GPR3, GPR6, GPR12 and GPR12L) in Chickens and Ducks and Regulation of GPR12 Expression in Ovarian Granulosa Cells by Progesterone

G C A T T A C G G C A T genes

Article Characterization of Four Orphan Receptors (GPR3, GPR6, GPR12 and GPR12L) in Chickens and Ducks and Regulation of GPR12 Expression in Ovarian Granulosa Cells by Progesterone

Zejiao Li 1, Biying Jiang 1, Baolong Cao 1, Zheng Zhang 1, Jiannan Zhang 1 , Juan Li 1, Yan Huang 2,* and Yajun Wang 1,*

1 Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, China; [email protected] (Z.L.); [email protected] (B.J.); [email protected] (B.C.); [email protected] (Z.Z.); [email protected] (J.Z.); [email protected] (J.L.) 2 China Conservation and Research Centre for the Giant Panda, Wolong Nature Reserve, Wolong 623006, China * Correspondence: [email protected] (Y.H.); [email protected] (Y.W.); Tel.: +86-8541-2650 (Y.W.); Fax: +86-8541-4886 (Y.W.)

Abstract: The three structurally related orphan G protein-coupled receptors, GRP3, GPR6, and GPR12, are reported to be constitutively active and likely involved in the regulation of many physiological/pathological processes, such as neuronal outgrowth and oocyte meiotic arrest in mammals.

However, the information regarding these orphan receptors in nonmammalian vertebrates is ex- tremely limited. Here, we reported the structure, constitutive activity, and tissue expression of these

Citation: Li, Z.; Jiang, B.; Cao, B.; receptors in two representative avian models: chickens and ducks. The cloned duck GPR3 and Zhang, Z.; Zhang, J.; Li, J.; Huang, Y.; duck/chicken GPR6 and GPR12 are intron-less and encode receptors that show high amino acid Wang, Y. Characterization of Four (a.a.) sequence identities (66–88%) with their respective mammalian orthologs. Interestingly, a novel Orphan Receptors (GPR3, GPR6, GPR12-like receptor (named GPR12L) sharing 66% a.a. identity to that in vertebrates was reported in GPR12 and GPR12L) in Chickens and the present study. Using dual-luciferase reporter assay and Western blot, we demonstrated that GPR3, Ducks and Regulation of GPR12 GPR6, GPR12, and GPR12L are constitutively active and capable of stimulating the cAMP/PKA Expression in Ovarian Granulosa signaling pathway without ligand stimulation in birds (and zebraﬁsh), indicating their conserved sig- Cells by Progesterone. Genes 2021, 12, naling property across vertebrates. RNA-seq data/qRT-PCR assays revealed that GPR6 and GPR12L 489. https://doi.org/10.3390/genes expression is mainly restricted to the chicken brain, while GPR12 is highly expressed in chicken 12040489 ovarian granulosa cells (GCs) and oocytes of 6 mm growing follicles and its expression in cultured

Academic Editor: Jun–Heon Lee GCs is upregulated by progesterone. Taken together, our data reveal the structure, function, and expression of GPR3, GPR6, GPR12, and GPR12L in birds, thus providing the ﬁrst piece of evidence

Received: 14 February 2021 that GPR12 expression is upregulated by gonadal steroid (i.e., progesterone) in vertebrates. Accepted: 22 March 2021 Published: 27 March 2021 Keywords: chickens; ducks; GPR3; GPR6; GPR12; GPR12L; ovary; progesterone

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. G protein-coupled receptor 3 (GPR3), GPR6, and GPR12 are the three orphan receptors, which share about 60% amino acid (aa) sequence identity with each other [1]. Structurally, the three receptors are related to melanocortin receptors (MCRs), cannabinoid receptors (CBR), adenosine receptor (AR), sphingosine 1-phosphate (S1PR), and lysophosphatidic Copyright: © 2021 by the authors. acid (LPAR) receptor [2,3]. To date, no endogenous ligands have been identified [4]. For Licensee MDPI, Basel, Switzerland. these receptors, it was reported that sphingosine 1-phosphate (S1P) and dihydrosphingo- This article is an open access article sine 1-phosphate (DHS1P) were ligands of GPR3, given that both could act on GPR3 to distributed under the terms and increase the intracellular cAMP level [5]. Paradoxically, several research teams could not conditions of the Creative Commons reproduce this result [6,7]. Similarly, S1P was demonstrated to be a ligand of GPR6 [5], Attribution (CC BY) license (https:// however, Yin et al. [6] failed to detect the agonistic activity of SIP on GPR6 [6]. Thus, the creativecommons.org/licenses/by/ endogenous ligands for these receptors remain to be identified. 4.0/).

Genes 2021, 12, 489. https://doi.org/10.3390/genes12040489 https://www.mdpi.com/journal/genes Genes 2021, 12, 489 2 of 19

Although their endogenous ligands have not yet been identified, all three receptors are found to be constitutively active in mammals. It is reported that about 25% of the constitutively active GPCRs identified so far are found coupled to Gαs and Gαq proteins, and about 50% of these GPCRs couple to Gαi/o proteins. In 1995, Eggerickx et al. [8] first discovered that GPR3 has constitutive activity and is coupled to Gs protein and can increase the intracellular cAMP level in the absence of ligands. Furthermore, they found that GPR3-induced cAMP level is equivalent to that of other ligand-activated Gs-coupled receptors. Subsequently, GPR6 and GPR12 also have been shown to constitutively activate the Gs-cAMP signaling pathway [5,9]. Together with the reports that many diseases are related to the constitutive activity of orphan GPCRs [10], increasing evidence showed that GPR3, GPR6, and GPR12 are likely coupled to the Gαs-cAMP/PKA signaling pathway and play important roles in the regulation of many physiological/pathological processes in mammals. For example, GPR3 is reported to play a part in mammalian Alzheimer’s disease, obesity, and neuronal axonal growth [11–14]. GPR6 is implicated in Parkinson’s disease, Alzheimer’s disease, and neuronal cell survival [14–16]. GPR12 is reported to play vital roles in neurite outgrowth and neuronal development [14]. In addition to being related with the many central nervous system-related diseases, these GPRs are also reported to be involved in the meiosis arrest. In oocytes, a high cAMP level can continuously activate PKA, which phosphorylates and activates nuclear kinase Weel/MytI, which in turn inactivates cell division cycle 25B (CDC25B). Thus, as the activator of cyclin-dependent kinase 1 (CDK1), CDC25B can ultimately maintain the M- phase promoting factor (MPF) in an inactive state and prevent meiosis resumption [17,18]. In 2004, Mehlmann et al. [13] reported that GPR3 can maintain meiotic arrest in mouse oocytes through the Gαs signaling pathway [13]. Later, in 2005, Mary Hinckley et al. [19] also found that rat oocytes only expressed GPR12, which is involved in the regulation of meiotic arrest [19], including preventing oocyte maturation and downregulation of oocyte GPR12 expression to promotes meiotic resumption. In addition, in 2008, Deng et al. [20] found that overexpression of GPR3 in Xenopus laevis can increase cAMP levels to maintain meiotic arrest and the overexpression of GPR12 can prevent progesterone-induced meiosis resumption [20]. In 2012, Yang et al. [21] found that injection of specific small interfering double-stranded RNA (siRNA) complementary to GPR3 into pig oocytes can resume meiosis in early pre-antral follicles, in contrast, the overexpression of GPR3 by reinjecting of GPR3 mRNA can block this process again [21]. Although the constitutive activity of these orphan receptors seems to regulate the functions of the central nervous system (CNS) and oocyte meiotic arrest in mammals, our knowledge regarding the expression, function, and regulation of GPR3, GPR6, and GPR12 in nonmammalian vertebrate species is rather limited. Hence, using chickens and ducks as animal models, our present study aims to address (1) whether GPR3, GPR6, and GPR12 exist and function in birds; (2) whether these orphan receptors are expressed in the CNS and ovary. The results from this study will undoubtedly help to reveal the conserved roles of GPR3, GPR6, and GPR12 signaling across vertebrates.

2. Materials and Methods 2.1. Chemicals, Enzymes, Primers and Antibodies All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All primers used in this study were synthesized by Youkang Biotechnology Co., Ltd. (Chengdu, China) and are listed in Supplemental Table S1. The anti-CREB and anti-β-actin antibodies were purchased from Cell Signaling Technology Inc (CST, Beverly, MA, USA). Genes 2021, 12, 489 3 of 19

2.2. Animal Tissues The adult chickens (1-year-old) or chicks (4-week-old) (Lohmann layer) were purchased from a local commercial company in Chengdu. Chickens were killed, and various tissues were collected. Granulosa cells (GCs) of 6 mm ovarian follicles from laying hens were collected for primary cell culture [22,23]. All animal experimental protocols used in this study were approved by the Animal Ethics Committee of College of Life Sciences, Sichuan University, and the assurance number is 2020030808 (8 March 2020).

2.3. RNA Extraction, RT-PCR, and Quantitative Real-Time PCR Assays Total RNA was extracted from chicken tissues and cultured cells by RNAzol (Molec- ular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instruction. Reverse transcription (RT) was performed by Moloney murine leukemia virus (MMLV) reverse transcriptase (Takara, Dalian, China). In brief, oligodeoxythymide (0.5 µg) and total RNA (2 µg) were mixed in a total volume of 5 µL, incubated at 70 ◦C for 10 min, and cooled at 4 ◦C for 2 min. Then, the ﬁrst step buffer, 0.5 mM each of deoxynucleotide triphosphate and 100 U MMLV reverse transcriptase were added into the reaction mix, for a total volume of 10 µL. RT was performed at 42 ◦C for 90 min. RT-PCR assay was performed to examine mRNA expression of cGPR12 and β-actin genes in chicken 6–8 mm follicle oocytes, 6–8 mm follicle GCs, F5 follicle GCs, and F1 follicle GCs. PCR was performed under the following conditions: 2 min at 94 ◦C denaturation, followed by 39 cycles (30 sec at 98 ◦C, 30 s at 62 ◦C, and 15 s at 68 ◦C) of reaction, ending with a 20 min extension at 68 ◦C. The PCR products were visualized on a UV-transilluminator (Bio-Rad Laboratories, Inc. Hercules, CA, USA) after running electrophoresis on 2% agarose gel containing ethidium bromide. According to a previously established method [24], quantitative real-time PCR (qRT- PCR) assay was performed to examine the mRNA expression of target genes in chicken tissues.

2.4. Cloning the cDNAs of Chicken, Duck, Zebrafish, and Pig GPR3, GPR6, GPR12, and GPR12L According to predicted cDNA sequences of chicken GPR6 (XM_004940310.3), GPR12 (XM_025146842.1), and GPR12L (XM_015278663.2) deposited in the GenBank, gene-specific primers were designed to amplify the 50-cDNA and 30-cDNA ends of GPR6, GPR12, and GPR12L from adult chicken brain. The amplified PCR products were cloned into pTA2 vector (TOYOBO, Osaka, Japan) and sequenced. Finally, the full-length cDNAs of GPR6, GPR12, and GPR12L were determined based on the sequences of 50- and 30-cDNA ends with overlapping regions. Using RT-PCR, we also cloned the coding regions of GPR3, GPR6, GPR12, and GPR12L from duck, zebrafish, and pig brain tissues based on their predicted cDNA sequences deposited in the GenBank. Using genomic DNA extracted from human embryonic kidney 293 cells (HEK293) as the template, we also designed gene-specific primers and cloned the coding region of human GPR3, GPR6, and GPR12 by PCR.

