Abstract
Melanocortin-3 receptor (MC3R) is a regulator of energy homeostasis, and interaction of MC3R and melanocortin-2 receptor accessory protein 2 (MRAP2) plays a critical role in MC3R signaling of mammals. However, the physiological roles of MC3R in teleosts are not well understood. In this study, qRT-PCR was used to measure gene expression. Radioligand binding assay was used to study the binding properties of topmouth culter MC3R (caMC3R). Intracellular cAMP generation was determined by RIA, and caMC3R expression was quantified with flow cytometry. We showed that culter mc3r had higher expression in the CNS. All agonists could bind and stimulate caMC3R to increase dose dependently intracellular cAMP accumulation. Compared to human MC3R, culter MC3R showed higher constitutive activity, higher efficacies, and Rmax to alpha-melanocyte-stimulating hormone (α-MSH), des-α-MSH, and adrenocorticotrophic hormone. Both caMRAP2a and caMRAP2b markedly decreased caMC3R basal cAMP production. However, only caMRAP2a significantly decreased cell surface expression, Bmax, and Rmax of caMC3R. Expression analysis suggested that MRAP2a and MRAP2b might be more important in regulating MC3R/MC4R signaling during larval period, and reduced mc3r, mc4r, and pomc expression might be primarily involved in modulation of MC3R/MC4R in adults. These data indicated that the cloned caMC3R was a functional receptor. MRAP2a and MRAP2b had different effects on expression and signaling of caMC3R. In addition, expression analysis suggested that MRAP2s, receptors, and hormones might play different roles in regulating culter development and growth.
Introduction
Melanocortin receptors (MCRs) belong to rhodopsin-like family A G-protein-coupled receptors (GPCRs). Five MCRs (named MC1R–MC5R), with diverse ligand affinities (including α-, β-, γ-melanocyte-stimulating hormones (MSHs) and adrenocorticotropic hormone (ACTH)) and multiple physiological roles, have been extensively studied in mammals (1, 2, 3, 4). MC3R and MC4R are known as neural MCRs with high expression in the CNS (5, 6, 7, 8). These two MCRs play vital roles in modulation of energy homeostasis. Mutations in MC3R and MC4R are associated with obesity (9, 10, 11). Mc4r knockout mice show obesity phenotype with increased food intake and decreased energy expenditure (12, 13). Targeted deletion of Mc3r in mice show a moderate obesity phenotype with decreased lean mass, increased fat mass, normal food intake, and metabolism, suggesting that MC3R could regulate feed efficiency and alterations in nutrient partitioning (14, 15, 16). In addition, studies found that MC3R plays a key role in anomalous metabolic adaption to restricted feeding (17, 18). A recent study showed that MC3R is a critical regulator of boundary controls on melanocortin signaling, providing rheostatic control on energy storage (19).
In addition to the CNS expression, MC3R is also expressed in several peripheral tissues, including the intestine, placenta, heart, gut, kidney, and macrophages (5, 20, 21, 22, 23). Owing to its wide expression, MC3R has been shown to have other potential physiological functions in the periphery, including involvement in immune response and inflammation (20, 23, 24, 25, 26), regulating cardiovascular function (27, 28), and natriuresis (29). The MC3R primarily couples to the stimulatory G protein to stimulate adenylyl cyclase activity, leading to increased production of the intracellular second messenger cAMP to trigger downstream signaling.
MCRs have been shown to interact with small single transmembrane proteins – melanocortin-2 receptor accessory proteins (MRAPs, including MRAP1 and MRAP2) (30, 31, 32, 33) (reviewed in (34, 35)). MRAP2 has high expression in CNS and plays a crucial role in regulating energy homeostasis. Targeted deletion of Mrap2 in mice shows severe obesity (36, 37). MRAP2 interacts with and modulates MC4R signaling in mammals and other species (33, 38, 39, 40, 41, 42). Additionally, the function of MRAP2 in regulating energy homeostasis through MC3R has also been reported (32, 38, 43).
