Pharmacological modulation of two melanocortin-5 receptors by MRAP2 proteins in zebrafish

in Journal of Molecular Endocrinology
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  • 1 Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China

Correspondence should be addressed to C Zhang: zhangchao@tongji.edu.cn

*(M Zhu and M Wang contributed equally to this work)

Melanocortin receptor accessory protein 2 (MRAP2) plays an important role in regulating melanocortin receptors. In zebrafish, MRAP2a and MRAP2b show distinct pharmacological effects on MC4R activity, but how MRAP2 protein regulates other zebrafish melanocortin receptors is barely studied. Zebrafish have two mc5r genes: mc5ra and mc5rb, it is still vague which one is the homologous isoform to the mammalian paralog. Here, we utilize synteny and phylogenetic analysis to demonstrate the evolutionary conservation of zebrafish MC5Ra and MC5Rb among different species. We also show that MRAP2a and MRAP2b could interact and regulate surface expression of two MC5R receptors. Bimolecular fluorescence complementation (BiFC) studies suggest that zebrafish MC5Rs could form homo- and heterodimers, which are suppressed by co-expression with MRAP2 proteins. In comparison with mammalian MC5R-MRAP2 system and different pharmacological effects of zMRAP2 protein on MC5Rs, zmc5ra is identified as the evolutionary homologous paralog to the mammals, and it is regulated by metabolic state in zebrafish brain region.

Abstract

Melanocortin receptor accessory protein 2 (MRAP2) plays an important role in regulating melanocortin receptors. In zebrafish, MRAP2a and MRAP2b show distinct pharmacological effects on MC4R activity, but how MRAP2 protein regulates other zebrafish melanocortin receptors is barely studied. Zebrafish have two mc5r genes: mc5ra and mc5rb, it is still vague which one is the homologous isoform to the mammalian paralog. Here, we utilize synteny and phylogenetic analysis to demonstrate the evolutionary conservation of zebrafish MC5Ra and MC5Rb among different species. We also show that MRAP2a and MRAP2b could interact and regulate surface expression of two MC5R receptors. Bimolecular fluorescence complementation (BiFC) studies suggest that zebrafish MC5Rs could form homo- and heterodimers, which are suppressed by co-expression with MRAP2 proteins. In comparison with mammalian MC5R-MRAP2 system and different pharmacological effects of zMRAP2 protein on MC5Rs, zmc5ra is identified as the evolutionary homologous paralog to the mammals, and it is regulated by metabolic state in zebrafish brain region.

Introduction

The melanocortin receptor family consists of five G-protein coupled receptors, MC1R~MC5R, which are regulated by pro-opiomelanocortin (POMC) and agouti protein families (Logan et al. 2003). MC1R plays a role in dermal pigmentation; MC2R mediates the effect of adrenocorticotropic hormone (ACTH) on steroid secretion; MC3R and MC4R regulate energy homeostasis in the central nerve system (Cone 2006). In chickens, MC3R and MC4R can be activated by α-MSH or ACTH. But AgRP can antagonize α-MSH or ACTH actions on MC3R/MC4R and lower the constitutive activity of MC3R and MC4R (Zhang et al. 2017). In mice, knockout of MC5R exhibit a severe dysfunction of exocrine secretion (Chen et al. 1997). In adipocytes, MC5R regulates lipolysis and re-esterification through the cAMP/PKA and MAPK/ERK1/2 pathways (Rodrigues et al. 2013). MC5R also participates in the energy homeostasis since it can increase glucose uptake in skeletal muscle through the PKA pathway (Enriori et al. 2016). MC5R showed a hepatic lipolysis function in sea bass (Sanchez et al. 2009). Heterodimerization of MC1R and MC5R may be involved in the control of color change in teleosts (Kobayashi et al. 2016). The mc5r gene is duplicated in zebrafish: mc5ra and mc5rb (Vastermark & Schioth 2011), while there is only one mc5r gene in fugu or goldfish (Cerda-Reverter et al. 2003, Klovins et al. 2004). In zebrafish, mc5ra is highly expressed in ovary, brain and gastrointestinal tract, while mc5rb is highly expressed in ovary, brain, gastrointestinal tract and eye, both of them can be stimulated by α-MSH (Ringholm et al. 2002).