2.5. Sequence Alignment and Phylogenetic Analysis We searched protein sequences of GPR3, GPR6, GPR12, and GPR12L genes in several vertebrates listed in Supplemental Table S2 (https://www.ncbi.nlm.nih.gov/). The deduced amino acid sequences were aligned using the ClustalW program (BioEdit, Carls- bad, CA, USA) [25]. The putative transmembrane (TM) domains were predicted by using an online protein topology prediction tool uniprot (https://www.uniprot.org/). To analyze the evolutionary relationship among vertebrate GPR3, GPR6, GPR12, and GPR12L genes (Supplemental Table S3), phylogenetic analysis was computed by using the program MEGA7 [26], in which the phylogenetic tree was constructed with maximum likelihood method, and conﬁdence was estimated with 500 bootstrap replicates. Genes 2021, 12, 489 4 of 19

2.6. Detection of the Basal Constitutive Activity of Human, Pig, Chicken, Duck, and Zebrafish GPR3, GPR6, GPR12, and GPR12L The expression plasmids encoding chicken (c) GPR6, cGPR12, and cGPR12L were prepared by cloning their complete open reading frames (ORFs) into the pcDNA3.1 (+) expression vector (Invitrogen, Waltham, MA). According to the cloned cDNA sequences of human GPR3 (hGPR3), GPR6 (hGPR6), and GPR12 (hGPR12), pig GPR3 (pGPR3), GPR6 (pGPR6), and GPR12 (pGPR12), duck GPR3 (dGPR3), GPR6 (dGPR6), GPR12 (dGPR12), and GPR12L (dGPR12L), zebrafish GPR3 (zfGPR3), GPR6 (zfGPR6), GPR12 (zfGPR12), and GPR12La (zfGPR12a) deposited in the GenBank, the expression plasmids for these receptors were also prepared by cloning their ORFs into the pcDNA3.1 (+) expression vector (Invitrogen). According to our previously established methods [27,28], all these receptors were tran- siently expressed in HEK293 cells and their basal constitutive activities were detected by dual-luciferase reporter assays. In brief, HEK293 cells were cultured in Dulbecco minimal Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (Thermo Fisher Scientific Inc, Waltham, MA, USA), 100 U/mL penicillin G, and 100 µg/mL strep- tomycin (Life Technologies Inc., Grand Island, NY, USA) in a Corning Cell BIND 48-well ◦ plate (Corning, Tewksbury, MA, USA) and incubated at 37 C with 5% CO2 for 24 h. A mixture containing 700 ng of pGL3-CRE-luciferase reporter construct, 700 ng of receptor expression plasmid or empty pcDNA3.1 (+) vector, 50 ng pL-TK vector, and 3 µL of jet- PRIME (Polyplus-trans-fection SA, Illkirch, France) were prepared in 200 µL of jetPRIME buffer solution for 4 wells. Transfection was performed according to the manufacturer’s instruction when the cells reached 70% confluence. The cells were incubated for an ad- ditional 24 h at 37 ◦C before being harvested for dual-luciferase reporter assay. After the removal of culture medium, HEK293 cells were lysed by adding 100 µL of 1 × Cell Culture Lysis Buffer (Promega) per well, and the luciferase activity of 20 µL cellular lysates was determined with the luciferase assay kit (Promega, Madison, WI, USA). The luciferase activities in experimental groups were expressed as the relative fold increase compared to the control group transfected with empty pcDNA3.1(+) vector.

2.7. Western Blot As described in our previous studies [29], HEK293 cells transfected with cGPR6, cGPR12, or cGPR12L expression plasmid were cultured on a 48-well plate at 37 ◦C for 24 h. Then, the cells were lysed and the phosphorylated CREB (pCREB), β-actin, phosphorylated ERK1/2 (pERK), and total ERK1/2 (tERK) were assayed by Western blot. pCREB levels were quantiﬁed using Image J program v1.8 (National Institutes of Health, Bethesda, MD, USA), normalized by that of intracellular β-actin, and then expressed as the relative fold increase compared to respective controls.

2.8. qPCR Detection of GPR12 Expression in Chicken Granulosa Cell (GC) and Oocytes Total RNA was extracted from granulosa cells of 6–8 mm follicles, F1 follicles, and F5 follicles, according to our previously established method [30]. To extract the total RNA from oocytes of 6–8 mm follicles, four follicles were cut open by a pair of ﬁne scissors to collect ooplasm (including the yolk) in a 2 mL tube pre-cooled by ice. The total RNA was then extracted from ooplasm by RNAzol. To examine the purity of total RNA extracted from the oocytes, the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GFD9) (GenBank accession no.: NC_006091.5 and NC_006100.5, respectively) in oocyte (and granulosa cells) was examined by qRT-PCR assay. As expected, BMP15 and GDF9 were detected in the oocyte nearly exclusively, but not in the granulosa cell layer, indicating the high purity of the total RNA extracted from oocytes. Genes 2021, 12, 489 5 of 19

2.9. Effect of E2, P4 and DHT5α on GPR12 Expression in Cultured 6–8 mm Follicle GCs Granulosa cells (GCs) of 6–8 mm follicles were collected and digested by Collagenase 1 (Hyclone) at 37 ◦C for 20 min [30]. The dispersed GCs were cultured in Medium 199 supplemented with 15% fetal bovine serum in a Corning Cell BIND 48-well plate (Corning) at ◦ 37 C with 5% CO2. After 4 h culture, the cells were treated with 0 nM, 1 nM, 10 nM, or 100 nM of gonadal steroids (E2, P4, DHT5α (dihydrotestosterone, an endogenous androgen sex steroid and hormone)) for 4 h and 24 h. Then, the total RNA was extracted with RNAzol reagent (Molecular Research Center) from cultured GCs and used for qPCR assay of GPR12 mRNA levels. β-actin mRNA level was also examined as an internal control.

2.10. Promoter Analysis of Chicken GPR12 Gene To determine whether the 50-flanking region (near exon 1) of cGPR12 displays promoter activities, we designed gene-specific primers and amplified their 50-flanking regions (near exon 1) with high-fidelity KOD DNA polymerase (TOYOBO). The PCR products were cloned into pGL3-Basic vector (Promega) and sequenced. Finally, two promoter-luciferase reporter constructs of cGPR12 (−3050/+277Luc and −1962/+277Luc) were prepared. In this experiment, the transcription start site (TSS) on exon 1 of cGPR12 determined by 50-RACE was designated as “+1”, and the first nucleotide upstream of TSS was designed as “−1”. Finally, the promoter activities of these constructs were examined in cultured chicken fibroblast cells line (DF-1) by the dual-luciferase reporter assay (Promega), as described in our previous study [31].

2.11. Detection of the Basal Constitutive Activity of GPR12 in Cultured GCs To detect the basal constitutive activity of GPR12 in cultured GCs, 100 ng of cGPR12 expression plasmid, 100 ng of pGL3-CRE-luciferase reporter construct, and 10 ng pL-TK plasmid were co-transfected into GCs cultured in the Corning Cell BIND 48-well plate at a density of 5 × 104 per well. After 24 h transfection, the luciferase activity was detected by dual-luciferase reporter assay, as described in our previous study [27,28].

2.12. Data Analysis The mRNA levels of chicken GPR12/GDF9/BMP15 were ﬁrst calculated as the ratio to that of β-actin and then expressed as the fold difference compared to that of control/chosen group. The data was analyzed by one-way ANOVA followed by the Dunnett’s test in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). To validate the results, all experiments were repeated at least twice.

3. Results 3.1. Cloning the Full-Length cDNAs of GRP3, GPR6, GPR12, and GPR12L in Chickens and Ducks Using 50- and 30-RACE, we ampliﬁed and cloned the full-length cDNAs of cGPR6 (Accession no. MW310573) and cGPR12 (Accession no. MW310572) from adult chicken brain. In addition, we cloned a novel GPR12-like receptor from chicken brain and designated it as GPR12L (Accession no. MW310571). The coding regions of cGPR6, cGPR12, and cGPR12L genes are 987 bp, 999 bp, and 1053 bp long, respectively, which are predicted to encode receptors of 329, 333, and 351 amino acids (a.a.), respectively. Comparison of these cDNA sequences with the chicken genome database (GRCg6a, Ensembl release 101, http://www.ensembl.org/Gallus_gallus) revealed that cGPR6, cGPR12, and cGPR12L are intron-less (Figure1E). Genes 2021, 12, x FORGenes PEER2021 REVIEW, 12, 489 6 of 19 6 of 19

TMD1 TMD2 hGPR3 1 MMWGAGSPLAWLSAGSGNVNVSSVGPAEGPTGPAAPLPSPKAWDVVLCISGTLVSCENALVVAIIVGTPAFRAPMFLLVGSLAVADLLAGLGLVLHFAAVFCIGSAEMSLVLVGVLAMAF 120 pGPR3 1 ...... P....M..I.ST....D.D...... R...... M...... LM.... 120 gpGPR3 1 ...... S..A.....T.....R...... T...... 120 dGPR3 1 ..EDGPPNSSAGQR.WFEARNG.G.SLDLESVVQPLALN.--...... V...VI.....V..VV.FY...... I....T...... I....F.YLVPWEAL..LT..L.VTS. 118 zfGPR3 1 -.DQNS.EVGLEVILAM.QLLDLPPSQMP.KPLE.SVMT.--..IT..V..V.IC....I...T.FSSSSL...... I....W..F...V..L...LSRR.VY.EALE.GS..L.VS.L 117 sgGPR3 1 .NENFSKSSGESEQ.IWLESAEDG.SLDLTSV.RE.MLN.--...... VIA....I...V.FY..SL.T.....I....T...... I...VFLY.VQ.EAVN.IT..L.VAS. 118 TMD3 TMD4 TMD5 hGPR3 121 TASIGSLLAITVDRYLSLYNALTYYSETTVTRTYVMLALVWGGALGLGLLPVLAWNCLDGLTTCGVVYPLSKNHLVVLAIAFFMVFGIMLQLYAQICRIVCRHAQQIALQRHLLPASHYV 240 pGPR3 121 ...... APA...... V...... 240 gpGPR3 121 ...V...... ARA...... 240 dGPR3 119 ...VC...... I...... R...... I..V.T..ASMCY.....MG....KEPSA.S..K..T....LI.SVS...... V.....V...... V...F.GS..D. 238 zfGPR3 118 S..VF...G..L..F...HH....G.QR.H.Q.RCA..VG.TL.GLQ.A..A.G....HDGDS.S.LR..TRV..AT.CGG.LLTLALL...N.R...V.L..SH...... T..SAR-T 236 sgGPR3 119 ...VS...... I...... H...... I..I.T..VS.C..M...VG....KDTAS.SI.R..T.TN..I.SVS.....AL...... K...... H...... F..T.... 238 TMD6 TMD7 hGPR3 241 ATRKGIATLAVVLGAFAACWLPFTVYCLLGDAHSPPLYTYLTLLPATYNSMINPIIYAFRNQDVQKVLWAVCCCCSSSKIPFRSRSPSDV 330 pGPR3 241 ...... Q...... 330 gpGPR3 241 ...... I...... S...... 330 dGPR3 239 ...... I..T..S.....A...... SS..A....V...... L..L...... EI...... G.V.PTM...... 328 zfGPR3 237 H..RRVH...LI.AT..S..I..AL.G....GS..A....A..V.V.G..LL..L...Y..THI.R..RQTV...LPNTPNKNTHT.... 326 sgGPR3 239 T.....S....I..T..S.....AI...... YTY..I...I...... EI...... A..G.M...LSC...... 328

