 amino acids, and a 3′-UTR of
sol 776 bp with the addition of poly (A) tail. The tongue sole LPXRFa-R had
e seven hydrophobic TM domains structure, which is a characteristic of
an GPCRs. In addition, several potential sites for post-translational mod-
d t ification (N-linked glycosylation and protein phosphorylation) were
he present (Fig. 1).
nuc Multiple amino acid sequence alignments of LPXRFa-R from dif-
leot ferent teleost species are shown in Fig. 2. At the protein level, the
ide tongue sole LPXRFa-R displayed a high degree of identity with the order
seq Perciformes (71.90–72.41%), followed by puffer fish (65.32–66.22%).
ue However, the sequence homology droped to relatively lower levels
nc when compared to LPXRFa-R reported in cyprinid fish (47.33–57.81%).
A phylogenetic tree of the tongue sole LPXRFa-R with previously

LPXRFa-R/18S mRNA levels

(fold expression)
B. Wang et al. Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32

(A) 2 b b b
0 0.1 b b
1 10 1001000
1.5 LPXRF
a a a a b -1
ba b a
1 a b

0.5
(B) 0 0.1 1 10 1001000
0 concentrations (nM) 2 LPXRFa-2 concentrations (nM)

1.5

1
(C)
3
0.5
2.5

2 0
1.5

0.5 (D)

0 3

2.5

1.5

0.5

0
FSK (10 μM) - + + + + +
d d FSK (10 μM) - + + + + +
cd LPXRFa-1 (nM) --1 10 100 1000
LPXRFa-2 (nM) - - 1 10 100 1000

Fig. 5. Differential regulation of CRE-luc activity by LPXRFa peptides in COS-7 cells transfected with tongue sole LPXRFa-
b b R. Cells were treated with LPXRFa-1 (A) and LPXRFa-2 (B) alone or co-
c
incubated with forskolin (FSK) in the absence or presence of various doses of LPXRFa-1 (C) and LPXRFa-
2 (D) for 24 h and then collected for the analysis of CRE-
luc activity. Data are presented as the mean ± SEM (n = 8). Groups with different letters are significantly different from each oth
er (p < 0.05; ANOVA followed by Duncan's multiple range test).
p eraction of LPXRFa peptides with LPXRFa-R cloned
a e in this study and the possible signal transduction pathways involved,
identified LPXRFa-
p CRE and SRE reporter gene assays were performed in COS-7 cells. No
R sequences was constructed, dem
ti response in CRE-luc and SRE-
onstrating that the
d luc activity was observed when cells
tongue sole LPXRFa-
e transfected with empty pcDNA3.1 were stimulated with a high dose
R clustered together with the branch of fish LPXRFa-
s (1 μM) of LPXRFa peptides (data not shown), indicating that COS-7 cells
R (Fig. 3). Phylogenetically, it was closely related to the
do not naturally express endogenous LPXRFa receptors. Similarly, cells
counterparts of orange-spotted grouper and Nile tilapia, less related to
transfected with LPXRFa-
the LPXRFa-R reported in other teleosts, and distally related to the o R did not respond to various doses of LPXRFa- 1 and LPXRFa-2 in CRE-
LPXRFa-R family of tetrapods (Fig. 3). c luc activity (Fig. 5A and B). However, forskolin
l significantly increased CRE-luc activity, and this stimulatory effect was
3.2. Tissue distribution of LPXRFa receptor mRNA a not altered in the presence of various concentrations of LPXRFa-1
r (Fig. 5C). Conversely, LPXRFa-2 suppressed forskolin-induced CRE-
To gain insight into the potential physiological roles of LPXRFa-R, if luc activity in a dose-dependent manner (Fig. 5D). The inhibitory effect of
the expression pattern of LPXRFa-R mRNA in various tissues were de- y
termined by quantitative real-time PCR. LPXRFa-R transcripts were t
most abundant in the brain, to a lesser extent in the pituitary, and at h
low levels in the ovary and other peripheral tissues (Fig. 4). e
i
n
3.3. Functional characterization of LPXRFa-R in response to LPXRFa
t
 LPXRFa-2 on forskolin-evoked CRE-luc activity became significant at
28 activity (fold increase)
CRE-luc100 nM and was maximal at 1000 nM.
On the other hand, both LPXRFa-1 and LPXRFa-2 stimulated SRE-
luc activity in COS-7 cells expressing tongue sole LPXRFa-R in a dose-
dependent manner (Fig. 6A and B). A significant induction of SRE-luc
activity was observed only at 1000 nM LPXRFa-1, and lower con-
centrations did not markedly affect SRE-luc activity (Fig. 6A). In con-
trast, doses of LPXRFa-2 of 1 nM and lower were ineffective in stimu-
lating SRE-luc activity, while doses of 10 nM and above progressively
stimulated SRE-luc activity and reached maximal levels at 1000 nM
(Fig. 6B). However, these stimulatory effects were evidently attenuated
by PLC inhibitor U73122 (Fig. 6C and D) and totally abolished by PKC
inhibitor GF109203X (Fig. 6E and F).