Melanocortin receptor accessory proteins (MRAPs) are single-transmembrane proteins, several studies confirmed that they could form antiparallel homodimers and heterodimers (Metherell et al. 2005, Sebag et al. 2007). The MRAP system is involved in the regulation of trafficking and signaling of melanocortin receptors (Asai et al. 2013, Cerda-Reverter et al. 2013). Two main forms MRAP1 and MRAP2 exist in zebrafish genome. As in mammals, MRAP1 is essential for adrenocorticotropic hormone receptor (MC2R) trafficking to the cell membrane, ligand binding and downstream signaling (Agulleiro et al. 2010). MRAP2 (melanocortin receptor accessory protein 2) was found as a homolog of MRAP1. MRAP2 interacts with all five MCRs and modulates receptors trafficking to the membrane, agonist binding and cAMP production (Chan et al. 2009, Cerda-Reverter et al. 2013). Recent studies demonstrated that MRAP2 could also interact with non-melanocortin receptors and modulate their signaling (Chaly et al. 2016, Srisai et al. 2017). In mammals, MRAP2 enhances MC4R activity and loss of function of MRAP2 causes early-onset obesity (Asai et al. 2013). The mrap2 gene is present in the genome of lampreys (Vastermark & Schioth 2011). In zebrafish, mrap2 has two homologs: mrap2a and mrap2b. Both of MRAP2a and MRAP2b can interact with zebrafish MC4R: MRAP2a inhibit the binding of MC4R to its agaonist α-MSH, whereas MRAP2b stimulates MC4R activation by increase ligand-binding ability and surface expression (Sebag et al. 2013). Josep Agulleiro et al. (2013) also found that MC4R could be activated by ACTH when co-expressed with MRAP2a. MRAP2b can decrease the constitutive activity of MC4R during fasting which suggests that the deletion of MRAP2b may induce growth changes or obesity. As in zebrafish, MRAP2 can enhance the sensitivity of MC4R for ACTH and block the constitutive activity of MC4R in chickens (Zhang et al. 2017). Unlike MC4R, the physiological functions between MC5Rs and MRAP2 proteins are largely unknown in zebrafish.

In this study, we examined the interaction and pharmacological modulation between two pairs of zebrafish proteins: MC5Ra, MC5Rb and MRAP2a, MRAP2b. MRAP2a prevents both MC5Ra and MC5Rb expression on cell membrane, thus inhibits their activity. MRAP2b inhibits the efficacy of MC5Ra but stimulates MC5Rb. In addition, we compared the regulation of MC5R and MRAP2 between zebrafish and mice. Our results revealed the fact that zebrafish MC5Ra was the functional homologous to mammals.

Methods

Homology and phylogenetic analysis of MC5R

Preparatory multiple sequence alignments were performed using MUSCLE 3.8.31 with default parameters (Edgar 2004). The percentage of similarity between the amino acid sequences were calculated with DNAMAN. Putative TMDs of MC5Rs were predicted with TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), GenBank or NCBI Reference Sequence accession numbers for MC5Rs: human Homo sapiens (AAH95531.1), western gorilla Gorilla gorilla (XP_004059266.1), chimpanzee Pan troglodytes (NP_001009119.1), hamadryas baboon Papio hamadryas (ACO90015.1), rabbit Oryctolagus cuniculus (CCX35395.1), American mink Neovison vison (AGT56096.1), red fox Vulpes vulpes (ABG48760.1), dog Canis lupus familiaris (NP_001074193.1), raccoon dog Nyctereutes procyonoides (ABO38182.1), Norway rat Rattus norvegicus (NP_037231.1), house mouse Mus musculus (NP_058673.2), pig Sus scrofa (NP_999338.1), sheep Ovis aries (NP_001119842.1), goat Capra hircus (NP_001272520.1), cattle Bos taurus (NP_776535.1), water buffalo Bubalus bubalis (AHN49803.1), domestic yak Bos grunniens (ADH51715.1), wild yak Bos mutus (ADZ24287.1), American alligator Alligator mississippiensis (KYO35523.1), Ring-necked pheasant Phasianus colchicus (ACF35264.1), chicken Gallus gallus (NP_001026685.1), hazel grouse Tetrastes bonasia (ACF35263.1), common carp Cyprinus carpio (CBX89936.1), goldfish Carassius auratus (CAD58853.1), Ya-fish Schizothorax prenanti (AGF80338.1), zebrafish Danio rerio (NP_775387.1) and (NP_775386.1). Phylogenetic tree based on the amino acid sequences was constructed by neighbor-joining (NJ) methods with 1000 bootstrap resampling and Poisson correction model, pairwise-deletion option using Mega 7.0 software. To determine whether mc5ra or mc5rb is more orthologous to mammals, synteny analysis was performed between zebrafish, medaka, spotted gar, human, mouse and rat with ensemble (http://www.ensembl.org/index.html). The adjacent genes of zebrafish mc5ra or mc5rb were as follow: rnmt, mRNA cap guanine-N7 methyltransferase; fam210aa, uncharacterized protein C18orf19 homolog A; cthrc1a, collagen triple helix repeat containing 1a; slc25a32a, Slc25a32a protein; fam210ab, uncharacterized protein C18orf19 homolog B; ldlrad4b, low-density lipoprotein receptor class A domain-containing protein 4-like; mc2r, melanocortin 2 receptor; lmna, lamin.