(A)

TMD1 TMD2 cGPR6 1 MEPTA-VLNESSAAVPWLPA------RAGNASLELASRPPAP------TAINPWDVMLCVSGTAIACENAIVVAIICYSPTLRTPMFVLIGSLATAD 84 hGPR6 1 .NAS.AS..D.QVV.VAAEGAAAAATAAGGPDTGEWGPP--AAAALGAGGGA.G....S.QLS.GPPGLLLP.V.....L...... V..G...L...L.AST.A...... V...... 118 pGPR6 1 .NAS.SS..D.QVVAVAAEGVAAVATAARAPDVGEWGPPAAAAAALGAGG.A...... S.QL..GPPGLLLS.V.....L...... V..G...L...L.AST.A...... V...... 120 gpGPR6 1 .NAS.SS....QVVAVTAEG-AAAATAAGARETGEWGPP--AAAALGGGG.V.T....S.QL..GPPGLLLS.V.....L...... V..G...L...L.AST.A...... V...... 117 dGPR6 1 ...EP-A..D.L.....M..------.G...... ------A...... V...... 84 zfGPR6 1 .NESESLT...AMGF.SSSWLEAETF------.S.GT.A.S.AS.NF------HV....I...L...V...... F.T....N...... 89 sgGPR6 1 .NGS-LAS..TA.PA.---WIEAT------D..ST...S.ELLDF------...... I...I...V...... F.T...... 83 TMD3 TMD4 TMD5 cGPR6 85 LLAGLGLILNFVFQYVIRSETVSLLTVGFLVASFAASVSSLLAITVDRYLSLYNALTYYSERTVLCIHTMLVCTWGVSLCLGLLPVLGWNCLHDRAACSVVRPLTKSNVTLLSASFFLIF 204 hGPR6 119 ....C....H.....LVP...... R..L.GV.LL.AA..T...G...... AE...... AR.H.A....A..MV. 238 pGPR6 121 ....C....H...... VP...... R..L.GV.FL.AA..T...G...... AE...... H...R.H.A....T..AV. 240 gpGPR6 118 ....C....H...... VP...... R..L.GV.LL.AA..T...G...... AE..T...... R.H.A....A..AV. 237 dGPR6 85 ...... G...... SV...... 204 zfGPR6 90 ....M...... A...LVS...I..I...... T..I...... F..K.LHYV.L...G...A...... D.AST..I....KR..L...AT...I.. 209 sgGPR6 84 ....M...... L.P...I..I...... T..I...... F...... F...... Y..M...I...... I...... DEPLS..I.K...... AIL..V.. 203 TMD6 TMD7 cGPR6 205 IIMLHLYIEICKIVCRHAHQIALQQHFLTASHYVATKKGVSTLAIILGTFGASWLPFAIYCVVGDPDYPSVYTYATLLPATYNSMINPIIYAYRNQEIQRSMWVLFCGCFQSKVSFRSRS 324 hGPR6 239 G...... VR..QV.W...... C.APP.LA..R...G...VV...... SHED.A...... F...... AL.L.L...... P..... 358 pGPR6 241 G...... VR..QV.W...... C.APP.LA..R...G...VV...... SHED.A...... F...... AL.L...... P..-W. 359 gpGPR6 238 G...... VR..QV.W...... C.APP.LA..R...G...VV...... SRED.A...... F...... AL.L...... P..... 357 dGPR6 205 ...... 324 zfGPR6 210 .L..S..FK...... F.T...... L..ERE..P...... T.....IHM...... NG.Y.... 329 sgGPR6 204 M...... FK...... F...... L..ER...P...... IF...... TN..Y.... 323

cGPR6 325 PSDV 328 hGPR6 359 ..E. 362 pGPR6 360 ..E. 363 gpGPR6 358 ..E. 361 dGPR6 325 .... 328 zfGPR6 330 ..E. 333 sgGPR6 324 ..E. 327

(B)

TMD1 TMD2 cGPR12 1 MNEEPTVNASWLPQDR----VEASSTENAS--GSSLVPVVEPEP--ELWVNPWDIVLCTSGTLISCENAVVVLIIFHNPSLRAPMFLLIGSLALADLLAGVGLIVNFVFAYLLRSEATKL 112 hGPR12 1 ...DLK..L.G..R.Y----LD.AAA..I.AAV..R..A.....--..V...... I...... I...T...... Q...... 114 pGPR12 1 ...DLK..G.RP.R.Y----LD.GAAV.V.AAV..QG...... --..V...... I...... I...I...... Q...... 114 gpGPR12 1 ...DLQ..L.G..R.Y----LD.GAA..V.AAV..Q...... --..V...... I...... I...I...... Q...... 114 dGPR12 1 ...DLK..L...... H----.....P....AA...... D...--..L...... I...I...... Q...... 114 zfGPR12 1 .S..VS.SP...A.EPTWGSSG.G.I..ITVGTY.PAVILPAR.PT..L...... S.....A....L...V.WQ..A...... L..VLH.T...... DSAQ. 120 sgGPR12 1 ....AQ...... YSH--A.GGAA..VTAAV...... DT..--..I...... A....I...... L..VI...... Q.DSA.. 116 TMD3 TMD4 TMD5 cGPR12 113 VTVGLIVASFSASVGSLLAITVDRYLSLYYALTYNSERTVTFTYVMLILLWGAAICIGLLPVMGWNCLRDESTCSVIRPLTKNNAAVLSVSFLLMFALMLQLYIQICKIVMRHAHQIALQ 232 hGPR12 115 ..I...... C...... H...... VM...TS..L...... V...... I...... F...... 234 pGPR12 115 ..I...... C...... H...... VM...TS..L.....L...... V...... I..I...F...F...... 234 gpGPR12 115 ..I...... C...... H...... VM...TS..L...... V...... I...... F...... 234 dGPR12 115 ..I...... S...... 234 zfGPR12 121 L....V...... F...... I...... AA...T..V....VSL.L.....T.V...GQ.VN...VW...... V...... L.G...... V...... 240 sgGPR12 117 ...... C...... I...... LV...GSV.L.....T.V...... I...... V...... G...... V...... L...... 236 TMD6 TMD7 cGPR12 233 HHFLATS-HYVTTRKGVSTLAIILGTFAACWMPFTLYSLIADYTYPSIYTYATLLPATYNSIINPVIYAFRNQEIQKALWLVCCGCVPSNLSQRARSPSDV 332 hGPR12 235 ...... -...... C.I....I..S.A...... 334 pGPR12 235 ...... -...... C.I....I..S...... 334 gpGPR12 235 ...... -...... C.I....I..S...... 334 dGPR12 235 ...... -...... I....I...... 334 zfGPR12 241 .....A.P...... V...... PL...... V...... V...... I.ASVAH...T.... 341 sgGPR12 237 ...... -...... V...... R....I...... ASV...... 336

(C)

TMD1 cGPR12L 1 MLHGPAAMGEPWQP-QSQQQRLPALGNASAPSARPSAAAEE------GGGPAGTAAPGGAGPAAAVSPWDIALCATGTAVAGENALVLAVLFYTPSLRAPSFV 96 dGPR12L 1 .....G.L.G....P.Q...... G..T..V...W.....AAAAGGGPVPATPGGGGGGGDGDGG...AG.GG..A.G.VGGP...... A...... T.. 120 zfGPR12La 1 ------MNDLLQNSSL.N----WDVLEE.NASL.PP------WEVEPV.V------FIN...VL..V...LMSC...V.I.LIA...T....M.. 73 zfGPR12Lb 1 .I.---SLAAAMSNQLQNTSS.STA.TWNLLDV.N.SPVRT------TELRPL.------...... V...LISC...I.I.I.....T....M.I 83 sgGPR12L 1 .I.---TLAAAMSNQLPNTSS.NS.N-WNFQDG.N.SLVRA------SDLRQL.------...... V...LISC...I.I.I.....T....M.I 82 TMD2 TMD3 TMD4 cGPR12L 97 LIGSLALADLLAGLGLVGNFAVRYLLRPPSEAAELGAAGLLLAAFSASVCSLLAITVDRYLSLYNALTYHSERTLGFTCGAVLLMWLLCLGVGLLPPLGWHCLRDQSACSVLRPVTKDNA 216 dGPR12L 121 ...... AT.....A...... L...N...EP...... 240 zfGPR12La 74 ...... F...... IL..IIT.IID--.GLVT.LS....IT...... LNI...... T...MT..YVTLIF..IISAAL.S..V...N..E.E.T..IC...N.N.. 191 zfGPR12Lb 84 ...... F...... IL..VFI.M.N--T.FVT.ISV.M.I...... LNI...... T...VT..YVV.V.I..V.ITL....V...N....EAS..IC.....N.. 201 sgGPR12L 83 ...... F...... IL..VFI.M.N--..FIS.IS....I...... NI....I...... T...VT..YMMLVFI..V.ISL....LM..N..DNE.S...... N.. 200 TMD5 TMD6 TMD7 cGPR12L 217 AVLAVTFLLLFALMMQLYLQICKIAFRHAQQIAVQHQFIATAQATSTRKGLSTLSLILGTFALCWIPFAIYSLVADSSYPAVYTYSLALPAACNSLINPIIYAFRNPDIQKSLWLACCGC 336 dGPR12L 241 .....A...... L...... L...... T...... 360 zfGPR12La 192 .A...S...V...IL...... R..MTASH.S..T..V...TT...... F....L.M...... TRS.VI...VT....I.H.I...MV...... E.LR..RI..... 311 zfGPR12Lb 202 V...... V...... M.IS----.T..V....V..C...V..M...M..I...... MI...ATV...T...V...... Y...... 317 sgGPR12L 201 ...... I...... M..SH.S..T..V....I...... M.A...... MI...ATV...T...V...... M...... 320

cGPR12L 337 GP-ALSSRPRTSSDV 350 dGPR12L 361 V.S.F...... 375 zfGPR12La 312 M.YSF.V....P... 326 zfGPR12Lb 318 V.SNF.L...... 332 sgGPR12L 321 V.SNF.L...... 335

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Figure 1. Cont.