Considering the crucial importance of energy metabolism, understanding the endocrine modulation of energy homeostasis is important for economically important fishes and may potentially lead to novel approaches to manipulate fish growth, feed efficiency, and final product quality in cultured fish. Hence, it is not surprising that MC3R has also attracted some attention in fish. Our mining of NCBI database and literature search revealed that the mc3r gene is found in some fish, including Holocephali (elephant shark Callorhinchusmilii), Elasmobranchii (spiny dogfish Squalusacanthias, thorny skate Amblyrajaradiata, red stingray Hemitrygonakajei, velvet belly lantern shark Etmopterusspinax), Polypteriformes (reedfish Erpetoichthyscalabaricus, gray bichir Polypterussenegalus), Lepisosteiforme (spotted gar Lepisosteusoculatus), Coelacanths (coelacanth Latimeriachalumnae), and teleosts including zebrafish Daniorerio, common carp Cyprinuscarpio, Mexican tetra Astyanaxmexicanus, red-bellied piranha Pygocentrusnattereri, yellow catfish Tachysurusfulvidraco, channel catfish Ictaluruspunctatus, striped catfish Pangasianodonhypophthalmus, electric eel Electrophorus electricus, Chinook salmon Oncorhynchustshawytscha, coho salmon Oncorhynchuskisutch, river trout Salmotrutta, rainbow trout Oncorhynchusmykiss, Arctic char Salvelinusalpinus, northern pike Esoxlucius, denticle herring Denticepsclupeoides, and Asian bonytongue Scleropagesformosus, although it is absent in other species including lungfish Dipnomorpha, Acipenseriformes (Yangtze sturgeon Acipenserdabryanus and American paddlefish Polyodonspathula), cichlid Simochromisdiagramma, medaka Oryziaslatipes, fugu Takifugurubripes, stickleback Gasterosteusaculeatus, ricefield eel Monopterusalbus, and orange-spotted grouper Epinepheluscoioides (39, 44, 45, 46, 47). Since MC3R is considered a specific receptor for γ-MSH in higher vertebrates (5, 7) and γ-MSH is absent in teleosts (48), the absence of MC3R in some fish might be considered as one example of co-evolution of ligand and receptor.
Only three studies have investigated the pharmacological properties of fish MC3Rs so far (43, 44, 49). Of interest, two studies reported high constitutive activities in zebrafish and channel catfish MC3Rs (43, 49), similar to the results in teleost MC4Rs (39, 40, 41, 47, 50, 51, 52, 53) and MC1R (54). In this study, topmouth culter (Culteralburnus) was used as an animal model to explore the physiology and pharmacology of culter MC3R. Topmouth culter is an important species of freshwater fish with wide distribution in reservoirs, rivers, and lakes in China (55, 56). We cloned culter mc3r and explored its tissue distribution. We also investigated the pharmacology of caMC3R and modulation by caMRAP2s. The potential functions of MC3R, MC4R, and MRAP2s in embryo development and adult were also studied.
Materials and methods
Ligands and plasmids
[Nle4,D-Phe7]-α-MSH (NDP-MSH) was purchased from Peptides International (Louisville, KY, USA). Human α-MSH was obtained from Pi Proteomics (Huntsville, AL, USA). Human ACTH (1–24) was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). Human des-acetyl-α-MSH was obtained from GenScript (Piscataway, NJ, USA). Culter α-MSH and ACTH are 100 and 87.5% identical with the corresponding human counterparts, respectively (40). [125I]-cAMP and [125I]-NDP-MSH were iodinated using chloramine T method (57, 58). The human MC3R (hMC3R) subcloned into pcDNA3.1 vector (pcDNA3.1-hMC3R) was generated as previously described (59). N-terminal Flag-tagged caMRAP2a and N-terminal Flag-tagged caMRAP2b were reported before (40). N-terminal myc-tagged caMC3R was commercially synthesized and subcloned into pcDNA3.1 by GenScript to generate the plasmid used for transfection.
Animal studies
All animal experiments were approved by Animal Care Committee of Hunan Normal University and was in strict accordance with the Management Rule of Laboratory Animals (Chinese Order No. 676 of the State Council, revised March 1, 2017). Culters were provided and fed in the Engineering Center of Polyploid Fish Breeding of the Ministry of Education (Hunan Normal University, China). After 3 days of acclimation, fish were used for experimentation. Fish were reared in tanks (height of 100 cm and diameter of 95 cm) and fed twice daily at 09:00 and 17:00 h. Experimental conditions were as follows: natural light, water temperature (22–28°C), and dissolved oxygen (approximately 8 mg/L). All fish were anesthetized with MS222 (1:10,000, Sangon Biotech, China) and sampled immediately. Brain was collected and stored at −80°C for RNA extraction and qRT-PCR.
Gene cloning and sequence alignment
Gene cloning was performed according to the procedure described previously (40). Briefly, total RNA was purified using Trizol™ reagent (Invitrogen). The first-strand cDNA was synthesized using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). Primers were designed via Primer Premier 5.0 (Supplementary Table 1, see section on supplementary materials given at the end of this article). PCR products were separated through 1.2% agarose gels, ligated to pMD18-T vector (Takara), and sequenced (Sangon Biotech).
Tissue distribution of mc3r
To explore the tissue distribution of mc3r, the mesencephalon, olfactory bulb, cerebellum, telencephalon, medulla, hypothalamus, pituitary gland, muscle, heart, gonads (ovaries and testes), liver, head kidney, spleen, skin, gill, and kidney were collected from three females or males. Three pairs of primers for each gene were designed by AlleleID 6. Each pair of primers was tested for amplification efficiency and melting curve. The primers were selected in qRT-PCR with 95–105% amplification efficiency and single peak melting curve (Supplementary Table 1). The same trend was found when β-actin, hprt, and gapdh were used as housekeeping genes to normalize target gene expression. Thus, β-actin was used as the internal reference for normalization in this study. The qRT-PCR was performed using Prism 7500 Sequence Detection System (ABI, Foster City, CA, USA). The amplification was performed in a total volume of 10 μL, containing 5 μL SYBR green PCR Master Mix, 0.5 μL each primer, 3 μL water, and 1 μL cDNA. The RT-qPCR program was set as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 61°C for 45 s. Each sample was added to a 96-well plate repeated thrice. The relative expression of genes was calculated using the 2−ΔΔCT method (60).