Animal care

WT zebrafish (TU) were raised at 26–28°C, with 14-h light and 10 h night cycle. Fish aged from 5 to 10 dpf were fed twice a day with paramecia, fish aged from 10 to 15 dpf were fed with paramecia and brine shrimp, adult fish were fed with brine shrimp in system water. Zebrafish care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Tongji University.

Plasmids and peptides

3HA-zMC5Ra, 3HA-zMC5Rb, 2Flag-zMRAP2a and 2Flag-zMRAP2b, cloned from WT zebrafish, were constructed into vector pcDNA3.1 (+) (Invitrogen). α-MSH and SHU9119 were purchased from GenScrpipt Corporation Ltd. (China). AgRP (83–132) were synthesized by Chinese Peptide Company (Hangzhou, China).

Cell culture and transfection

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium contain 10% fetal bovine serum and 1% penicillin/streptomycin. Chinese hamster ovary (CHO) cells were cultured in DMEM/F12 medium contain 5% fetal bovine serum and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere consisting of 5% CO2 at 37°C. For transfection, when cells growth to 70–80% confluence, plasmids were transfected using ViaFect Transfection Reagent (Promega).

Co-immunoprecipitation and Western blotting

1 × 106 HEK 293T cells were seeded into 60 mm dishes and transfected next day. After 48 h of transfection, cells were washed with PBS and lysed with lysis buffer (0.75% Triton-X, 50 mM Tris–HCl pH 7.9, 150 mM NaCl and proteinase inhibitor cocktail from Roche) for 1 h at 4°C and centrifuged. Supernatants were incubated with anti-HA (Abcam) overnight at 4°C. Protein A/G agarose beads were added to cell lysate next day, and rotated for 4 h at 4°C, beads were washed three times using lysis buffer and centrifuged, suspended in loading buffer and boiled for 15 min. 10% gels were used for SDS–PAGE. Anti-HA (Abcam) and anti-Flag (abclonal) antibodies were used at a dilute of 1:4000 for immunoblotting.

cAMP assays

HEK 293T cells were plated into 24-well plates, and transfected with indicated plasmids next day. After 24 h of transfection, cells were treated with α-MSH or SHU9119 in DMEM medium supplemented with 0.1% bovine serum albumin for 4 h at 37°C, concentration of α-MSH ranged from 10−12 M to 10−6 M, concentration of SHU9119 ranged from 10−13 M to 10−7 M signaling assay was tested in HEK 293T cells stimulated by 2 × 10-9 M α-MSH and AgRP with final concentration ranging from 10−13 M to 10−7 M. The cAMP level was measured using Dual-Glo Luciferase Assay System (Promega). Luminescence was measured using a Spectramax M5 plate reader. All data were normalized to Renilla luminescence.

Cell-surface ELISA

Twelve-well plates were treated with Poly-l-lysine Solution before used, HEK 293T cells were seeded onto treated plates and transfected next day. On the third day, cells were washed with D-PBS three times, fixed with 4% poly-formaldehyde for 20 min at room temperature. After fixation, cells were washed, blocked for 30 min in 5% milk in D-PBS for surface groups or in 5% milk in lysis buffer (Beyotime) for whole-cell groups and incubated with anti-HA antibody (Abcam) for 2 h at room temperature. After washed three times with D-PBS, cells were incubated with secondary antibody for 1 h. Then, cells were washed three times with D-PBS and incubated with TMB solution for 15–30 min, the reaction was stopped by adding 2 M H2SO4. Spectramax M5 plate reader was used to record OD at 450 nm. Then all liquid in plate wells were removed, and added 200 μL 1× Janus Green per well to stain for 5 min, plates were washed three to five times with deionized water. After removal of all the water, added 200 μL 0.5 M HCl to shake for 10 min, record OD at 595 nm. All data were normalized to OD 595 nm before used. Statistical analyses were performed using Graph Pad Prism, version 6 (GraphPad Software).