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Figure 1. AminoFigure acid1. Amino sequence acid alignment sequence of alignment G protein-coupled of G protein receptor-coupled 3 (GPR3), receptor GPR6, 3 GPR12, (GPR3 and), GPR6, GPR12L GPR12 in vertebrates., (A) Alignmentand GPR12L of the cloned in vertebrates. duck (d-)/zebrafish (A) Alignment (zf-)/pig of (p-)the GPR3cloned with duck that (d of-)/zebrafish humans (hGPR3, (zf-)/pig NP_005272.1), (p-) GPR3 giant pandas (gpGPR3,with XP_034503372.1) that of humans and (hGPR3, spotted NP_005272.1), gars (sgGPR3, giant XP_015204111.1); pandas (gpGPR3, (B) Alignment XP_034503372.1) of the cloned and chicken(c-)/duck(d- spotted )/zebrafish(zf-)/pig(p-)gars (sgGPR3, GPR6 XP_015204111.1); with that of humans (B) Alignment (hGPR6, of NP_005275.1), the cloned chicken(c giant pandas-)/duck(d (gpGPR6,-)/zebrafish(zf XP_002925693.2),- and spotted gars)/pig(p (sgGPR6,-) GPR6 XP_006626355.1); with that of humans (C) Alignment (hGPR6, of NP_005275.1), the cloned chicken gian (c-)/duckt pandas (d-)/zebrafish(gpGPR6, (zf-)/pig (p-) GPR12 XP_002925693.2), and spotted gars (sgGPR6, XP_006626355.1); (C) Alignment of the cloned with that of humans (hGPR12, NP_005279.1), giant pandas (gpGPR12, XP_002924260.1), and spotted gars (sgGPR12, chicken (c-)/duck (d-)/zebrafish (zf-)/pig (p-) GPR12 with that of humans (hGPR12, NP_005279.1), XP_015196669.1); (D) Alignment of the cloned chicken (c-)/duck (d-)/zebrafish (zf-) GPR12L with that of anole lizard giant pandas (gpGPR12, XP_002924260.1), and spotted gars (sgGPR12, XP_015196669.1); (D) (lGPR12L, XP_028571577.1) and spotted gars (sgGPR12L, XP_015206591.1). The seven transmembrane domains (TMD1–7) Alignment of the cloned chicken (c-)/duck (d-)/zebrafish (zf-) GPR12L with that of anole lizard and N-glycosylation sites (NXT/S) are shaded; the XXXWD motif is boxed; conserved motifs (DRY, XWXP, and NPXXY) (lGPR12L, XP_028571577.1) and spotted gars (sgGPR12L, XP_015206591.1). The seven transmem- are markedbrane by arrowheads; domains (TMD1 (E) Exon–intron–7) and N-glycosylation organization ofsites chicken (NXT/S)GPR6 are, GPR12 shaded;, and theGPR12L XXXWD. The motif red is boxes indicate the codingboxed; region, conserved and the numbers motifs (DRY, inside denoteXWXP, the and size NPXXY) (in bp); are while marked the blue by boxarrowheads; represents (E the) Exon untranslated–intron regions, (UTRs) andorganization the number of within chicken specifies GPR6 the, GPR12 size (in, and bp). GPR12L. The red boxes indicate the coding region, and the numbers inside denote the size (in bp); while the blue box represents the untranslated regions, GPR3 GPR6 GPR12 (UTRs) and the numberUsing within RT-PCR, specifies we the also size cloned (in bp). the coding region sequences of , , , and GPR12L(s) from brain tissues of duck (dGPR3 (MW310577), dGPR6 (MW310576), dGPR12 (MW310575), and dGPR12L (MW310574)), zebrafish (zfGPR3 (MW310585), zfGPR6 Using RT-PCR, we also cloned the coding region sequences of GPR3, GPR6, GPR12, (MW310584), zfGPR12 (MW310583), zfGPR12La (MW310581), and zfGPR12Lb (MW310582)), and GPR12L(s) from brain tissues of duck (dGPR3 (MW310577), dGPR6 (MW310576), and pig (pGPR3 (MW310580), pGPR6 (MW310579), and pGPR12 (MW310578)). dGPR12 (MW310575), and dGPR12L (MW310574)), zebrafish (zfGPR3 (MW310585), Amino acid sequence alignment showed that 1) cGPR6 shows high a.a. sequence zfGPR6 (MW310584),identity zfGPR12 with GPR6 (MW310583), of ducks (96.3%), zfGPR12La zebrafish (MW310581), (74.8%), pigs (67.0%), and zfGPR12Lb humans (67.1%), giant (MW310582)), pandasand pig (67.9%), (pGPR3 and spotted(MW310580), gars (79.8%); pGPR6 2) cGPR12(MW310579), shows highand a.a. pGPR12 sequence identity (MW310578)). with GPR12 of ducks (94.9%), zebrafish (74.4%), pigs (87.1%), humans (88.3%), giant pandas Amino acid(88.9%), sequence and alignment spotted gars showed (85.7%); that 3) cGPR12L 1) cGPR6 shows shows high high a.a. a.a. sequence sequence identity with identity with GPR6GPR12L of ducks of ducks (96.3% (84%),), zebrafish zebrafish (74.8% (GPR12La,), pigs 54.5%; (67.0% GPR12Lb,), humans 61.60%), (67.1% and), gi- spotted gars ant pandas (67.9%),(63.6%) and (Supplemental spotted gars ( Table79.8% S4).); 2) LikecGPR12 human shows GPR6 high and a.a. GPR12, sequence chicken/duck/zebrafish identity with GPR12 of ducksGPR6, ( GPR12,94.9%), and zebrafish GPR12L (74.4% have), many pigs conserved(87.1%), humans motifs and (88.3% amino), giant acid pan- residues, charac- das (88.9%), andteristic spotted of gars family (85.7% A GPCR,); 3) cGPR12L such as a DRYshows motif high for a.a. G sequence protein coupling identity and with two cysteine GPR12L of ducksresidues (84%), for zebrafish a disulfide (GPR12La, bond formation 54.5%; GPR12Lb, [32]. In addition, 61.60% we), and noted spotted a XXXWD gars motif near (63.6%) (Supplementalthe first transmembraneTable S4). Like domain,human GPR6 which and is conserved GPR12, chicken/duck/zebrafish across vertebrates (Figure 1A–D). GPR6, GPR12, and GPR12LGPR3 was have cloned many in ducks conserved and zebrafish motifs and only, amino as this geneacid residues, seems to be char- lost in chickens. Duck GPR3 is 329 a.a. long and shares high a.a. identity with GPR3 of humans (65.7%), acteristic of family A GPCR, such as a DRY motif for G protein coupling and two cysteine pigs (66.3%), giant pandas (65.4%), zebrafish (49.6%), and spotted gars (74%) and a compar- residues for a disulfide bond formation [32]. In addition, we noted a XXXWD motif near atively lower degree of sequence identity with chicken GPR6 (61.4%), GPR12 (62.1%), and the first transmembrane domain, which is conserved across vertebrates (Figure 1A–D). GPR12L (48.2%) (Supplemental Table S4). Likewise, many structural features such as DRY, GPR3 was XWXP,cloned andin ducks NPXXY and motifs zebrafish are present only, as in duckthis gene GPR3 seems [32] (Figure to be lost1A). in chickens. Duck GPR3 is 329 a.a. long and shares high a.a. identity with GPR3 of humans (65.7%), pigs (66.3%),3.2. GPR3, giant GPR6, pandas GPR12, (65.4%), and GPR12Lzebrafish in (49.6%) Birds and, and Other spotted Vertebrates gars (74%) and a comparatively lowerTo tracedegree the of evolutionary sequence identity origin of with chicken/duck chicken GPR6GPR3 ,(61.4%),GPR6, GPR12, GPR12and GPR12L, (62.1%), and GPR12Lwe performed (48.2%) (Supplemental synteny analyses Table by searching S4). Likewise, the genes many adjacent structural to GPR3, features GPR6, GPR12, such as DRY, XWXPand GPR12L, and NPXXYin chickens, motifs ducks, are present humans, in pigs,duck mice, GPR3 turtles, [32] (Figure zebrafish, 1A). and spotted gars. As shown in Figure2, GPR6 and GPR12 exist in all vertebrate species examined, 3.2. GPR3, GPR6,indicating GPR12, thatand GPR12L chicken and in Birds duck andGPR6 Otherand VertebratesGPR12 are orthologous to GPR6 and GPR12 in To trace thehumans evolutionary and other origin vertebrates. of chicken/duck Duck GPR3 GPR3is orthologous, GPR6, GPR12 to GPR3, andof GPR12L humans,, zebrafish, and spotted gars, however, it is likely lost in chickens. GPR12L, which is a novel recep- we performed synteny analyses by searching the genes adjacent to GPR3, GPR6, GPR12, tor identified in this study, exists in chickens, ducks, and other nonmammalian species, and GPR12L in chickens, ducks, humans, pigs, mice, turtles, zebrafish, and spotted gars. including zebrafish, however, it is likely lost in humans, pigs, and mice. As shown in Figure 2, GPR6 and GPR12 exist in all vertebrate species examined, indicating that chicken and duck GPR6 and GPR12 are orthologous to GPR6 and GPR12 in humans and other vertebrates. Duck GPR3 is orthologous to GPR3 of humans, zebrafish, and spotted gars, however, it is likely lost in chickens. GPR12L, which is a novel receptor

Genes 2021, 12, x FOR PEER REVIEW 8 of 19

Genes 2021, 12, 489 8 of 19 identified in this study, exists in chickens, ducks, and other nonmammalian species, including zebrafish, however, it is likely lost in humans, pigs, and mice.

(A) (B)

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FigureFigure 2. 2.SyntenySynteny analysis analysis of of GPR3GPR3, ,GPR6GPR6,, GPR12GPR12, and GPR12LGPR12Lin in chickens chickens and and other other vertebrates. vertebratesGPR3. GPR3(A ),(AGPR6), GPR6(B), (B), GPR12GPR12 (C()C, and), and GPR12L GPR12L (D (D) )are are located located in in four four syntenic syntenic regions conservedconserved in in vertebrate vertebrate species species including including chickens, chickens, ducks, ducks, humans,humans, pigs, pigs, turtles, turtles, spotted spotted gars gars,, andand zebrafish.zebrafish. Dashed Dashed lines lines denote denote the the genes genes of interest, of interest, while while dotted dotted lines indicate lines indicate the thesyntenic syntenic genes genes identified identified inthese in these species. species. Chr, Chr, chromosome. chromosome. (E) Schematic (E) Schem diagramatic diagram showing showing the existence the existence of GPR3, GPR6of GPR3, , GPR6, GPR12, and GPR12L in vertebrate species including spotted gars, teleosts (e.g., zebrafish), turtles, chicken, and GPR12, and GPR12L in vertebrate species including spotted gars, teleosts (e.g., zebrafish), turtles, chicken, and mammals. mammals. GPR3, GPR6, GPR12, and GPR12L were likely duplicated from a common ancestral gene (denoted as “GPR”), GPR3, GPR6, GPR12, and GPR12L were likely duplicated from a common ancestral gene (denoted as “GPR”), which had which had undergone the two rounds of genome duplication event (2R) during early vertebrate evolution. The two undergone the two rounds of genome duplication event (2R) during early vertebrate evolution. The two GPR12L genes in GPR12L genes in zebrafish likely originated from the 3rd round (3R) genome duplication event occurred in teleost lineage. zebrafish likely originated from the 3rd round (3R) genome duplication event occurred in teleost lineage.

ToTo analyze analyze the the evolutionary evolutionary relationship relationship among among vertebrate vertebrate GPR3GPR3, ,GPR6GPR6, GPR12, GPR12,, and GPR12Land GPR12L genes,genes, using using the m theaximum maximum likelihood likelihood method method of MEGA7of MEGA7 software, software, we we con- structedconstructed a phylogenetic a phylogenetic tree treeusing using the thesequences sequences deposited deposited in inthe the GenBank GenBank (Figure (Figure 33 and Supplementaland Supplemental Table Table S4). The S4). results The results showed showed that that GPR3GPR3, GPR6, GPR6, GPR12, GPR12, and, and GPR12LGPR12L form a formcluster, a cluster, which which is evolutionarily is evolutionarily distant distant from from another another cluster cluster formed formed by melanocortinmelanocortin 4 receptor4 receptor (MC4R (MC4R), ),cannabinoid cannabinoid receptor receptor 1 1 (CNR1),), andand lysophosphatidic lysophosphatidic acid acid receptor receptor 1 1 (LPAR1(LPAR1),), an andd d dopamineopamine receptorsreceptors 1A 1A and and 1B 1B (D1A (D1Aand andD1B D1B) of) of vertebrate vertebrate species. species.