4. Discussion

CRE-luc activity (fold increase) In the

CRE-luc activity presen
(fold t study, we cloned the LPXRFa-R gene from the brain of
increase)
tongue sole. To the best of our knowledge, this is the first reported
identifi cation of LPXRFa receptor in the order Pleuronectiformes. The
tongue sole LPXRFa-R gene contained an ORF of 1365 bp that resulted
in a predicted protein of 454 amino acids. Sequence alignment revealed
that the tongue sole LPXRFa-R had a high degree of identity (> 70%)
with the counterparts of orange-spotted grouper (Wang et al., 2015)
and Nile tilapia (Biran et al., 2014). Analysis of the predicted amino
acid sequence of tongue sole LPXRFa-R revealed seven putative trans-
membrane domains, which are a typical characteristic of GPCRs (Yin
et al., 2005). In addition, it contained several putative glycosylation
sites and phosphorylation sites, similar to that observed in chicken
(Ikemoto and Park, 2005), grass puffer (Shahjahan et al., 2011), and
tilapia (Biran et al., 2014). The presence of phosphorylation sites sug-
gests that the receptor potentially conveys its signal via the PKA and/or
B. Wang et al. Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32

(A) 6 d
0
5 0.1 1 10 1001000 d
LPXRFa-
4 c
1
3 c b c
a
2 b
b
1 ab ab a a a
a a
0 (B) 0 0.1
1
6
concentrations (nM) 10 100 1000
5 LPXRFa-2 concentrations (nM)

3 4
(C)
3

2
2
1

0
1

(D)
3
0

0
LPXRFa-1 (1 μM) - + + -
LPXRFa-2 (1 μM) - + + -
d U73122 (5 μM) - - + +
U73122 (5 μM) - -

c
c

b
a
b

a a
(E)
3

2
3

1 2

1
0

0
LPXRFa-1 (1 μM) - + + -
LPXRFa-2 (1 μM) - + + -
c GF109203X (10 μM) - - + +
GF109203X (10 μM) - - + +

Fig. 6. LPXRFa-induced SRE-luc activity in COS-7 cells expressing tongue sole LPXRFa-
R. Cells were treated with LPXRFa-1 (A) and LPXRFa-2 (B) alone or co- incubated with LPXRFa-1 (C, E) and LPXRFa-
2 (D, F) in the presence of PLC inhibitor U73122 (C, D) and PKC inhibitor GF109203X (E, F) for 24 h and then collected
for the analysis of SRE-luc activity. Data are presented as the mean ± SEM (n = 6–
b 8). Groups with different letters are significantly different from each other