Cell culture and transfection
Human embryonic kidney (HEK) 293T cells (ATCC) were cultured in an incubator (37°C in a 5% CO2-humidified atmosphere) (61). Briefly, the medium contained Dulbecco’s Modified Eagle’s medium (Invitrogen), 10% newborn calf serum, 0.25 µg/mL of amphotericin B, 100 IU/mL of penicillin, 100 µg/mL streptomycin, 50 µg/mL of gentamicin, and 10 mM HEPES. Cells were plated into 6-well or 24-well plates pre-coated with 0.1% gelatin. At approximately 70% confluency, cells were transfected with 0.25 µg/μL MC3R with or without MRAP2 plasmids using calcium phosphate precipitation method (62). Empty vector pcDNA3.1 was used to normalize the total DNA in each well.
Flow cytometry assay
The expression of the caMC3R was quantified with flow cytometry as described earlier (53, 63), carried out by the C6 Accuri Cytometer (Accuri Cytometers, Ann Arbor, MI, USA). Four ratios (1:0, 1:1, 1:3, and 1:5) of caMC3R and caMRAP2a/caMRAP2b plasmids were co-transfected into cells in six-well plates. Fluorescence of cells transfected with empty vector (pcDNA3.1) was used for background staining. The expression of the caMC3R was calculated as the percentage of 1:0 group was set as 100% expression. The expression levels of other groups were calculated as % of the 1:0 group (63).
Ligand binding assays
Binding assay was described previously (43, 61). The ligands and their final concentrations used in this study were NDP-MSH (from 10−12 to 10−6 M), α-MSH (from 10−12 to 10−6 M), des-acetyl-α-MSH (from 10−12 to 10−6 M), or ACTH (1–24) (from 10−12 to 10−6 M). To investigate the modulation of caMRAP2a or caMRAP2b on the binding property of caMC3R, caMC3R (0.25 μg/μL) and caMRAP2a or caMRAP2b plasmids in two ratios (1:0 and 1:5) were applied to co-transfect cells (six-well plate), and ACTH (1–24) (from 10−11 to 10−6 M) and α-MSH (from 10−10 to 10−5 M) were used.
cAMP assays
cAMP signaling assay was performed as described previously (57, 61). The final concentration of ligands used was 10−12 to 10−6 M. To explore the effects of caMRAP2a and caMRAP2b on caMC3R signaling, cells (24-well plate) were transfected with caMC3R (0.25 μg/μL) and caMRAP2a or caMRAP2b plasmids in two ratios (1:0 and 1:5), and two ligands, α-MSH (from 10−13 to 10−7 M) and ACTH (1–24) (from 10−13 to 10−7 M) were used. To investigate the dose-dependent modulation of caMRAP2a or caMRAP2b on maximal response (Rmax) of cAMP signaling of caMC3R to α-MSH stimulation (10−6 M), four ratios (1:0, 1:1, 1:3, and 1:5) of caMC3R (0.25 μg/μL) and caMRAP2a or caMRAP2b were co-transfected into cells (24-well plate). To study the constitutive activity of cAMP signaling, caMC3R plasmid in increasing concentrations (0, 0.007, 0.015, 0.030, 0.060, 0.125, and 0.250 µg/μL) were transfected into cells (six-well plate).
Physiological functions in the embryos and adults
To explore the roles of MC3R, MC4R, and MRAP2s in the embryo development and adult brain, the embryos at 1, 2, 3, 4, and 5 days post-fertilization (dpf) and the brain of adult culter in different weights (20.02 ± 1.38 g, 50 ± 3.25 g, 100 ± 8.12 g, 200 ± 13.81 g, 500 ± 30.56 g, and 800 ± 40.93 g) were obtained. Each stage had at least three fish.
Statistical analysis
All data were shown as mean ± s.e.m. GraphPad Prism 8.3 software (GraphPad) was used to calculate the parameters of ligand binding and cAMP signaling assays. The significance of differences in expression levels, ligand binding, and cAMP signaling parameters between caMC3R and hMC3R, as well as vehicle and ligand-treated groups, were all determined by Student’s t-test. F test was first analyzed to compare variances for each study. If P value for F test is less than 0.05, an unpaired t-test with Welch's correction would be performed. If P value for F test is more than 0.05, a normal unpaired t-test would be performed. One-way ANOVA was used to analyze the significance of differences in binding, cAMP, flow cytometry, and gene expression between multiple groups. For one-way ANOVA (more than two groups), if P value for F test is less than 0.05, the transformation would be performed to meet the P value for F test. Statistical significance was set at P < 0.05.