Immunofluorescence

YFP fragments YFP-F1 and YFP-F2 (Sebag & Hinkle 2009) were constructed into C-terminal of GPCRs respectively CHO cells were seed into a 12-well plates with slides and transfected next day. After 24 h of transfection, cells were fixed with paraformaldehyde for 20 min, and then washed 3 times with PBS. To detect surface expression of MRAP2s, cells were incubated with anti-V5 antibody (Abcam) at 1:1000 for 2 h at 37°C. Then washed 3 times and incubated with 1:1000 secondary antibody Alexa Fluor594 (Abcam) for 2 h at 37°C. For FACS analysis, MRAP2:MC5R-F1:MC5R-F2 were transfected at ratio 2:1:1, after 36 h of transfection, cells were digested into single cell using 0.02% EDTA and centrifuged, 5 × 105 cells were dispensed into 500 μL DMEM/F12 medium, filtered through a 40 μm mesh sieve into a 5 mL Polystyrene Round-Bottom Tube (FALCON), and then analyzed by BD FACSVerse.

Fasting and real-time quantitative PCR

Male WT zebrafish (TU) were fasted 0, 2, 10, 15 days respectively, keeping water change every day. On indicated day, the brain RNA of adult zebrafish was extracted using RNAiso Plus (TaKaRa), according to the manufacturer’s instructions. 1 μg RNA was reverse transcribed using a TIANGEN FastKing RT kit (Beijing, China). cDNA reaction was diluted 1:10 as a template for qPCR. Real-time PCR was performed using LightCycler 96 (Roche), the PCR conditions were: preincubation at 95°C for 600 s; 45 cycles of 95°C 10 s, 60°C for 10 s, and 72°C for 10 s; melting at 95°C for 10 s, 65°C for 60 s and 97°C for 1 s. Primers for qPCR: mc5ra, forward 5′-TCCTCATCCTTGGCATCGTCAGT-3′,reverse 5′-TGGCGATTGGTGAGCAGGTAGAT-3′; mc5rb, forward 5′-AGAACAAGAACCTCCACTCACCAATG-3′, reverse 5′-TTGCCAGCAGATGAATGACGATTGT-3′; ef1α, forward 5′-CCTGCCAGTGTTGCCTTCGT-3′, reverse 5′-CCTCCTTGCGCTCAATCTTCC-3′. All reactions were run in triplicate and repeated three times.

Statistical analyses

All experiments were performed at least three times. Data were analyzed using GraphPad Prism 6 (GraphPad Software, Inc.). Results of cAMP assays were analyzed by two-way ANOVA with Tukey post-test. Results of surface ELISA and qPCR were analyzed by two-tailed t test. All Results were shown as mean ± s.e.m.

Results

Amino acid sequences of MC5R

The amino acid sequences of MC5R consisted of seven transmembrane domains connected by alternating extracellular and intracellular loops with an extracellular N-terminus and an intracellular C-terminus (Supplementary Fig. 1, see section on supplementary data given at the end of this article). Alignment of the amino acid sequences of MC5R with that of other species demonstrated that zebrafish MC5Ra and MC5Rb were homologs to tetraodon (78.53 and 78.85%, respectively), and fugu (78.53 and 75.00%, respectively) MC5Rs, whereas the homology of zebrafish MC5R to mammalian MC5Rs, such as human and mouse MC5Rs, was lower (61.83–75%). As shown in Supplementary Fig. 1, the amino acid sequences in the transmembrane domains and intracellular loops of MC5Ra and MC5Rb were found to be highly identical to that of other species while lower homology was identified at the N-termini and extracellular loops. A NJ phylogenetic tree was analyzed based on amino acid sequences of MC5Rs from different species and our results indicated that MC5Ra of zebrafish was more evolutionarily related to tetraodon and fugu MC5Rs. However, the zebrafish MC5Rb out-groups to all bony vertebrate MC5R orthologs (Fig. 1A).

Figure 1
Figure 1

Phylogenetic and synteny analysis of zebrafish MC5R receptors. (A) Phylogenetic tree of MC5Rs constructed by NJ method with Mega 6.0 software. Jones–Taylor–Thornton (JTT) model was used. The strength of branch relationships was assessed by bootstrap replication (N = 1000 replicates). Asterisk (*) indicates zebrafish mc5ra and mc5rb. (B) Synteny analysis of MC5Rs. Genes in solid black square are conserved among zebrafish, spotted agar, human, mouse, and rat.

Citation: Journal of Molecular Endocrinology 62, 1; 10.1530/JME-18-0104

To determine which mc5r is more orthologous to mammals, synteny analysis was performed between zebrafish, spotted gar, human, mouse and rat (Fig. 1B). Our results showed that adjacent genes of mc5ra were highly conserved between zebrafish and human, mouse and rat MC5Rs. The adjacent genes of mc5ra, including rnmt, fam210aa, ldlrad4a, sehil, ptpn2b and psmg2 were identical with those of spotted gar, human, mouse and rat MC5Rs. Interestingly, the adjacent genes of mc5rb: ldlrad4b, fam210ab and especially mc2r were conserved between zebrafish mc5rb and mouse, rat and human MC5Rs.