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Genes 2021, 12, 489 9 of 19

FigureFigure 3. The The phylogenetic phylogenetic tree of vertebrate tree GPR3, of vertebrate GPR6, GPR12, GPR12L, GPR3, melanocortin GPR6, 4GPR12, receptor (MC4R), GPR12L, cannabinoid melanocortin 4 recep- receptor 1 (CNR1), and lysophosphatidic acid receptor 1 (LAPR1), dopamine receptor 1A (D1A), and dopamine receptors tor(D1B) (MC4R constructed), cannabinoid by MEGA7 software receptor (maximum 1 likelihood (CNR1 method).), and The lysophosphatidic GPR3 cluster is highlighted acid in deep receptor green; the 1 (LAPR1), dopa- mineGPR6 receptor cluster in orange; 1A the(D1A GPR12), clusterand indopamine brown; the GPR12L receptors cluster in purple.(D1B) constructed by MEGA7 software (maxi-

mum likelihood method)3.3. Detection. The of GPR3 the Constitutive cluster Activity is highlighted of GPR3, GPR6, GPR12in deep and GPR12Lgreen; in the Birds GPR6 cluster in orange; the GPR12 clusterIt in is reportedbrown; that the mammalian GPR12L GPR3, cluster GPR6, in andpurple. GPR12 have basal constitutive activity and can activate the Gs-cAMP signaling pathway [5,8,9]. To determine whether avian GPR3, GPR6, GPR12, and GPR12L have basal constitutive activity, we detected the 3.3. Detection of theconstitutive Constitutive activity Activity of these receptors of GPR3, expressed GPR6, in HEK293 GPR12 cells using and dual-luciferase GPR12L in Birds reporter assays. As shown in Figure4A–D, like human and pig GPR3, GPR6, and GPR12, It is reported chickenthat GPR6,mammalian GPR12, and GPR12LGPR3, and GPR6 duck GPR3,, and GPR6, GPR12 GPR12, and have GPRL12L basal have constitutive ac- high basal constitutive activity and the luciferase activity of HEK293 cells expressing these tivity and can activatereceptors theis more Gs than-cAMP 30-fold higher signaling than that of the pathway control group. [5,8,9] Similarly,. five To zebrafish determine whether avian GPR3, GPR6receptors, GPR12 (zfGPR3,, and zfGPR6, GPR12L zfGPR12, have zfGPR12La, basal and constitutive zfGPR12Lb) also displayactivity, strong we detected the constitutive activity under the same condition (Figure4E). All these findings indicate constitutive activitythat of GPR3, these GPR6, GPR12,receptors and the expressed novel GPR12L are in constitutively HEK293 active cells in vertebrates using dual-luciferase reporter assays. Asincluding shown birds. in Figure 4A–D, like human and pig GPR3, GPR6, and GPR12, chicken GPR6, GPR12, and GPR12L and duck GPR3, GPR6, GPR12, and GPRL12L have high basal constitutive activity and the luciferase activity of HEK293 cells expressing these receptors is more than 30-fold higher than that of the control group. Similarly, five zebrafish receptors (zfGPR3, zfGPR6, zfGPR12, zfGPR12La, and zfGPR12Lb) also display strong constitutive activity under the same condition (Figure 4E). All these findings indicate that GPR3, GPR6, GPR12, and the novel GPR12L are constitutively active in vertebrates including birds.

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(A) (B) (C) (D)

(E) (F) (G) (H)

Figure 4. Constitutive activity of GPR3, GPR6, GPR12 GPR12,, and GPR12L(s) in in chicken ( A),), duck duck ( (B),), human ( (C),), pig ( D)),, and zebrafishzebrafish ( E)).. The activity of each receptor expressed in human embryonic kidney 293 ( (HEK293)HEK293) cells was detected by dualdual-luciferase-luciferase reporter assay. Each datadata pointpoint representsrepresents thethe meanmean± ± SEM of 4 replicates ((NN = 4).4). ***, p < 0 0.001.001 vs. control ((cellscells transfected with empty pcDNA3.1(+) vector vector).). Western Western blot detection of the phosphorylated CREB (pCREB, ( F)))) and ERK (pERK, (H)) in HEK293 cells expressing cGPR6, cGPR12, and cGPR12L. pCREB and pERK levels were first normal- ERK (pERK, (H)) in HEK293 cells expressing cGPR6, cGPR12, and cGPR12L. pCREB and pERK levels were first normalized ized by that of actin and total ERK (tERK), respectively, and then expressed as the fold increase compared with the control by that of actin and total ERK (tERK), respectively, and then expressed as the fold increase compared with the control group group (transfected with empty pcDNA3.1(+) vector). ***, p < 0.001 vs. control. (G) The levels of pCREB increase dose- p G dependently(transfected with in HEK293 empty pcDNA3.1(+)cells transfected vector). with ***,increasing< 0.001 amount vs. control. of cGPR6, ( ) The cGPR12 levels, ofor pCREBcGPR12L increase expression dose-dependently plasmid (50 ng,in HEK293 100 ng, cellsand 200 transfected ng). Note. with zebrafish increasing has amounttwo GPR12L of cGPR6, genes cGPR12, (GPR12La or cGPR12Land GPR12Lb expression). plasmid (50 ng, 100 ng, and 200 ng). Note. zebrafish has two GPR12L genes (GPR12La and GPR12Lb). Using Western blot, we found that transfection of chicken GPR6, GPR12, and GPR12L Using Western blot, we found that transfection of chicken GPR6, GPR12, and GPR12L into HEK293 cells for 24 h can dose-dependently enhance phosphorylation level of CREB into HEK293 cells for 24 h can dose-dependently enhance phosphorylation level of CREB (pCREB), a downstream mediator of the cAMP/PKA signaling pathway (Figure 4F,G). All (pCREB), a downstream mediator of the cAMP/PKA signaling pathway (Figure4F,G). All these findings support the hypothesis that all the orphan receptors identified in chickens these findings support the hypothesis that all the orphan receptors identified in chickens can activate the Gαs-cAMP/PKA/CREB signaling pathway. can activate the Gα -cAMP/PKA/CREB signaling pathway. Interestingly, wes found that chicken GPR6, GPR12, and GPR12L expressed in Interestingly, we found that chicken GPR6, GPR12, and GPR12L expressed in HEK293 HEK293 cell can enhance ERK phosphorylation (Figure 4H) [33]. cell can enhance ERK phosphorylation (Figure4H) [33].

3.4.3.4. Tissue Tissue Expression Expression of GPR3, GPR6, GPR12 GPR12,, and GPR12L in Chickens and Ducks ToTo examine the tissue distribution of of GPR6,, GPR12GPR12,, and GPR12LGPR12L inin adult adult chickens, chickens, we analyzed analyzed the the expression expression of of the the three three receptors receptors in in37 37 chicken chicken tissues tissues with with reference reference to theto theRNA RNA-seq-seq data data previously previously obtained obtained in our in ourlab. lab.We Wefound found that thatGPR6GPR6 andand GPR12LGPR12L are wiaredely widely expressed expressed in the in central the central nervous nervous system system (CNS). (CNS). cGPR6cGPR6 showsshows a high a mRNA high mRNA level inlevel the inhypothalamus, the hypothalamus, cerebrum cerebrum,, and pineal and pineal body, body,while whilecGPR12L cGPR12L has a hashigh a mRNA high mRNA level inlevel the inhypothalamus, the hypothalamus, cerebrum, cerebrum, cerebellum, cerebellum, hindbrain, hindbrain, midbrain, midbrain, and retina. and In retina.addition, In GPR6addition, andGPR6 GPR12Land areGPR12L also expressedare also expressed in several in peripheral several peripheral tissues, including tissues, including the adrenal the glandadrenal (GPR6 gland), ( GPR6 pituitary), pituitary (GPR6 (GPR6, GPR12L, GPR12L), uterus), uterus (GPR12L (GPR12L), and), and parathyroid parathyroid gland ((GPR12LGPR12L).). Weak Weak or nondetectable expression expression of of GPR6 and GPR12L was found in other tissuestissues examined. examined. In In contrast, contrast, c cGPR12GPR12 isis predominantly predominantly expressed expressed in in the the ovary, ovary, testes testes,, and anterioranterior pituitary pituitary ( (FigureFigure 55).).

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Figure 5. RNA-seq data analysis showing the expression of GPR6 (A), GPR12 (B), and GPR12L (C) in adult chicken tissues. Each data point represents the mean ± SEM of 6 individual adult chickens (3 males and 3 females) (N = 6). Figure 5. RNA-seq data analysis showing the expression of GPR6 (A), GPR12 (B), and GPR12L (C) 3.5. Regulation of GPR12 Expression in Ovarian Granulosa Cell (GC) by Steroid Hormones in adult chicken tissues. Each data point represents the mean ± SEM of 6 individual adult chickens Since only GPR12 is predominantly expressed in chicken ovary, we further examined (3 males and 3 females)its expression (N = 6). in the GC layer of developing ovarian follicles, including 6–8 mm prehierarchy follicles, F5 and F1 preovulatory follicles by qRT-PCR assay. As shown in Figure6A, 3.5. Regulation of GPR12cGPR12 expression Expression in the in GC Ovarian is stage-dependent. Granulosa It is highlyCell (GC) expressed by inSteroid the GC ofHormones 6 mm follicles, and its expression decreases gradually in the GCs of F5 and F1 follicles. Since only GPR12 is predominantly expressed in chicken ovary, we further examined its expression in the GC layer of developing ovarian follicles, including 6–8 mm prehierarchy follicles, F5 and F1 preovulatory follicles by qRT-PCR assay. As shown in Figure 6A, cGPR12 expression in the GC is stage-dependent. It is highly expressed in the GC of 6 mm follicles, and its expression decreases gradually in the GCs of F5 and F1 follicles.

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FigureFigure 6. (A 6.) (qPCRA) qPCR of GPR12 of GPR12 mRNA mRNA expression expression in in oocytes oocytes (6–8(6–8 mm mm Oo) Oo) of of chicken chicken 6–8 6 mm–8 mm follicles follicles and and granulosa granulosa cells cells of 6–of8 mm 6–8 mm(6–8 (6–8mm mmGC), GC), F5 (F5 F5- (F5-GC)GC) and and F1 F1follicles follicles (F1 (F1-GC).-GC). (B (B,C,C) )Predominant Predominant expression expression ofof BMP15BMP15 ( B(B)) and and GDF9 GDF9 (C) mRNA(C) mRNAin 6–8 mm in 6–8 GC, mm 6 GC,–8 mm 6–8 mmOo, Oo,F5-GC F5-GC and and F1 F1-GC.-GC. In In graphs graphs A–C,A–C, differentdifferent letter letter indicates indicates the the statical statical difference difference betweenbetween two twogroups. groups. (D) (RTD)-PCR RT-PCR detection detection of ofGPR12 GPR12 mRNA mRNA expressed expressed in in chicken chicken 6 6–8–8 mm mm GC, GC, 6 6–8–8 mm Oo, F5-GC,F5-GC, and F1-GC.and The F1-GC. GPR12 The band GPR12 intensity band intensity is the strongest is the strongest in 6–8 in mm 6–8 Oo mm, and Oo, its and signal its signal in GC in GCcells cells decreases decreases along along follicle follicle development.development. No PCR Noband PCR was band de wastected detected in all in reverse all reverse transcription transcription (RT (RT)-negative)-negative controls.controls. In In graphs graphs A–D, A– eachD, each data data point point representsrepresents the themean mean ± SEM± SEM of 4 of replicates 4 replicates (N (N == 4). 4). (E (E) )The The constitutive constitutive activity activity of of GPR12 GPR12 in culturedin cultured 6 mm 6 mm GC transfectedGC transfected withwith cGPR12 cGPR12 plasmid plasmid as asdetected detected by by dual dual-luciferase-luciferase reporter assay.assay. Each Each data data point point represents represents the meanthe mean± SEM ± SEM of four of four replicatesreplicates (N = ( N4).= *** 4). p *** < p0.001< 0.001 vs. vs. control. control.