Results
Nucleotide and deduced amino acid sequences of caMC3R
The cloned culter mc3r (GenBank: MW419813) had a 984-bp open reading frame, encoding a protein of 327 amino acids with an estimated molecular mass of 36.01 kDa (Fig. 1A). Multiple alignment of MC3Rs revealed that the predicated caMC3R had the classical characteristic of Family A GPCRs, with seven hydrophobic transmembrane domains (TMDs) and several conserved motifs (PMY, DRY, and DPxxY) at homologous positions with MC3Rs of other species (Supplementary Fig. 1). Three potential N-linked glycosylation sites (Asn2, Asn16, and Asn23) in N-terminus and consensus sequence for protein kinase C phosphorylation (Thr313Phe314Lys315) in the C-terminus were found in caMC3R (Fig. 1A). The identities between caMC3R and other piscine MC3R orthologs were 99% to Wuchang bream, 98% to goldfish, 97% to common carp, 95% to zebrafish, 81% to coho salmon, as well as high homology to mammalian MC3Rs (with 83% to pig and mouse and 82% to human) (Supplementary Fig. 1). Phylogenetic tree showed that caMC3R nested with Wuchang bream, goldfish, and zebrafish MC3Rs (Fig. 1B).
Nucleotide and deduced amino acid sequences (A) and phylogenetic tree (B) of caMC3R. Positions of nucleotide and amino acid sequences are indicated on both sides. N-linked glycosylation sites are present in open boxes. Shaded boxes show putative TMD1-7. Oval frame denotes potential phosphorylation site. The conserved motifs (PMY, DRY, and DPxxY) are underlined. Asterisk (*) shows stop codon. The tree was constructed by the neighbor-joining ethod. Numbers at nodes indicate the bootstrap value, as percentages, obtained for 1000 replicates. Black dot denotes culter MC3R. MC3Rs: Culteralburnus (topmouth culter, MW419813), Megalobramaamblycephala (Wuchang bream, AWA81517.1), Carassiusauratus (goldfish, BAJ83473.1), Daniorerio (zebrafish, AAO24744.1), Oncorhynchuskisutch (coho salmon, XP_020360426.1), Homo sapiens (human, NP_063941.3), Musmusculus (mouse, AAI03670.1), Gallus gallus (chicken, XP_004947293.1), Susscrofa (pig, AFK25142.1), Rattusnorvegicus (rat, NP_001020441.3), Equuscaballus (horse, NP_001243901.1), Pangasianodonhypophthalmus (iridescent shark, XP_026770221.1), Astyanaxmexicanus (Mexican tetra, XP_007231215.1), Oryx gazella (gemsbok, AFH58734.1), Pteropusvampyrus (large flying fox, XP_011368476.1), Pteropusalecto (black flying fox, XP_006921991.1), Dasypusnovemcinctus (nine-banded armadillo, XP_004447768.1), Feliscatus (cat, XP_023106851.1), Loxodontaafricana (African bush elephant, XP_003419952.1), Oncorhynchustshawytscha (chinook salmon, XP_024229914.1), Salvelinusalpinus (Arctic char, XP_023994975.1), Amazonaaestiva (turquoise-fronted amazon, KQL61336.1), Scleropagesformosus (Asian arowana, XP_018615783.1), Pelodiscussinensis (Chinese softshell turtle, XP_006129463.1), Terrapenecarolinatriunguis (common box turtle, XP_024059166.1), and Alligator sinensis (Chinese alligator, XP_006018246.1).
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
Tissue distribution of culter mc3r
The tissue distribution of culter mc3r was determined by qRT-PCR (Fig. 2). Culter mc3r expression showed sexual dimorphism. In male culter, mc3r was primarily expressed in the brain, including telencephalon, cerebellum, medulla, mesencephalon, and hypothalamus, as well as highly expressed in the periphery (testis, liver, and head kidney) (Fig. 2A). In female culter, mc3r was highly expressed in the telencephalon, olfactory bulb, medulla, mesencephalon, and hypothalamus and also expressed in skin and ovary (Fig. 2B).
Tissue expression of mc3r in male (A) and female (B) culter. The mRNA levels of mc3r were measured by qRT-PCR. Data are presented as the mean ± s.e.m. (n = 3). Mc: mesencephalon; Ob: olfactory bulb; Ce: cerebellum; Tc: telencephalon; Hp: hypothalamus; Me: medulla; Pit: pituitary gland; Lv: liver; He: heart; St: stomach; Kd: kidney; Int: intestine; Hk: head kidney; Gd: gonad; Mu: muscle; Sk: skin; Gi: gill; Sp: spleen.