Interaction of zebrafish MRAP2s with MC5Rs

To verify the protein–protein interactions between two MRAP2s and two MC5Rs, four groups of HEK 293T cells were transfected with 2Flag-zMRAP2a and 3HA-zMC5Ra, 2Flag-zMRAP2b and 3HA-zMC5Ra, 2Flag-zMRAP2a and 3HA-zMC5Rb, 2Flag-zMRAP2b and 3HA-zMC5Rb, respectively. Co-immunoprecipitation results showed that zebrafish MRAP2a (Fig. 2A and B) and MRAP2b (Fig. 2C and D) could interact with MC5Ra and MC5Rb, suggesting that MRAP2a and MRAP2b could modulate the signaling of MC5Ra and MC5Rb in zebrafish.

Figure 2
Figure 2

MRAP2s interact and regulate surface expressions of MC5Rs. Co-immunoprecipitation assay exhibits the interactions between zMC5Ra and zMRAP2a (A), zMRAP2b (B), also between zMC5Rb and zMRAP2a (C), zMRAP2b (D). *IgG heavy chain. Surface expression of zMC5Ra and zMC5Rb measured by whole-cell ELISA in 293T cells transfected with zMC5Ra or zMC5Rb and different amounts of zMRAP2a or zMRAP2b (E, F, G and H). Data are shown as mean ± s.e.m. and analyzed by two-tailed t test. **P < 0.01; ***P < 0.001. Surface expression of mammalian MC5R measured by whole-cell ELISA in 293T cells transfected with mammalian MC5R and different amounts of mammalian MRAP2 (I and J).

Citation: Journal of Molecular Endocrinology 62, 1; 10.1530/JME-18-0104

Surface expression of MC5Rs was influenced by MRAP2s

To investigate the influences of MRAP2s on the MC5R trafficking, we next measured the surface expression of MC5Ra and MC5Rb in the absence or presence of MRAP2a or MRAP2b. 293T cells were transfected with MC5R alone (1:0) and MRAP2 at progressive ratio (1:3, 1:6) as used in previous studies (Liang et al. 2018). The results showed that MRAP2a decreased the surface expression of both MC5Ra and MC5Rb, whereas MRAP2b had no significant effect (Fig. 2E, F, G and H). Since the results were different from previous studies in human (Chan et al. 2009, Sebag & Hinkle 2009), then we validated the surface expression of mammalian MC5Rs in the absence or presence of mammalian MRAP2s. These results showed that human MRAP2 decreased the surface expression of human MC5R like MRAP2a, but mouse MRAP2 had no significant influence on the surface expression of mouse MC5R, which was similar to the results of MRAP2b in zebrafish (Fig. 2I and J).

MRAP2a and MRAP2b modulate signaling of MC5Ra and MC5Rb in different ways

To determine how MRAP2a and MRAP2b affect MC5Rs signaling, CRE-luciferase reporter assay was performed to detect the cAMP level stimulated by α-MSH through MC5Rs. We found that MRAP2a dose-dependently inhibited the efficacy of both MC5Ra and MC5Rb, MRAP2b inhibited MC5Ra efficacy but increased the maximal stimulated efficacy of MC5Rb (Fig. 3A, B, C and D). To test whether this effect was stable with other agonist, similar results were seen when stimulated with SHU9119, another reported MC5R agonist (Fig. 3E, F, G and H). We also tested the effect of mouse MRAP2 on MC5R and found that MRAP2 dose-dependently inhibited the efficacy of mouse MC5R (Fig. 3I and J), which was similar to zebrafish MC5Ra. However, both MRAP2a and MRAP2b did not affect the agonists and antagonists signaling competing affinity (Fig. 3K, L, M and N). LogEC50 values of each curve were shown in Table 1.

Figure 3
Figure 3

Pharmacological modulation of MC5Rs by MRAP2s. Dose response curves of α-MSH induced cAMP production in 293T cells upon transfection with zMC5Ra (A and B) or zMC5Rb (C and D) in presence of different amount of zMRAP2a or zMRAP2b. Dose response curves of SHU9119 induced cAMP production in 293T cells transfected with zMC5Ra (E and F) or zMC5Rb (G and H) in presence of different amount of zMRAP2a or zMRAP2b. Dose response curves of α-MSH (I) and SHU9119 (J) induced cAMP production in 293T cells transfected with mouse MC5R and the indicated amount of mouse MRAP2. Binding competition of agonist (α-MSH) and antagonist (AgRP) of MC5Rs modulated by MRAP2s (K, L, M and N).

Citation: Journal of Molecular Endocrinology 62, 1; 10.1530/JME-18-0104

Table 1

Analyze data in Fig. 3.