Interestingly,Interestingly, using using qRT-PCRqRT-PCR or or RT-PCR RT-PCR assay, assay, we we found found that thatGPR12 GPR12has a has compara- a compar- ativelytively high high expression level level in in the the oocytes oocytes (of (of 6 mm 6 mm follicles) follicles) (Figure (Figure6B–D), 6B where–D), therewhere is there previous report of abundant expression of BMP15 and GDF9 [30]. is previous report of abundant expression of BMP15 and GDF9 [30]. The expression of GPR12 in gonads led us to speculate that its expression in the GCs mayThe be expression regulated by of gonadalGPR12 in steroids, gonads such led asus estradiolto speculate (E2), that androgen its expression (DHT5α ),in and the GCs mayprogesterone be regulated (P4). by To gonadal test this, steroids, the GCs such collected as estradiol from 6 mm(E2), follicles androgen were (DHT5α) cultured, andand pro- gesteronetreated by (P4). E2, DHT5To testα, this, and P4, the and GCsGPR12 collectedmRNA from levels 6 mm were follicles examined were by qRT-PCR. cultured and treatedAs shown by E2, in DHT5α Figure7,, P4and treatment P4, and forGPR12 4 h or mRNA 24 h can levels increase wereGPR12 examinedexpression by qRT dose--PCR. As showndependently, in Figure while 7, P4 E2 treatment and DHT5 αforseem 4 h capableor 24 h to can slightly increase increase GPR12GPR12 expressionexpression. dose-dependently, while E2 and DHT5α seem capable to slightly increase GPR12 expression.

Genes 2021, 12, x FOR PEER REVIEW 13 of 19

Genes 2021, 12Genes, 489 2021, 12, x FOR PEER REVIEW 13 of 19 13 of 19

(A) (B) (C)

(D) (E) (F)

Figure 7. The effects of androgen (DHT5α) (A,D), progesterone (P4) (B,E), and estradiol (E2) (C,F) treatment (0 nM, 1 nM,

10 nM, and 100 nM) for 4 h and 24 h on cGPR12 mRNA expression in cultured granulosa cells from 6–8 mm follicles as detected by qPCRFigure. Each 7. The data effectsFigure point of 7. representsandrogenThe effects (the ofDHT5α androgen mean) (±A SEM, (DHT5D), progesterone ofα 4)( replicatesA,D), progesterone (P4 (N) =(B 4,).E ),* (P4) pand < 0 ( Bestradiol.05;,E), ** and p < estradiol (0E2.01;) ( C***,F (E2) )p treatment< (0C.001,F) vs. (0 nM, 1 nM, control. 10 nM, and 100treatment nM) for (04 h nM, and 1 24 nM, h 10on nM,cGPR12 and 100mRNA nM) expression for 4 h and in 24 cultured h on cGPR12 granulosamRNA cells expression from 6 in–8 mm follicles as detected by qPCRcultured. Each granulosa data point cells represents from 6–8 mmthe folliclesmean ± asSEM detected of 4 replicates by qPCR. Each(N = data4). * pointp < 0.05; represents ** p < 0 the.01; *** p < 0.001 vs. control. mean3.6. Identification± SEM of 4 replicates of the Promoter (N = 4). * p Regions< 0.05; ** ofp

3.6. IdentificationSince the3.6. 5′ ofIdentification- theuntranslated Promoter of Regions theregions Promoter of Chicken (5′- UTR)Regions GPR12 of of cGPR12 Chicken was GPR12 determined by 5′-RACE PCR (Figure 1D),0 it led us to speculate0 that the promoter region(s) driving0 GPR12 tran- Since the 5 -untranslatedSince the 5′ regions-untranslated (5 -UTR) ofregionscGPR12 (was5′-UTR) determined of cGPR12 by 5 -RACE was determined PCR by 5′-RACE (Figurescription1D), may it led be us located to speculate upstream that the of promoter the 5′-UTR. region(s) To drivingtest thisGPR12 notion,transcription we cloned the 5′- PCR (Figure 1D), it led us to speculate that the promoter region(s) driving GPR12 tran- mayflanking be located regions upstream of cGPR12 of the into 50-UTR. the To pGL3 test this-Basic notion, vector we and cloned test theed 5 0their-flanking promoter regions activities scription may be located upstream of the 5′-UTR. To test this notion, we cloned the 5′- ofin cGPR12culturedinto DF the-1 pGL3-Basiccells. As shown vector in and Figure tested 8, their the promoter 5′-flanking activities regions in cultured(from −3050 DF-1 to +277) flanking regions of0 cGPR12 into the pGL3-Basic vector and tested their promoter activities cells.of cGPR12 As shown display in Figure promoter8, the 5 activity-flanking in regions DF-1 cells. (from −3050 to +277) of cGPR12 display promoter activityin cultured in DF-1 DF cells.-1 cells. As shown in Figure 8, the 5′-flanking regions (from −3050 to +277) of cGPR12 display promoter activity in DF-1 cells.

0 FigureFigure 8. 8.Detection Detection of of promoter promoter activities activities of theof the 5 -flanking 5′-flanking region region of chicken of chickenGPR12 GPR12in cultured in cultured 0 chickenchicken fibroblast fibroblast cells cells line line (DF-1). (DF Various-1). Various lengths lengths of the 5of-flanking the 5′-flanking regions ofregionscGPR12 ofwere cGPR12 cloned were intocloned pGL3-Basic into pGL3Figure vector-Basic 8. for Detection thevector generation for of thepromoter of generation multiple activities promoter-luciferase of multiple of the 5′promoter-flanking constructs-luciferase region (−3050/+277 of constructschicken Luc GPR12 in cultured and(−3050/+277−1962/+277 Lucchicken Luc). and Thesefibroblast−1962/+277 promoter-luciferase cells Luc). line These (DF- constructs1promoter). Various- wereluciferase lengths then co-transfectedof constructs the 5′-flanking were into DF-1 thenregionscells co- trans-of cGPR12 were alongfected with into pRL-TK DFcloned-1 cells vector, into along andpGL3 with their-Basic promoterpRL vector-TK activitiesvector for the, and weregeneration their determined promoter of multiple by dual-luciferaseactivities promoter were reporter- luciferasedetermined constructs by 0 assays.dual-luciferase The transcriptional(−3 050/+277reporter assays.Luc start and site The identified−1962/+277 transcriptional by Luc). 5 -RACE These start was sitepromoter designated identified-luciferase as by “+1” 5′- (seeRACE constructs Figure was S1). designatedwere then co-trans- Eachas “+ value1” (see represents Figurefected intoS1). the meanDFEach-1 ±valuecellsSEM along represents of four with replicates pRL the -meanTK (N =vector 4).± SEM ***, pand of< 0.001 fourtheir vs.replicates promoter pGL3-Basic ( activitiesN = vector. 4). *** were p < determined by 0.001 vs. pGL3dual-Basic-luciferase vector. reporter assays. The transcriptional start site identified by 5′-RACE was designated as “+1” (see Figure S1). Each value represents the mean ± SEM of four replicates (N = 4). *** p < Using 0.001a promoter vs. pGL3 deletion-Basic vector. approach, we noted that the region from −1962 to +277 is capable to display strong promoter activities, hinting that the core promoter region of Using a promoter deletion approach, we noted that the region from −1962 to +277 is capable to display strong promoter activities, hinting that the core promoter region of

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Using a promoter deletion approach, we noted that the region from −1962 to +277 is capable to display strong promoter activities, hinting that the core promoter region of cGPR12 is likely located within this region. Using the AnimalTFDB 3.0 database [34], ﬁltered with Q values less than 0.01, binding sites for many transcription factors such as Sp1, USF, E2F, and AP2 were predicted to exist within this promoter region (−1962/+277) (Figure S1). How- ever, whether they are functional cis-regulatory elements requires further investigation.

4. Discussion In this study, the coding regions of chicken and duck (and zebraﬁsh) GPR3, GPR6, GPR12, and GPR12L were cloned. Functional assays demonstrated that avian GPR3, GPR6, GPR12, and GPR12L can constitutively activate the Gs-cAMP/PKA signaling pathway. RNA-Seq and qPCR assays indicated that GPR6 and GPR12L are highly expressed in the CNS, revealing their active involvement of CNS function. In the present study, GPR12 was found to be highly expressed in ovarian GCs and oocytes of 6 mm growing follicles in chickens. In combination with the observance that progesterone can upregulate GPR12 expression in the GCs, thus implying the active role of GPR12 in meiotic events. To our knowledge, our study represents the ﬁrst to report the expression and functionality of the four orphan receptors in birds.

4.1. Identification of GPR3, GPR6, GPR12, and GPR12L in Birds Although GPR3, GPR6, and GPR12 are predicted to exist in nonmammalian vertebrates, their structure, expression, and functionality have not been studied. The present study for the first time reported the cDNAs of GPR3, GPR6, GPR12, and the novel GPR12L from chicken, ducks, and zebrafish, confirming that these orphan receptors are expressed in nonmammalian vertebrate species, including birds. Interestingly, we can only identify GPR6, GPR12, and GPR12L from chicken brain, while GPR3 is likely lost in chickens, as revealed by synteny analysis. Considering the relatively low degree of a.a. sequence identity of GPR3 among vertebrates (Supplemental Table S4) and the functional redundance of these orphan receptors, it is not surprising that GPR3 might have been lost during speciation, resulting in its absence in some avian species, e.g., in chickens, quails, and turkeys. In the present study, sequence analyses revealed that all these receptors share common conserved motifs and structural features. For example, they all retain DRY, XWXP, and NPXXY motifs present in TM3, TM6, and TM7, respectively [32]. In this study, we also found that GPR12L, the novel receptor identified in chickens and ducks, also shares these conserved motifs. Despite the high structural similarity shared among these receptors (Supplemental Table S4), the differences in their N- and C-termini, extracellular loops, intracellular loops, and transmembrane regions were observed. In the present study, the sequence analyses indicated a relatively low a.a. sequence identity (Supplemental Table S4, 60% a.a. identity) among four structurally related orphan receptors, including the novel GPR12L. Although low sequence identity exists between orthologous GPR receptors, all these genes could be classified into a homologous subfamily, reference to research in olfactory receptors [35]. Together with the phylogenetic tree, our study showing the closer evolutionary relationship among GPR3, GPR6, GPR12, and GPR12L led us to propose that the four receptors are likely originated from a common ancestral GPR gene, which had experienced two rounds of genome duplication (2R) during early vertebrate evolution, thus resulting in the current receptor repertoire in higher vertebrate species such as spotted gars, turtles, and birds. In addition, the third round of genome duplication (3R) in the teleost lineage likely resulted in the presence of two GPR12L genes (GPR12La, GPR12Lb) in some teleosts (e.g., zebrafish) (Figure2E). In this study, regardless of the absence or presence of their ligands, all the four receptors from chickens, ducks, zebrafish, pigs, and humans are proven to be constitutively active. As shown in Figure4, without stimulation by any ligands, the expression of GPR3, GPR6, GPR12, and GPR12L caused more than 30-fold increase in luciferase activities of HEK293 cells when compared with the control group. The present study is in accor- Genes 2021, 12, 489 15 of 19

dance with the reports from mouse and rat [13,16,19], supporting their active roles across species. Interestingly, we noted that chicken GPR6, GPR12, and GPR12L expression can also enhance ERK phosphorylation levels. Similar signal pathways have been detected in mouse, showing that GPR3 and GPR6 are able to mediate the anti-apoptotic effect of PC cells through extracellular signal-regulated kinase 1/2 (ERK1/2) [36,37]. In addition, overexpression of GPR12 may induce PC12 cells to differentiate into neuron-like cells by activating the ERK1/2 signaling pathway [38]. The constitutive activities shared by these receptors, including GPR3, GPR6, GPR12, and GPR12L, suggest that these receptors may exert important roles in target tissues, similarly to their mammalian counterparts [39].