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
Ligand binding properties of caMC3R
Binding assay was performed using multiple MC3R ligands, including NDP-MSH, α-MSH, des-α-MSH, and ACTH (1–24). hMC3R was used for comparison in the same experiments. The maximal binding value (Bmax) of caMC3R was 627.02 ± 68.52% of that of the hMC3R (set as 100%) (P < 0.001) (Fig. 3 and Table 1). caMC3R had significantly higher affinity to ACTH (1–24) (P < 0.05) (Fig. 3 and Table 1). IC50s were similar between the two MC3Rs when NDP-MSH, α-MSH, or des-α-MSH was used (Fig. 3 and Table 1).
Ligand binding properties of caMC3R. Different concentrations of unlabeled NDP-MSH (A), α-MSH (B), des-α-MSH (C), and ACTH (1–24) (D) were used to displace the binding of 125I-NDP-MSH. Results are expressed as % of hMC3R binding ± range from duplicate determinations within one experiment. All experiments were repeated at least three independent times.
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
The ligand binding properties of caMC3R.
| MC3R | caMC3R | hMC3R | |
|---|---|---|---|
| Bmax (%) | 627.02 ± 68.52b | 100 | |
| NDP-MSH | IC50 (nM) | 2.78 ± 0.71 | 1.78 ± 0.15 |
| α-MSH | IC50 (nM) | 20.81 ± 3.88 | 30.73 ± 1.31 |
| des-α-MSH | IC50 (nM) | 123.97 ± 11.48 | 117.95 ± 14.72 |
| ACTH (1–24) | IC50 (nM) | 19.66 ± 5.58a | 43.61 ± 3.44 |
Results are presented as the mean ± s.e.m. (n = 3–4).
aSignificant difference from the parameter of hMC3R, P < 0.05. bsignificant difference from the parameter of hMC3R, P < 0.001.
cAMP signaling properties of caMC3R
cAMP signaling properties were measured to investigate whether caMC3R could respond to NDP-MSH, α-MSH, des-α-MSH, or ACTH (1–24) stimulation. The results indicated that all agonists could stimulate caMC3R and dose dependently increase intracellular cAMP generation (Fig. 4 and Table 2). caMC3R had higher maximal responses (Rmax) to all agonists than those of hMC3R (Fig. 4 and Table 2). EC50s were remarkably decreased when caMC3R was stimulated by ACTH (1–24) and des-α-MSH (Fig. 4 and Table 2).
Signaling properties of caMC3R. HEK293T cells were transiently transfected with hMC3R or caMC3R plasmids. Different concentrations of NDP-MSH (A), α-MSH (B), des-α-MSH (C), and ACTH (1–24) (D) were used to stimulate the cells. (E) Constitutive activities of caMC3R in cAMP pathway. Increasing concentrations of caMC3R plasmid were transfected into HEK293T cells. Cells transfected with empty pcDNA3.1 vector were used as the control group. Data are mean ± s.e.m. from triplicate measurements within one experiment. All experiments were performed at least three times independently.
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
The signaling properties of caMC3R.
| MC3R | caMC3R | hMC3R | |
|---|---|---|---|
| Basal (%) | 406.89 ± 50.49c | 100 | |
| NDP-MSH | EC50 (nM) | 0.42 ± 0.13 | 0.28 ± 0.08 |
| Rmax (%) | 208.87 ± 15.15a | 100 | |
| α-MSH | EC50 (nM) | 0.22 ± 0.04 | 1.70 ± 0.42 |
| Rmax (%) | 171.91 ± 15.40a | 100 | |
| des-α-MSH | EC50 (nM) | 0.29 ± 0.05a | 3.14 ± 0.38 |
| Rmax (%) | 168.17 ± 11.07a | 100 | |
| ACTH (1–24) | EC50 (nM) | 0.44 ± 0.12b | 4.82 ± 0.79 |
| Rmax (%) | 140.92 ± 7.05a | 100 |
Results are presented as the mean ± s.e.m. (n = 3–4).
aSignificant difference from the parameter of hMC3R, P < 0.05. bSignificant difference from the parameter of hMC3R, P < 0.01. csignificant difference from the parameter of hMC3R, P < 0.001.
This study also showed that caMC3R had four times higher basal cAMP levels than that of hMC3R (Table 2). To further study whether caMC3R could be constitutively active, increasing concentrations (from 0 to 0.25 µg/μL) of caMC3R plasmid were transfected into cells. The data indicated that a low amount of caMC3R plasmid transfection could increase basal cAMP signaling, starting at 0.03 μg/μL (Fig. 4E).
Regulation of caMC3R expression and pharmacology by caMRAP2s
This study and our previous study showed that the mRNA of culter mc3r, mrap2a, and mrap2b was detected in the same tissues (40), indicating that MRAP2s might affect MC3R pharmacology. Therefore, we further investigated the potential modulation of caMRAP2s on MC3R expression and pharmacology.