a
a
(p < 0.05; ANOVA followed by Duncan's multiple range test).
PKC pathways (Bedecarrat
SRE-luc activity s(fold
et al., 2009). Phylogenetic analysis showed SRE-luc activity
increase)
that the tongue sole LPXRFa-R grouped into the LPXRFa-R branch of
other teleosts, which further substantiated the nature of the cloned
sequence. It should be noted that cyprinid fishes are the only teleost
species possessing three LPXRFa-R forms investigated so far, while only
one LPXRFa-R form was identifi ed in other teleost species, birds and
mammals (Munoz-Cueto et al., 2017; Ogawa and Parhar, 2014; Tsutsui
et al., 2015; Ubuka et al., 2016). Although we have obtained one type of
LPXRFa-R in the current study, further investigation is warranted to
clarify whether other LPXRFa-R forms/variants exist in the tongue sole.
The distribution of LPXRFa-R mRNA varied greatly among verte-
brates. Generally, a substantial degree of LPXRFa-R mRNA expression
was evident in the brains of pigs (Li et al., 2012), chicken (Ikemoto and
Park, 2005), Japanese quail (Yin et al., 2005), zebrafish (Zhang et al.,
SRE-luc activity (fold increase)
2010), grass puffer (Shahjahan et al., 2011), tilapia (Biran et al., 2014), orange-
spotted grouper (Wang et al., 2015), and cinnamon clownfish
(Choi et al., 2016). Moreover, the expression of LPXRFa-R mRNA was
also evident in the pituitary, ovary, and other peripheral tissues in a
species-specific manner (Biran et al., 2014; Ikemoto and Park, 2005; Li
SRE-luc activity (fold increase)
et al., 2012
(fold; increase)
Maddineni et al., 2008; Shahjahan et al., 2011; Wang et al.,
2015; Zhang et al., 2010). In the current study, the predominant ex-
pression of LPXRFa-R mRNA in the brain, pituitary and ovary suggests
that LPXRFa may serve as a key player of reproduction in tongue sole
and regulate the reproductive axis by acting not only at the brain and
pituitary levels but also on gonadal physiology. Our recent study
showed that tongue sole LPXRFa-1 increased hypothalamic expression
of gnrh2 and lpxrfa mRNAs, whereas LPXRFa-2 inhibited gnrh3 mRNA
expression in vitro (Liu et al., 2017). Further studies are needed to
clarify the physiological role of LPXRFa in pituitary and gonadal
functions in this species. It should be noted that the widespread dis-
tribution of LPXRFa-R mRNA indicates that tongue sole LPXRFa may
participate not only in reproductive functions but also in other phy-
siological functions. Indeed, LPXRFa has been implicated in the mod-
SRE-luc activity (fold increase)
ulation of feeding and reproductive behavior in birds and mammals,
while these actions have yet to be fully elucidated in teleosts (Munoz-
Cueto et al., 2017; Tsutsui et al., 2015; Ubuka et al., 2016). Interest-
ingly, the expression of LPXRFa mRNA in tongue sole largely matches
with the expression pattern of LPXRFa-R mRNA, notably most abundant
29
SRE-luc activity (fold increase)
B. Wang et al. Comparative Biochemistry and Physiology, Part A 223 (2018) 23–32

Fig. 7. Working model for action of LPXRFa peptides via

the LPXRFa-R in tongue sole. In this case, LPXRFa-R ac-
tivation by LPXRFa-2 can exert its functions through PKA
and PKC pathways. However, only PKC pathway med-
iates the action of LPXRFa-1. Gαq and Gαi, hetero-
trimeric G proteins; PLC, phospholipase C; DAG, dia-
cylglycerol; IP3, inositol 1,4,5-trisphosphate; PIP2,
phosphatidylinositol 4,5-bisphosphate; PKC, protein ki-
nase C; AC, adenylate cyclase; PKA, protein kinase A;
U73122 PLC U73122, a PLC inhibitor; GF109203X, a PKC inhibitor;
PIP2 forskolin, an activator of AC.
forskolin AC