4.2. Tissue Expression of GPR3, GPR6, GPR12, and GPR12L in Chickens and Ducks Understanding the tissue distribution of genes is important to probe their physiological roles in a species. In the present study, GPR6 was detected to be highly expressed in the adrenal gland and CNS, including the hypothalamus in chickens. Our findings are in part consistent with those findings in mammals where GPR6 is mainly located in the striatum and hypothalamus and participates in neurite outgrowth, thus being related with Alzheimer’s disease, Parkinson’s disease, and instrumental learning [14–16,40,41]. In the present study, the expression of GPR6 was not detected in stomach and testes, in contrast with the observance from Morales et al. [1]. In the present study, the expression of GPR12 was found to be expressed in a wide range of tissues, including pituitary, testis, ovary, and the brain subregions. Of the multiple tissues, the GPR12 was found to be expressed abundantly in pituitary, testis, and ovary. The tissue distribution of GPR12 in chicken was partially in accordant with that in human, where GPR12 were detected in the eyes, breast, liver, and skin and the brain subregions, including cerebral cortex, striatum, pituitary, and cerebellum [42,43]. It was reported that GPR12 expression is associated with neurite outgrowth and neuronal development, cell survival, proliferation, and carcinogenesis [14,19]. In this study, using qRT-PCR, we found that cGPR12 is highly expressed in oocytes and GCs of 6 mm prehierarchy follicles and its expression decreases gradually towards the follicle maturation and is the lowest in F1 follicles. Our data are similar to the finding in rodents, in which GPR12 and GPR3 are expressed in oocytes [19]. In the present study, the novel gene GPR12L was detected to be highly expressed in brain subregions including cerebrum, midbrain, cerebellum, hindbrain, hypothalamus, spinal cord, pineal body, retina, and pituitary. In combination with the observance that GPR3 is lost in chicken and there is a low abundance of GPR12 in brain subregions, the novel gene GPR12L may partially replace the function of GPR3 in the system. Similarly to the expression profiles of GRP6, GPR12, and GPR12L detected in chickens, GPR6 are highly expressed in the brain, while GPR12 and GPR12L are highly expressed in the gonads and brain in ducks (Figure S2C). Since GPR3 is found to exist in ducks, tissue expression analysis revealed that GPR3 is highly expressed in tissue brain and gonads, similarly to the study by Tanaka et al., [11–14,44,45]. In spotted gars and zebrafish, we also noted that GPR3, GPR6, GPR12, and GPR12L are highly expressed in the brains and gonads (Figure S2). The conserved expression of these receptors in the brain and gonads among vertebrates strongly suggests that the four “orphan” receptors play crucial roles in these tissues.

4.3. GPR12 is Expressed in Oocytes and GCs of 6-mm Growing Follicles In the present study, the high abundance of cGPR12 is found to be expressed in oocytes and the GCs of 6 mm prehierarchy follicles and its expression decreases gradually towards the follicle maturation. Together with the observance that GPR3 is likely lost in the chicken genome, the predominant expression of cGPR12 in oocytes and the surrounding GCs of chicken growing follicles (6 mm), in combination with its mediation of cAMP/PKA signaling pathway (Figure6E), strongly supports its involvement in oocyte meiosis arrest. In vertebrates, oocyte meiosis will arrest at the diplotene stage for a long time. Then, Genes 2021, 12, 489 16 of 19

oocyte meiosis will resume at the stage of sexual maturity when pituitary luteinizing hormone (LH) surges to trigger the events. The high cAMP level within the oocytes is reported to be necessary to maintain the meiosis arrest [46]. It is reported that the high cAMP concentration may be from the cumulus cells that enter into the oocyte through gap junctions [47]. In the later study, a report showed that the cAMP may be produced by the oocytes themselves through the Gαs signaling under hormone stimulation [48]. In 2004, Kalinowski et al. [49] found that the injection of negative-domain form of Gαs into oocytes of mice, Xenopus, and zebrafish can lead to the resumption of meiosis in oocytes [49]. The abundant expression of GPR12 in GCs led us to examine whether gonadotropin and gonadal steroids can regulate GPR12 expression in GCs. Our study showed that FSH or LH cannot regulate GPR12 expression in vitro. However, we found that P4 can significantly increase GPR12 expression in cultured GCs from 6–8 mm follicles in a time- and dose-dependent manner, while E2 and DHT5α only increase GPR12 expression slightly. Considering that P4 is predominantly expressed in GCs of the largest preovulatory F1 follicles, the upregulation of GPR12 induced by P4 in these follicles may enhance cAMP levels, which will further maintain the high cAMP levels within the oocytes through gap junction [50,51]. Our data provide the first piece of evidence that gonadal steroid, i.e., P4, can upregulate GPR12 expression in the gonads of vertebrates. The identification of the core promoter region of chicken GPR12 is also helping to explore the detailed regulation mechanism by P4 in further investigation.

5. Conclusions In summary, four orphan receptors, namely, GPR3, GPR6, and GPR12, and a novel GPR12-like receptor (GPR12L), were cloned in chickens/ducks and other vertebrate species in this study. Functional study elucidated that GPR3, GPR6, GPR12, and GPR12L are constitutively active and capable of increasing intracellular cAMP levels. RNA-seq assays revealed that avian GPR3, GPR6, and GPR12L are mainly expressed in the brain and GPR12 is highly expressed in the pituitary and gonads. Moreover, we observed that GPR12 is highly expressed in the oocytes and GCs of growing follicles and its expression in GC is likely regulated by P4. Our data provide valuable insights into the function, tissue expression, and roles of these orphan receptors in vertebrates under a comparative endocrinology perspective.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/1 0.3390/genes12040489/s1, Figure S1: Analysis of the promoter region of cGPR12, Figure S2: RNA- seq data analysis showing the tissue expression of GPR3, GPR6, GPR12, GPR12La (GPR185a), and GPR12Lb (GPR185b) in spotted gars (A) and zebraﬁsh (B) duck (C) tissues, Table S1: Primers used in this study, Table S2. Lists of the GPCR sequences, their GenBank accession numbers and species used in amino acid sequence alignment, Table S3. Lists of genes and their GenBank accession numbers used to generate the phylogenetic tree in this study, Table S4. Amino acid sequence identity of GPR3, GPR6, GPR12, GPR12L among vertebrate species including chickens, humans, ducks, zebraﬁsh, and spotted gars. Author Contributions: Conceptualization, Z.L. and Y.W.; methodology, Z.L.; validation, B.C., B.J. and Z.Z.; formal analysis, B.C., B.J. and J.Z.; investigation, J.L. and J.Z.; resources, J.L., Y.H. and Y.W.; writing—original draft preparation, Z.L. and Y.W.; writing—review and editing, Y.H. and Y.W.; visualization, Z.L. and J.Z.; supervision, Y.W.; funding acquisition, Y.H. and Y.W. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by grants from the National Natural Science Foundation of China (31771375, 31772590, 32072706), the Science and Technology Support Program of Sichuan (2016NYZ0042), and Open projects of Key Laboratory SFGA on conservational biology of rare animals in the giant panda national park (KLSFGAGP2020.001, KLSFGAGP2020.027). Genes 2021, 12, 489 17 of 19