Flow cytometry was used to measure caMC3R expression. We found that caMRAP2a significantly decreased the cell surface expression in 1:5 group but had no effect on total expression of caMC3R (Fig. 5A and B). However, caMRAP2b did not significantly affect the cell surface and total expression of caMC3R (Fig. 5A and B).
Regulation of caMC3R expression and signaling by caMRAP2a or caMRAP2b. Cell surface expression (A) and total expression (B) of caMC3R modulated by caMRAP2a or caMRAP2b were measured by flow cytometry. Basal (C) and maximal signaling (D) of caMC3R regulated by MRAP2a or MRAP2b were determined by RIA. HEK293T cells were co-transfected with different ratios of caMC3R/caMRAP2a or caMC3R/caMRAP2b (1:0, 1:1, 1:3, and 1:5). The empty vector pcDNA3.1 fluorescence was used for background staining. The results are calculated as % of 1:0 group. Each data point represented as the mean ± s.e.m. (n = 3 – 4). Different letters indicate significant difference (P < 0.05) (one-way ANOVA followed by Tukey test).
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
Ligand binding assays with α-MSH and ACTH indicated that only caMRAP2a significantly decreased the Bmax of caMC3R in 1:5 group, but caMRAP2b did not (Fig. 6A, B and Table 3). caMRAP2a and caMRAP2b had no significant effect on affinities of caMC3R to α-MSH and ACTH (1–24) (Fig. 6A, B and Table 3).
Modulation of caMC3R pharmacology by caMRAP2a or caMRAP2b. Ligand binding (A, B) and signaling (C, D) properties of caMC3R to α-MSH or ACTH (1–24) upon co-expression of caMC3R with caMRAP2a or caMRAP2b were measured. HEK293T cells were co-transfected with caMC3R/caMRAP2a or caMC3R/caMRAP2b in two different ratios (1:0 and 1:5). Results of binding properties were calculated as % of hMC3R binding ± range from duplicate determinations within one experiment. All experiments were measured at least three times independently.
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
The effect of caMRAP2a or caMRAP2b on ligand binding properties of caMC3R.
| caMC3R/caMRAP2s | Bmax | α-MSH, IC50 (nM) | ACTH, IC50 (nM) |
|---|---|---|---|
| caMC3R/caMRAP2 (1:0) | 100 | 40.97 ± 8.63 | 83.73 ± 13.89 |
| caMC3R/caMRAP2a (1:5) | 48.71 ± 5.03a | 33.97 ± 7.18 | 57.43 ± 12.43 |
| caMC3R/caMRAP2b (1:5) | 86.57 ± 6.61 | 49.20 ± 2.97 | 107.13 ± 27.48 |
Results are expressed as the mean ± s.e.m. (n = 3–4).
aSignificant difference from the parameter of 1:0, P < 0.001.
Modulation of caMRAP2a or caMRAP2b on caMC3R signaling was also studied. Results showed that both MRAP2a and MRAP2b did not markedly affect EC50s in response to α-MSH and ACTH; MRAP2a significantly decreased Rmax, but MRAP2b did not (Fig. 6C, D and Table 4). Additionally, increasing ratios of caMC3R/caMRAP2a or caMRAP2b (1:0, 1:1, 1:3, and 1:5) were co-transfected into cells. Results showed that both MRAP2a and MRAP2b dose dependently decreased basal cAMP generation (Fig. 5C). The cAMP generation of caMC3R stimulated by 10−6 M α-MSH were dose dependently decreased by MRAP2a but not MRAP2b (Fig. 5D).
The effect of caMRAP2a or caMRAP2b on cAMP signaling of caMC3R.
| caMC3R/caMRAP2a or caMRAP2b | α-MSH | ACTH | ||
|---|---|---|---|---|
| EC50 (nM) | Rmax | EC50 (nM) | Rmax | |
| caMC3R (1:0) | 0.41 ± 0.02 | 100 | 2.09 ± 0.17 | 100 |
| caMC3R/caMRAP2a (1:5) | 0.29 ± 0.04 | 50.86 ± 7.13b | 1.91 ± 0.57 | 42.49 ± 9.45a |
| caMC3R/caMRAP2b (1:5) | 0.57 ± 0.15 | 111.62 ± 6.28 | 1.87 ± 0.38 | 99.36 ± 13.22 |
Results are expressed as the mean ± s.e.m. (n = 3–4).
aSignificant difference from the parameter of 1:0, P < 0.05. bSignificant difference from the parameter of 1:0, P < 0.001.
Expression of mc3r, mc4r, mrap2a, and mrap2b in culter embryos and adults
qRT-PCR was used to analyze developmental expression kinetics of pomc, agrp, mc3r, mc4r, mrap2a, and mrap2b in the culter embryo at 1, 2, 3, 4, or 5 dpf. All genes could be detected from 1 dpf to 5 dpf (Fig. 7). Compared to 1 dpf, expression of pomc, mc4r and mrap2a was increased at 3, 4, or 5 dpf; argp and mc3r expression was decreased at 2, 3, 4, or 5 dpf (Fig. 7).