ATP
IP3
DAG

cAMP

GF109203X PKC

PKA
30

gene expression

in the brain (Wang et al., 2018). Similar phenomena were also observed
in zebrafish and grass puffer (Shahjahan et al., 2011; Zhang et al.,
2010). The concomitant expression of the ligand and its receptor in the
brain of tongue sole may be indicative of the physiological significance
of the LPXRFa/LPXRFa-R system in this species, which is supported by
our recent study (Liu et al., 2017).
To date, the intricate web of intracellular signals mediating the
action of LPXRFa is still far from being fully understood, though the
physiological relevance and functions of LPXRFa system were evaluated
from fish to mammals (Munoz-Cueto et al., 2017; Ogawa and Parhar,
2014; Tsutsui et al., 2015; Ubuka et al., 2016). Initially, Hinuma et al.
(2000) showed that LPXRFa peptides efficiently suppressed forskolin-
induced production of cAMP in CHO cells transfected with LPXRFa-R,
indicating that LPXRFa-R may couple to G protein. Similarly, activa-
αi

tion of chicken and orange-spotted grouper LPXRFa-Rs also inhibited

the forskolin-evoked CRE-luc activity, suggesting that both receptors
appear to couple to G protein (Shimizu and Bedecarrats, 2010; Wang
αi

et al., 2015). However, tilapia LPXRFa-R and zebrafish LPXRFa-R2 and

LPXRFa-R3 were probably coupled to G protein (Biran et al., 2014;
αs

Spicer et al., 2017). These results indicate that differential activation of

signaling pathways via LPXRFa-Rs may contribute to the functional
divergence of LPXRFa on the reproductive axis in teleosts (see In-
troduction for details). Members of the GPCR family typically couple to
either G or G , and signaling through G or G results in the acti-
αs αi αs αi