Institutional Review Board Statement: All animal experimental protocols used in this study were approved by the Animal Ethics Committee of College of Life Sciences, Sichuan University, and the assurance number is 2020030808 (8 March 2020). Informed Consent Statement: “Not applicable” for studies not involving humans. Data Availability Statement: Data sharing not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Morales, P.; Isawi, I.; Reggio, P.H. Towards a better understanding of the cannabinoid-related orphan receptors GPR3, GPR6, and GPR12. Drug Metab. Rev. 2018, 50, 74–93. [CrossRef] 2. Fredriksson, R.; Lagerstrom, M.C.; Lundin, L.G.; Schioth, H.B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 2003, 63, 1256–1272. [CrossRef] 3. Fredriksson, R.; Schioth, H.B. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 2005, 67, 1414–1425. [CrossRef] 4. Sun, Q.Y.; Miao, Y.L.; Schatten, H. Towards a new understanding on the regulation of mammalian oocyte meiosis resumption. Cell Cycle 2009, 8, 2741–2747. [CrossRef] 5. Uhlenbrock, K.; Gassenhuber, H.; Kostenis, E. Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors. Cell. Signal. 2002, 14, 941–953. [CrossRef] 6. Yin, H.; Chu, A.; Li, W.; Wang, B.; Shelton, F.; Otero, F.; Nguyen, D.G.; Caldwell, J.S.; Chen, Y.A. Lipid G protein-coupled receptor ligand identification using β-arrestin PathHunter assay. J. Biol. Chem. 2009, 284, 12328–12338. [CrossRef][PubMed] 7. Valverde, O.; Celerier, E.; Baranyi, M.; Vanderhaeghen, P.; Maldonado, R.; Sperlagh, B.; Vassart, G.; Ledent, C. GPR3 receptor, a novel actor in the emotional-like responses. PLoS ONE 2009, 4, e4704. [CrossRef] 8. Eggerickx, D.; Denef, J.F.; Labbe, O.; Hayashi, Y.; Refetoff, S.; Vassart, G.; Parmentier, M.; Libert, F. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase. Biochem. J. 1995, 309 (Pt 3), 837–843. [CrossRef] 9. Martin, A.L.; Steurer, M.A.; Aronstam, R.S. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors. PLoS ONE 2015, 10, e0138463. [CrossRef] 10. Seifert, R.; Wenzel-Seifert, K. Constitutive activity of G-protein-coupled receptors: Cause of disease and common property of wild-type receptors. Naunyn. Schmiedebergs Arch. Pharmacol. 2002, 366, 381–416. [CrossRef][PubMed] 11. Godlewski, G.; Jourdan, T.; Szanda, G.; Tam, J.; Cinar, R.; Harvey-White, J.; Liu, J.; Mukhopadhyay, B.; Pacher, P.; Ming Mo, F.; et al. Mice lacking GPR3 receptors display late-onset obese phenotype due to impaired thermogenic function in brown adipose tissue. Sci. Rep. 2015, 5, 14953. [CrossRef][PubMed] 12. Huang, Y.; Skwarek-Maruszewska, A.; Horre, K.; Vandewyer, E.; Wolfs, L.; Snellinx, A.; Saito, T.; Radaelli, E.; Corthout, N.; Colombelli, J.; et al. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer’s disease mouse models. Sci. Transl. Med. 2015, 7, 309ra164. [CrossRef][PubMed] 13. Mehlmann, L.M.; Saeki, Y.; Tanaka, S.; Brennan, T.J.; Evsikov, A.V.; Pendola, F.L.; Knowles, B.B.; Eppig, J.J.; Jaffe, L.A. The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes. Science 2004, 306, 1947–1950. [CrossRef][PubMed] 14. Tanaka, S.; Ishii, K.; Kasai, K.; Yoon, S.O.; Saeki, Y. Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth. J. Biol. Chem. 2007, 282, 10506–10515. [CrossRef] 15. Oeckl, P.; Hengerer, B.; Ferger, B. G-protein coupled receptor 6 deficiency alters striatal dopamine and cAMP concentrations and reduces dyskinesia in a mouse model of Parkinson’s disease. Exp. Neurol. 2014, 257, 1–9. [CrossRef][PubMed] 16. Benoit, M.E.; Hernandez, M.X.; Dinh, M.L.; Benavente, F.; Vasquez, O.; Tenner, A.J. C1q-induced LRP1B and GPR6 proteins expressed early in Alzheimer disease mouse models, are essential for the C1q-mediated protection against amyloid-β neurotoxicity. J. Biol. Chem. 2013, 288, 654–665. [CrossRef] 17. Han, S.J.; Conti, M. New pathways from PKA to the Cdc2/cyclin B complex in oocytes: Wee1B as a potential PKA substrate. Cell Cycle 2006, 5, 227–231. [CrossRef] 18. Oh, J.S.; Han, S.J.; Conti, M. Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption. J. Cell Biol. 2010, 188, 199–207. [CrossRef] 19. Hinckley, M.; Vaccari, S.; Horner, K.; Chen, R.; Conti, M. The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes. Dev. Biol. 2005, 287, 249–261. [CrossRef] 20. Deng, J.; Lang, S.; Wylie, C.; Hammes, S.R. The Xenopus laevis isoform of G protein-coupled receptor 3 (GPR3) is a constitutively active cell surface receptor that participates in maintaining meiotic arrest in X. laevis oocytes. Mol. Endocrinol. 2008, 22, 1853–1865. [CrossRef] 21. Yang, C.R.; Wei, Y.; Qi, S.T.; Chen, L.; Zhang, Q.H.; Ma, J.Y.; Luo, Y.B.; Wang, Y.P.; Hou, Y.; Schatten, H.; et al. The G protein coupled receptor 3 is involved in cAMP and cGMP signaling and maintenance of meiotic arrest in porcine oocytes. PLoS ONE 2012, 7, e38807. [CrossRef][PubMed] 22. Zhu, G.; Fang, C.; Mo, C.; Wang, Y.; Huang, Y.; Li, J. Transcriptomic analysis of granulosa cell populations proximal and distal to the germinal disc of chicken preovulatory follicles. Sci. Rep. 2021, 11, 1–14. [CrossRef][PubMed] Genes 2021, 12, 489 18 of 19

23. Gilbert, A.; Evans, A.; Perry, M.; Davidson, M. A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus). Reproduction 1977, 50, 179–181. [CrossRef][PubMed] 24. Cai, G.; Mo, C.; Huang, L.; Li, J.; Wang, Y. Characterization of the Two CART Genes (CART1 and CART2) in Chickens (Gallus gallus). PLoS ONE 2015, 10, e0127107. [CrossRef][PubMed] 25. Hall, T.; Biosciences, I.; Carlsbad, C. BioEdit: An important software for molecular biology. GERF Bull. Biosci. 2011, 2, 60–61. 26. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [CrossRef] 27. Zhang, J.; Li, X.; Zhou, Y.; Cui, L.; Li, J.; Wu, C.; Wan, Y.; Li, J.; Wang, Y. The interaction of MC3R and MC4R with MRAP2, ACTH, α-MSH and AgRP in chickens. J. Endocrinol. 2017, 234, 155–174. [CrossRef] 28. Zhang, J.; Li, J.; Wu, C.; Hu, Z.; An, L.; Wan, Y.; Fang, C.; Zhang, X.; Li, J.; Wang, Y. The Asp298Asn polymorphism of melanocortin- 4 receptor (MC4R) in pigs: Evidence for its potential effects on MC4R constitutive activity and cell surface expression. Anim. Genet. 2020, 51, 694–706. [CrossRef] 29. He, C.; Zhang, J.; Gao, S.; Meng, F.; Bu, G.; Li, J.; Wang, Y. Molecular characterization of three NPY receptors (Y2, Y5 and Y7) in chickens: Gene structure, tissue expression, promoter identification, and functional analysis. Gen. Comp. Endocrinol. 2016, 236, 24–34. [CrossRef][PubMed] 30. Wang, Y.; Li, J.; Ying Wang, C.; Yan Kwok, A.H.; Leung, F.C. Epidermal growth factor (EGF) receptor ligands in the chicken ovary: I. Evidence for heparin-binding EGF-like growth factor (HB-EGF) as a potential oocyte-derived signal to control granulosa cell proliferation and HB-EGF and kit ligand expression. Endocrinology 2007, 148, 3426–3440. [CrossRef][PubMed] 31. Wang, Y.; Li, J.; Yan Kwok, A.H.; Ge, W.; Leung, F.C. A novel prolactin-like protein (PRL-L) gene in chickens and zebrafish: Cloning and characterization of its tissue expression. Gen. Comp. Endocrinol. 2010, 166, 200–210. [CrossRef][PubMed] 32. Morales, P.; Hurst, D.P.; Reggio, P.H. Methods for the Development of In Silico GPCR Models. Methods Enzymol. 2017, 593, 405–448. [CrossRef] 33. Cheng, Z.; Garvin, D.; Paguio, A.; Stecha, P.; Wood, K.; Fan, F. Luciferase Reporter Assay System for Deciphering GPCR Pathways. Curr. Chem. Genom. 2010, 4, 84–91. [CrossRef][PubMed] 34. Hu, H.; Miao, Y.-R.; Jia, L.-H.; Yu, Q.-Y.; Zhang, Q.; Guo, A.-Y. AnimalTFDB 3.0: A comprehensive resource for annotation and prediction of animal transcription factors. Nucleic Acids Res. 2019, 47, D33–D38. [CrossRef] 35. Olender, T.; Jones, T.E.; Bruford, E.; Lancet, D. A unified nomenclature for vertebrate olfactory receptors. BMC Evol. Biol. 2020, 20, 42. [CrossRef][PubMed] 36. Ignatov, A.; Lintzel, J.; Kreienkamp, H.J.; Schaller, H.C. Sphingosine-1-phosphate is a high-affinity ligand for the G protein- coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway. Biochem. Biophys. Res. Commun. 2003, 311, 329–336. [CrossRef] 37. Tanaka, S.; Miyagi, T.; Dohi, E.; Seki, T.; Hide, I.; Sotomaru, Y.; Saeki, Y.; Antonio Chiocca, E.; Matsumoto, M.; Sakai, N. Developmental expression of GPR3 in rodent cerebellar granule neurons is associated with cell survival and protects neurons from various apoptotic stimuli. Neurobiol. Dis. 2014, 68, 215–227. [CrossRef] 38. Lu, X.; Zhang, N.; Dong, S.; Hu, Y. Involvement of GPR12 in the induction of neurite outgrowth in PC12 cells. Brain Res. Bull. 2012, 87, 30–36. [CrossRef][PubMed] 39. Srinivasan, S.; Lubrano-Berthelier, C.; Govaerts, C.; Picard, F.; Santiago, P.; Conklin, B.R.; Vaisse, C. Constitutive activity of the melanocortin-4 receptor is maintained by its N-terminal domain and plays a role in energy homeostasis in humans. J. Clin. Invest. 2004, 114, 1158–1164. [CrossRef] 40. Song, Z.H.; Young, W.S., 3rd; Brownstein, M.J.; Bonner, T.I. Molecular cloning of a novel candidate G protein-coupled receptor from rat brain. FEBS Lett. 1994, 351, 375–379. [CrossRef] 41. Heiber, M.; Docherty, J.M.; Shah, G.; Nguyen, T.; Cheng, R.; Heng, H.H.; Marchese, A.; Tsui, L.C.; Shi, X.; George, S.R.; et al. Isolation of three novel human genes encoding G protein-coupled receptors. DNA Cell Biol. 1995, 14, 25–35. [CrossRef] 42. Saeki, Y.; Ueno, S.; Mizuno, R.; Nishimura, T.; Fujimura, H.; Nagai, Y.; Yanagihara, T. Molecular-Cloning of a Novel Putative G-Protein-Coupled Receptor (Gpcr21) Which Is Expressed Predominantly in Mouse Central-Nervous-System. FEBS Lett. 1993, 336, 317–322. [CrossRef] 43. Eidne, K.A.; Zabavnik, J.; Peters, T.; Yoshida, S.; Anderson, L.; Taylor, P.L. Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland. FEBS Lett. 1991, 292, 243–248. [CrossRef][PubMed] 44. Zhang, B.; Ding, J.; Li, Y.; Wang, J.; Zhao, Y.; Wang, W.; Shi, S.; Dong, F.; Zhang, Z.; Shi, F.; et al. The porcine Gpr3 gene: Molecular cloning, characterization and expression level in tissues and cumulus-oocyte complexes during in vitro maturation. Mol. Biol. Rep. 2012, 39, 5831–5839. [CrossRef][PubMed] 45. Iismaa, T.P.; Kiefer, J.; Liu, M.L.; Baker, E.; Sutherland, G.R.; Shine, J. Isolation and chromosomal localization of a novel human G-protein-coupled receptor (GPR3) expressed predominantly in the central nervous system. Genomics 1994, 24, 391–394. [CrossRef] 46. Cho, W.K.; Stern, S.; Biggers, J.D. Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J. Exp. Zool. 1974, 187, 383–386. [CrossRef] 47. Dekel, N.; Aberdam, E.; Sherizly, I. Spontaneous maturation in vitro of cumulus-enclosed rat oocytes is inhibited by forskolin. Biol. Reprod. 1984, 31, 244–250. [CrossRef] Genes 2021, 12, 489 19 of 19

48. Bornslaeger, E.A.; Schultz, R.M. Adenylate cyclase activity in zona-free mouse oocytes. Exp. Cell Res. 1985, 156, 277–281. [CrossRef] 49. Kalinowski, R.R.; Berlot, C.H.; Jones, T.L.; Ross, L.F.; Jaffe, L.A.; Mehlmann, L.M. Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway. Dev. Biol. 2004, 267, 1–13. [CrossRef][PubMed] 50. Webb, R.J.; Marshall, F.; Swann, K.; Carroll, J. Follicle-stimulating hormone induces a gap junction-dependent dynamic change in [cAMP] and protein kinase a in mammalian oocytes. Dev. Biol. 2002, 246, 441–454. [CrossRef] 51. Dekel, N.; Lawrence, T.S.; Gilula, N.B.; Beers, W.H. Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev. Biol. 1981, 86, 356–362. [CrossRef]