Expression of pomc (A), agrp (B), mc3r (C), mc4r (D), mrap2a (E), and mrap2b (F) in the first 5 days of culter embryos. Results are expressed as means ± s.e.m. (n = 3) and are analyzed by one-way ANOVA followed by Tukey’s test. Bars with the same letter are not significantly different (P > 0.05).
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
In adult culter, brains were collected from fish of different weights. Results showed that with increasing weight, expression of agrp, mc3r, mc4r, and mrap2a decreased (Fig. 8B, C, D and E). Compared to ~20 g fish, pomc expression was significantly increased at ~50 g and then decreased at ~500 and 800 g (Fig. 8A). Expression of mrap2b was markedly increased at ~50 g and ~800 g and decreased at ~500 g (Fig. 8F).
Expression of pomc (A), agrp (B), mc3r (C), mc4r (D), mrap2a (E), and mrap2b (F) in the brain of adult culter. I, II, III, IV, V, and VI indicated culter of different weights, at 20.02 ± 1.38 g, 50 ± 3.25 g, 100 ± 8.12 g, 200 ± 13.81 g, 500 ± 30.56 g, and 800 ± 40.93 g, respectively. Results are expressed as means ± s.e.m. (n = 3) and are analyzed by one-way ANOVA followed by Tukey’s test. Bars with the same letter are not significantly different (P > 0.05).
Citation: Endocrine Connections 10, 11; 10.1530/EC-21-0459
Discussion
In this study, we cloned culter mc3r and explored the pharmacological properties of caMC3R and its modulation by MRAP2a and MRAP2b. The expression of mc3r, mc4r, mrap2a, and mrap2b in embryos and adults was further investigated.
Culter mc3r had similar primary structure as MC3Rs of other species with seven TMDs and several highly conserved motifs (Supplementary Fig. 1). Phylogenetic tree analysis found that caMC3R was clustered with teleost MC3Rs (Fig. 1B). Culter mc3r was primarily expressed in the brain (Fig. 2). Differential expression patterns were observed in peripheral organs between the sexes. In males, culter mc3r was highly expressed in peripheral tissues, including liver, muscle, testis, and head kidney, whereas it was highly expressed in skin, ovary, and liver in females (Fig. 2). In chicken, mc3r mRNA is detected in the brain, muscle, and ovary (38). In red stingray, mc3r has high expression in the brain and inter-renal tissues (64). The wide expression (in the brain and peripheral tissues) of mc3r might be associated with its roles in regulating multiple physiological functions, including modulation of feed efficiency and nutrient partitioning, adaptation to fasting and overfeeding, as well as immune response.
Detailed pharmacological studies were performed on culter MC3R. Results showed that all agonists could bind and activate caMC3R (Figs 3 and 4). We found that caMC3R had high affinity and potency to ACTH (Tables 1 and 2), similar to dogfish (44) and channel catfish (43) MC3Rs. Fish MC4Rs also show high affinities and potencies to ACTH (40, 47, 51, 52, 65, 66). These indicated that ACTH may be the original ligand for the MC3R and MC4R (67). In addition, caMC3R showed decreased EC50s than hMC3R in response to α-MSH, des-α-MSH, and ACTH (Fig. 4 and Table 2). These results were similar to those of channel catfish MC3R (ipMC3R) where ipMC3R shows a significant decrease of EC50s in response to α-MSH and ACTH (43). In addition, caMC3R has higher Rmaxs than that of hMC3R to all agonist (Fig. 4 and Table 2). In channel catfish, MC3R shows lower Rmaxs than that of hMC3R to NDP-MSH, α-MSH, β-MSH, and ACTH (43).
We further explored the potential modulation of the trafficking, ligand binding, and signaling on caMC3R by caMRAP2s. Culter MRAP2a decreased cell surface expression but had no effect on total expression of caMC3R, and MRAP2b did not affect cell surface and total expression of caMC3R (Fig. 5). Both human MRAP1a and MRAP2 do not significantly affect hMC3R cell surface expression (32, 68). Zhang et al. showed that MRAP1 and MRAP2 have no effect on the cell surface expression of chicken MC3R (38). Collectively, the potential roles of MRAPs on MC3R cell surface expression vary in different species.