vation or inhibition of adenylyl cyclase (AC), which is associated with

an increase or decrease in intracellular cAMP levels, respectively. In
turn, cAMP activates cAMP-dependent protein kinase (PKA), and trig-
gers the binding of cAMP response element binding protein (CREB) to
cAMP response element (CRE) on target genes (Bedecarrats et al., 2009;
Shimizu and Bedecarrats, 2010). Thus, the possible targets of in-
tracellular mechanisms of action of the cloned receptor were further
analyzed in this study. No increase in CRE-luc activity was observed
 cep ly, LPXRFa peptides alone did not alter basal CRE-luc activity, indicating
tor that LPXRFa peptides exert their effects through Gαi by blocking the
ma activity of AC, thereby inhibiting the de novo production of cAMP rather
y than reducing pre-existing cellular levels (Shimizu and Bedecarrats,
cou 2010). In addition, the differential regulation of forskolin-elicited CRE-
ple luc activity by LPXRFa-1 and LPXRFa-2 may stem from difference in
to binding affinity to their receptor, demonstrating that LPXRFa-2 appears
Gαi to be the dominant ligand for the tongue sole LPXRFa-R, which is in
pr accordance with a recent study performed in European sea bass
ote (Paullada-Salmeron et al., 2016b).
in On the other hand, tilapia LPXRFa-2 stimulated SRE-luc activity in
when COS-7 cells expressing tongue sole LPXRFa-R were stimulated
an COS-7 cells expressing its cognate receptor, suggesting this receptor
with a wide range of concentrations of LPXRFa peptides, suggesting
d i may convey its signal through the PKC pathway (Biran et al., 2014).
that tongue sole LPXRFa-R dose not couple to G protein. Moreover,
αs
nhi Similarly, both LPXRFa-1 and LPXRFa-2 increased SRE-luc activity in COS-
forskolin is an activator of AC and inhibition of the effects of forskolin
bit 7 cells transfected with tongue sole LPXRFa-R in this study.
by LPXRFa would indicate coupling to G protein (Shimizu and
αi
AC However, contradictory results were observed in orange-spotted
Bedecarrats, 2010). Therefore, further studies were performed to verify
act grouper where grouper LPXRFa-1 inhibited SRE-luc activity in COS-7
whether the novel LPXRFa-R signals through G protein. In accordance
αi
ivit cells expressing its cognate receptor (Wang et al., 2015). Reasons for
with previous studies in orange-spotted grouper (Wang et al., 2015),
y. the apparent discrepancies between these earlier findings and ours are
chicken (Shimizu and Bedecarrats, 2010), and mammals (Son et al.,
No not known, perhaps due to LPXRFa-R coupling to different
2012), activation of tongue sole LPXRFa-R by LPXRFa-2 significantly
tab
inhibited the forskolin-induced CRE-luc activity, implying that this re-
B. Wang et al.
heterotrimeric G proteins. Interestingly, no dose response of the SRE
pathway was detected by any of the three LPXRFa peptides with any of
the three LPXRFa-Rs in zebrafish (Spicer et al., 2017). To further es-
tablish the possible participation of the PLC/PKC pathway in the action
of tongue sole LPXRFa peptides, we employed the specific PLC inhibitor
U73122 and the PKC inhibitor GF109203X. The LPXRFa-induced SRE-
luc activity was significantly reduced by these two inhibitors. Taken
together, these results suggest that tongue sole LPXRFa peptides utilize
the PLC/PKC pathway to exert their functions via the cognate receptor.
It is worth mentioning that differential activation of signaling pathways
by LPXRFa peptides observed in various species can be presumably due
to different experimental conditions of transfection assays such as
origin of the cells (COS-7 vs. GH ), expression vectors, plasmid con-
3 Vissio, P.G., 2016. Gonadotropin-inhibitory hormone in the cichlid fish Cichlasoma
centrations, transfection reagents, treatment time, and the luciferase
reporter assays tested.
The physiological and functional properties of LPXRFa peptides
vary greatly across vertebrates, depending on species, physiological
status, and route of administration (Munoz-Cueto et al., 2017; Ogawa
and Parhar, 2014; Tsutsui et al., 2015; Ubuka et al., 2016). Multiple
signals may underlie the function diversity of the LPXRFa system. It was
recently demonstrated that mouse LPXRFa peptides suppressed GnRH-
induced gonadotropin subunit gene expression by an inhibition of AC/
cAMP/PKA-dependent ERK (extracellular signal-regulated kinase)
pathway in a mouse gonadotrope cell line, LβT2 (Son et al., 2012). In
addition, the inhibitory action of ovine LPXRFa-3 on GnRH-stimulated
gonadotropin release from sheep pituitary cells in culture was likely
mediated by reducing cytoplasmic calcium levels and ERK phosphor-
ylation (Clarke et al., 2008; Sari et al., 2009). However, tilapia LPXRFa-
2 sigificantly increased gonadotropin secretion from primary cell cul-
tures of tilapia pituitaries probably through activation of PKA and/or
PKC pathways (Biran et al., 2014). Overall, research aiming to elucidate
the detailed signaling pathways mediating the actions of LPXRFa on the
hypothalamus-pituitary-gonadal axis could also contribute to under-
Moussavi, M., Wlasichuk, M., Chang, J.P., Habibi, H.R., 2012. Seasonal effect of GnIH on
standing the functional divergence of the LPXRFa system in teleosts.
Further studies are also being directed to ascertain whether LPXRFa can
interfere with signals induced by other reproduction-related factors,
such as GnRH (Shimizu and Bedecarrats, 2010; Son et al., 2012), and
kisspeptin (Son et al., 2016; Spicer et al., 2017; Wang et al., 2017c).
In summary, we have cloned the LPXRFa receptor gene in the
tongue sole and investigated its expression profiles and the possible
signaling pathways involved in the action of LPXRFa peptides (Fig. 7).
As far as we know, this study is the first to report the existence of
LPXRFa receptor in the order Pleuronectiformes and provides novel
information on the mechanisms of LPXRFa action in the tongue sole.
Acknowledgments
We thank Drs. Shuisheng Li and Caiyun Sun (School of Life Sciences,
Sun Yat-sen University, China) for providing SRE-luc and CRE-luc re-
porter plasmids, respectively. This work was supported by grants from
National Natural Science Foundation of China (31602133, 31502145),
Natural Science Foundation of Shandong Province (ZR2016CB02),
Central Public-interest Scientific Institution Basal Research Fund,
YSFRI, CAFS (20603022016018), Central Public-interest Scientific
Institution Basal Research Fund, CAFS (2017HY-XKQ01), and China
Agriculture Research System (CARS-47).
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