Analyze data in Fig. 3LogEC50P value for Vmax comparison
1:01:31:61:0 vs 1:31:0 vs 1:61:3 vs 1:6
AzMC5Ra:zMRAP2a−9.84 ± 0.16−9.58 ± 0.20−9.32 ± 0.200.0006<0.00010.0001
BzMC5Ra:zMRAP2b−9.87 ± 0.21−9.09 ± 0.18−8.73 ± 0.170.0009<0.00010.0085
CzMC5Rb:zMRAP2a−9.53 ± 0.12−8.91 ± 0.09−8.81 ± 0.160.18580.00050.0584
DzMC5Rb:zMRAP2b−9.11 ± 0.16−9.28 ± 0.22−9.54 ± 0.14<0.0001<0.0001<0.0001
EzMC5Ra:zMRAP2a−10.8 ± 0.32−10.88 ± 0.39−10.69 ± 0.260.038<0.00010.005
FzMC5Ra:zMRAP2b−10.49 ± 0.27−10.65 ± 0.30−10.66 ± 0.24<0.0001<0.00010.9981
GzMC5Rb:zMRAP2a−11.09 ± 0.12−10.54 ± 0.11−10.17 ± 0.14<0.0001<0.00010.3975
HzMC5Rb:zMRAP2b−10.82 ± 0.18−10.59 ± 0.30−10.41 ± 0.30<0.0001<0.0001<0.0001
ImMC5R:mMRAP2(a-MSH)−10.12 ± 0.41−9.03 ± 0.19−8.91 ± 0.200.02780.00050.3396
JmMC5R:mMRAP2(SHU9119)−9.82 ± 0.14−9.01 ± 0.09−8.79 ± 0.20<0.0001<0.0001<0.0001
KzMC5Ra:zMRAP2a−8.02 ± 0.17−8.31 ± 0.09−8.33 ± 0.12<0.0001<0.00010.4559
LzMC5Ra:zMRAP2b−8.15 ± 0.15−8.03 ± 0.12−7.99 ± 0.090.01160.2750.3085
MzMC5Rb:zMRAP2a−7.50 ± 0.29−7.87 ± 0.14−7.85 ± 0.15<0.0001<0.00010.0371
NzMC5Rb:zMRAP2b−7.73 ± 0.44−7.30 ± 0.42−7.42 ± 0.310.00390.2920.1425

Statistics were measured using a two-way ANOVA with Tukey post-test.

Dimerization of zebrafish MC5Rs

Human MC5R has been reported to form homodimers, which could be disrupted by MRAP (Sebag & Hinkle 2009). To assess the ability of dimerization of two zebrafish MC5Rs, YFP fragments were fused to C-terminal of zMC5Ra and zMC5Rb. zMC5R-YFP-F1 and zMC5R-YFP-F2 were co-expressed in CHO cells and the YFP fluorescence indicated the presence of zMC5Ra and zMC5Rb homodimers on cell surface (Fig. 4A and B). Unexpectedly, zMC5Ra and zMC5Rb could also form heterodimers (Fig. 4C). However, when co-expressed with MRAP2a or MRAP2b, the YFP fluorescence could barely be detected (Fig. 4D, E, F, G, H and I). Thus, we hypothesized that MRAP2a and MRAP2b could disrupt dimerization of zMC5Rs just like the effect of MRAP on human MC5R. To validate our hypothesis, we counted the fluorescent cells by FACS (Fluorescence Activated Cell Sorter). Compared to control groups that co-expressed with RAMP3, another reported transmembrane protein that did not regulate melanocortin receptor trafficking (Sebag & Hinkle 2009), zMRAP2a and zMRAP2b significantly decreased the percentage of YFP fluorescent cells (Supplementary Fig. 2). Thus, zMRAP2a and zMRAP2b could disrupt the dimerization of both zMC5Rs homodimers and heterodimers, similar to the effect of MRAP on human MC5R.

Figure 4
Figure 4

Zebrafish MC5R forms homo- and heterodimers. YFP fluorescence (green) of zMC5Ra homodimers (A), zMC5Rb homodimers (B) and zMC5Ra zMC5Rb heterodimers (C) (scale bar = 10 µm). Effect of zMRAP2a and zMRAP2b on zMC5Rs dimerization. CHO cells co-expressing zMC5Ra homodimers (D and E), zMC5Rb homodimers (F and G) or zMC5Rs heterodimers (H and I) with zMRAP2a (D, F and H) or zMRAP2b (E, G and I). Surface expression of zMRAP2a and zMRAP2b is shown in red, detected by anti-V5 antibody and secondary anti-mouse Alexa594 (abcam). MRAP2 suppressed the dimer formation of MC5R proteins.