In this study, we also showed that MRAP2a and MRAP2b did not affect affinities of caMC3R to α-MSH and ACTH (Fig. 6A, B and Table 3). MRAP2a decreased the Bmax of caMC3R, probably due to decreased cell surface expression. As for signaling, both MRAP2a and MRAP2b significantly decreased caMC3R basal activity (Fig. 5C), and only MRAP2a markedly decreased the Rmax (Table 4). Similar results were observed in channel catfish MC3R that MRAP2 decreases basal and ligand-stimulated cAMP signaling (43). In zebrafish, MRAP2a and MRAP2b do not modulate MC3R signaling (33). In hMC3R, MRAP2 significantly decreases NDP-MSH-induced cAMP generation, and MRAP1a increases agonist-stimulated cAMP signaling (32, 68). In chicken, MRAP2 increases agonist-induced cAMP production and reduces constitutive activity of MC3R, while MRAP1 has no effect on basal and agonist-induced cAMP generation (38). Furthermore, MRAP1 and MRAP2 increase sensitivity to ACTH of chicken MC3R (38). MRAP2a increases the sensitivity of zebrafish MC4R to ACTH (69, 70). MRAP2 also increases ACTH potency and makes hMC4R act as an ACTH-preferring receptor (69). However, this study and our previous reports on grouper MC4R do not find that MRAP2s could make MC3R or MC4R act as ACTH-preferring receptor (39, 40).
Our results demonstrated that caMC3R had high constitutive activity in cAMP signaling (Fig. 4 and Table 2), consistent with zebrafish, channel catfish, and chicken MC3Rs (38, 43, 49). The high basal activities of MC3R were decreased by MRAP2s in culter, channel catfish, and chicken (38, 43). AgRP (Agouti-related peptide), as an inverse agonist, decreases the constitutive activity of MC3R in channel catfish and chicken (38, 43). However, hMC3R has little or no basal activity in cAMP pathway (71, 72). hMC4R shows modest constitutive activity, and the defect in basal activity of MC4R mutations is considered as one cause of obesity (73, 74). MRAP2- and AgRP-suppressed basal activity of MC4R is essential for promoting zebrafish growth (33, 75). Thus, high basal activity of MC3R might provide new strategies to modulate MC3R signaling by AgRP or MRAP2 in these species. The potential physiological relevance of constitutive activity in teleost MC3Rs needs further study.
We next explored the potential roles of MC3R, MC4R, and MRAP2s in culter embryo development and adults. Sebag et al. reported that zebrafish MRAP2a (expressed from embryos to adults) stabilizes MC4R in an inactive conformation, decreases basal and ligand-stimulated signaling, and maximizes growth during the embryo period. MRAP2b is mainly expressed in adults, reducing basal activity and enhancing sensitivity of MC4R to agonist, and thus converting constitutive MC4R to ligand-dependent receptor (33). Similar to our previous study on caMC4R (40), this study showed that caMC3R had high basal activity; MRAP2a decreased basal and agonist-stimulated signaling, and MRAP2b only decreased basal activity of caMC3R. Our study showed that increased mrap2a and mrap2b expression was observed in culter embryos (Fig. 7) and might contribute to inhibit caMC3R and caMC4R signaling and thus maximize growth. In addition, culter mc3r had the highest expression at 1 dpf, but lower expression on other dpfs (Fig. 7), indicating that MC3R might lower its signaling by reducing its expression and further promoting growth. In adult culter, expression of pomc, mc3r, and mc4r was gradually decreased (Fig. 8). Lower mc4r expression, decreased stimulation by α-MSH/ACTH would reduce the anorexic action of MC4R, and further promoting food intake and rapid growth. Furthermore, decreased mc3r expression and ligand-induced signaling of MC3R might also affect feed efficiency and nutrient partitioning and further improve growth. Overall, MRAP2a and MRAP2b could block MC3R/MC4R functions and promote growth during larval period. Inhibition of MC3R and MC4R signaling by reduced m3cr, mc4r, and pomc expression would affect feeding, feed efficiency, and nutrient partitioning and further maximize growth in adults.
In summary, we cloned culter mc3r and investigated its expression patterns. Culter MC3R had high constitutive activity in cAMP pathway. Only caMRAP2a markedly decreased cell surface expression and Rmax of caMC3R. Both caMRAP2a and caMRAP2b decreased basal cAMP production. MRAP2a and MRAP2b might play a more important role in regulating MC3R/MC4R signaling during larval period. Reduced expression of mc3r, mc4r, and pomc might be mainly involved in adults. These findings laid the foundation for further physiological studies of culter MC3R that might provide new strategies for promoting growth and culture of culter.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-21-0459.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 31872551), the Natural Science Foundation of Hunan Province for Distinguished Young Scholars (Grant No. 2020JJ2022), 111 Project (D20007), and the China Agriculture Research System (Grant No. CARS-45) (to Min Tao). This study was also partially supported by Ocean University of China-Auburn University Joint Center Grants Program (to Ya-Xiong Tao). Ren-Lei Ji, Ting Liu, and Min Tao received fellowships from China Scholarship Council, People’s Republic of China.
Author contribution statement
Ren-Lei Ji: Writing – Original draft, Data curation, Methodology. Lu Huang: Data curation, Methodology, Formal analysis. Yin Wang: Software, Data curation, Methodology. Ting Liu: Software, review and editing. Si-Yu Fan: Methodology, Formal analysis. Min Tao: Project administration, Validation, Funding acquisition. Ya-Xiong Tao: Supervision, Funding acquisition, Conceptualization, Writing – review and editing.
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