Citation: Journal of Molecular Endocrinology 62, 1; 10.1530/JME-18-0104

Fasting increased the expression of mc5ra in zebrafish brain

The melanocortin system may be involved in regulating food intake of fish, but no experiments had been carried out to evaluate whether the progressive fasting could affect the expression of MC5Rs in zebrafish. We fasted WT zebrafish for 2, 10 and 15 days, and detected the mRNA level by qRT-PCR. After 2–10 days of fasting, the expression of mc5ra in zebrafish brain increased 4-fold, but mc5rb showed no significant differences (Fig. 5).

Figure 5
Figure 5

Fasting induced expression alternation of MC5R in zebrafish brain. Adult zebrafish (TU) were fasted 0, 2, 10, 15 days and the mc5ra and mc5rb expression in the brain were analyzed by qRT-PCR. Data are analyzed by two-tailed t test compared with Day 0, graphs are shown as mean ± s.e.m. **, P < 0.01; ***, P < 0.001; ns, not significant.

Citation: Journal of Molecular Endocrinology 62, 1; 10.1530/JME-18-0104

Discussion

The fish-specific genome duplication (FSGD or 3R) occurred about 350 million years ago; thus, zebrafish experienced an additional genome duplication than human (Meyer & Van de Peer 2005), which caused several gene duplications in zebrafish genome, such as mrap2a and mrap2b. Although MRAP2b seems more like mammalian MRAP2, MRAP2a can also regulate MC4R in a different way, both of them are functional in the control of zebrafish early development (Sebag et al. 2013). Following the three whole genome duplication event, chromosome reduction and gene loss, the only duplicated melanocortin receptor genes left in the zebrafish genome are the mc5ra and the mc5rb, and all the other zebrafish melanocortin receptor genes are present in single copy. Actually, the function of MC5Rs has not been elucidated in fish. Results of amino acid sequences demonstrated that the MC5Ra was more homologous to mammals (Supplementary Fig. 1). Zebrafish MC5Ra shared more than 78% amino acid identity with several other teleost MC5Rs, including tetraodon and fugu MC5Rs, and it was 61.27% identical to hMC5R. The amino acid sequences of MC5Rs were highly conserved at seven hydrophobic transmembrane domains. Phylogenetic analysis based on amino acid sequences revealed that MC5Ra was closer to hMC5R. Synteny analysis further demonstrated that genes locating in the upstream region of mc5ra in zebrafish were highly conserved compared with those in spotted gar, human, mouse and rat, we hypothesized that the transcriptional regulation of mc5ra could be more conserved than mc5rb. Sequence alignment of multiple MC5Rs could not differentiate them, thus we attempted to find the differences between MC5Ra and MC5Rb by exploring the regulating relationship between MRAP2s and MC5Rs. Previous studies have demonstrated that MRAP and MRAP2 can interact with MC5R, suppress MC5R activity by decreasing the cell-surface expression level (Chan et al. 2009, Sebag & Hinkle 2009). Chan et al. observed that hMRAP2 decreased hMC5R trafficking while we observed that mMRAP2 did not effect mMC5R trafficking. These data suggest that there appears to be two types of MRAP2 in mammals, and the alternative effects are species specific.

Our results suggested that the interactions between MRAP2s and MC5Rs were conserved in zebrafish. Although both MRAP2a and MRAP2b could interact with melanocortin receptors in zebrafish (Agulleiro et al. 2010, Sebag et al. 2013, Cortes et al. 2014), they had distinct functions on modulating the signaling of receptors. As same as mouse MRAP2, MRAP2b had no influence on the surface expression of MC5R. Previous studies showed that MRAP2b could inhibit the adult zebrafish MC4R constitutively activity but increase ligand-dependent activity (Sebag et al. 2013). Like MRAP2b, mouse MRAP2 could increase the signaling of mouse MC4R, so MRAP2b was considered as the zebrafish ortholog of MRAP2 (Asai et al. 2013).

We further verified that MRAP2a and MRAP2b could modulate the signaling of MC5Ra and MC5Rb in different ways. MRAP2a could inhibit maximal activity of both MC5Ra and MC5Rb, which might be explained by decreased cell surface expression. To our surprise, MRAP2b also inhibited MC5Ra activity in zebrafish without changing its surface expression, and unexpectedly increased MC5Rb activity. MC5R was reported to form homodimers and heterodimers with other GPCRs (Sebag & Hinkle 2009, Kobayashi et al. 2016). To elucidate the pharmacological mechanism of MC5Rs modulated by MRAP2s, we observed the dimerization of MC5Rs. zMC5Ra could form homodimers on cell membrane, but homodimers of zMC5Rb and heterodimers of zMC5Ra and zMC5Rb were trapped in endoplasmic reticulum. MRAP2 could disrupt MC5R homodimers (Sebag & Hinkle 2009), we obeserved the same phenomena, that both homodimers and heterodimers were decreased or disappeared when co-expressed with MRAP2a or MRAP2b. Thus, MRAP2a or MRAP2b could modulate MC5Rs signaling by regulating their dimerization. Recent studies suggested that α-MSH could regulate muscle glucose intake via MC5R-PKA pathway (Enriori et al. 2016), which might help us understanding why mc5ra mRNA level increased 4-fold during fasting. Promoter region is crucial in the transcription regulation; synteny analysis suggested that mc5ra had a more conserved far upstream sequence compared to mc5rb.

In summary, by parallel comparison of the evolution and regulation relationship between MRAP2 and MCRs family, our studies suggested that mc5ra was more homologous to mammalian MC5R. Further work is needed to elucidate the physiological roles of each mc5r by cas9 specific knockout in the zebrafish.

Supplementary data

This is linked to the online version of the paper at https://doi.org/10.1530/JME-18-0104.

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

The work was supported by grants from National Key Research and Development Program of China (Grant No. 2017YFA0103902 and 2016YFA0102200); The National Natural Science Foundation of China (Grant No. 81570760 and 31771283); One Thousand Youth Talents Program of China; The Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. A11323); The Shanghai Rising-Star Program (Grant No 15QA1403600); the Fundamental Research Funds for the Central Universities of Tongji University.

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    Phylogenetic and synteny analysis of zebrafish MC5R receptors. (A) Phylogenetic tree of MC5Rs constructed by NJ method with Mega 6.0 software. Jones–Taylor–Thornton (JTT) model was used. The strength of branch relationships was assessed by bootstrap replication (N = 1000 replicates). Asterisk (*) indicates zebrafish mc5ra and mc5rb. (B) Synteny analysis of MC5Rs. Genes in solid black square are conserved among zebrafish, spotted agar, human, mouse, and rat.

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    MRAP2s interact and regulate surface expressions of MC5Rs. Co-immunoprecipitation assay exhibits the interactions between zMC5Ra and zMRAP2a (A), zMRAP2b (B), also between zMC5Rb and zMRAP2a (C), zMRAP2b (D). *IgG heavy chain. Surface expression of zMC5Ra and zMC5Rb measured by whole-cell ELISA in 293T cells transfected with zMC5Ra or zMC5Rb and different amounts of zMRAP2a or zMRAP2b (E, F, G and H). Data are shown as mean ± s.e.m. and analyzed by two-tailed t test. **P < 0.01; ***P < 0.001. Surface expression of mammalian MC5R measured by whole-cell ELISA in 293T cells transfected with mammalian MC5R and different amounts of mammalian MRAP2 (I and J).

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    Pharmacological modulation of MC5Rs by MRAP2s. Dose response curves of α-MSH induced cAMP production in 293T cells upon transfection with zMC5Ra (A and B) or zMC5Rb (C and D) in presence of different amount of zMRAP2a or zMRAP2b. Dose response curves of SHU9119 induced cAMP production in 293T cells transfected with zMC5Ra (E and F) or zMC5Rb (G and H) in presence of different amount of zMRAP2a or zMRAP2b. Dose response curves of α-MSH (I) and SHU9119 (J) induced cAMP production in 293T cells transfected with mouse MC5R and the indicated amount of mouse MRAP2. Binding competition of agonist (α-MSH) and antagonist (AgRP) of MC5Rs modulated by MRAP2s (K, L, M and N).

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    Zebrafish MC5R forms homo- and heterodimers. YFP fluorescence (green) of zMC5Ra homodimers (A), zMC5Rb homodimers (B) and zMC5Ra zMC5Rb heterodimers (C) (scale bar = 10 µm). Effect of zMRAP2a and zMRAP2b on zMC5Rs dimerization. CHO cells co-expressing zMC5Ra homodimers (D and E), zMC5Rb homodimers (F and G) or zMC5Rs heterodimers (H and I) with zMRAP2a (D, F and H) or zMRAP2b (E, G and I). Surface expression of zMRAP2a and zMRAP2b is shown in red, detected by anti-V5 antibody and secondary anti-mouse Alexa594 (abcam). MRAP2 suppressed the dimer formation of MC5R proteins.

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    Fasting induced expression alternation of MC5R in zebrafish brain. Adult zebrafish (TU) were fasted 0, 2, 10, 15 days and the mc5ra and mc5rb expression in the brain were analyzed by qRT-PCR. Data are analyzed by two-tailed t test compared with Day 0, graphs are shown as mean ± s.e.m. **, P < 0.01; ***, P < 0.001; ns, not significant.