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Journal of Molecular M Zhu, M Wang et al. Pharmacological modulation of 62:1 27–36 Endocrinology MC5R by MRAP2 RESEARCH Pharmacological modulation of two melanocortin-5 receptors by MRAP2 proteins in zebrafish

Ming Zhu*, Meng Wang*, Yijun Chen and Chao Zhang

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: [email protected]

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

Abstract

Melanocortin receptor accessory protein 2 (MRAP2) plays an important role in regulating Key Words melanocortin receptors. In zebrafish, MRAP2a and MRAP2b show distinct pharmacological ff melanocortin 2 receptor effects on MC4R activity, but how MRAP2 protein regulates other zebrafish melanocortin accessory protein 2 receptors is barely studied. Zebrafish have twomc5r genes: mc5ra and mc5rb, it is ff melanocortin-5 receptor still vague which one is the homologous isoform to the mammalian paralog. Here, we ff zebrafish utilize synteny and phylogenetic analysis to demonstrate the evolutionary conservation ff pharmacology 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, mc5raz is identified as the evolutionary homologous paralog to the mammals, and it is regulated by metabolic state Journal of Molecular in zebrafish brain region. Endocrinology (2019) 62, 27–36

Introduction

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

-18-0104 Journal of Molecular M Zhu, M Wang et al. Pharmacological modulation of 62:1 28 Endocrinology MC5R by MRAP2 is duplicated in zebrafish: mc5ra and mc5rb (Vastermark & on cell membrane, thus inhibits their activity. MRAP2b Schioth 2011), while there is only one mc5r gene in fugu inhibits the efficacy of MC5Ra but stimulates MC5Rb. In or goldfish Cerda-Reverter( et al. 2003, Klovins et al. 2004). addition, we compared the regulation of MC5R and MRAP2 In zebrafish, mc5ra is highly expressed in ovary, brain and between zebrafish and mice. Our results revealed the fact gastrointestinal tract, while mc5rb is highly expressed in that zebrafish MC5Ra was the functional homologous ovary, brain, gastrointestinal tract and eye, both of them to mammals. can be stimulated by α-MSH (Ringholm et al. 2002). Melanocortin receptor accessory proteins (MRAPs) are single-transmembrane proteins, several studies Methods confirmed that they could form antiparallel homodimers Homology and phylogenetic analysis of MC5R and heterodimers (Metherell et al. 2005, Sebag et al. 2007). The MRAP system is involved in the regulation Preparatory multiple sequence alignments were of trafficking and signaling of melanocortin receptors performed using MUSCLE 3.8.31 with default parameters (Asai et al. 2013, Cerda-Reverter et al. 2013). Two main (Edgar 2004). The percentage of similarity between the forms MRAP1 and MRAP2 exist in zebrafish genome. As amino acid sequences were calculated with DNAMAN. in mammals, MRAP1 is essential for adrenocorticotropic Putative TMDs of MC5Rs were predicted with TMHMM hormone receptor (MC2R) trafficking to the cell Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), membrane, ligand binding and downstream signaling GenBank or NCBI Reference Sequence accession numbers (Agulleiro et al. 2010). MRAP2 (melanocortin receptor for MC5Rs: human Homo sapiens (AAH95531.1), western accessory protein 2) was found as a homolog of MRAP1. gorilla Gorilla gorilla (XP_004059266.1), chimpanzee MRAP2 interacts with all five MCRs and modulates Pan troglodytes (NP_001009119.1), hamadryas baboon receptors trafficking to the membrane, agonist binding Papio hamadryas (ACO90015.1), rabbit Oryctolagus and cAMP production (Chan et al. 2009, Cerda-Reverter cuniculus (CCX35395.1), American mink Neovison vison et al. 2013). Recent studies demonstrated that MRAP2 (AGT56096.1), red fox Vulpes vulpes (ABG48760.1), could also interact with non-melanocortin receptors dog Canis lupus familiaris (NP_001074193.1), raccoon and modulate their signaling (Chaly et al. 2016, Srisai dog Nyctereutes procyonoides (ABO38182.1), Norway et al. 2017). In mammals, MRAP2 enhances MC4R rat Rattus norvegicus (NP_037231.1), house mouse Mus activity and loss of function of MRAP2 causes early-onset musculus (NP_058673.2), pig Sus scrofa (NP_999338.1), obesity (Asai et al. 2013). The mrap2 gene is present in sheep Ovis aries (NP_001119842.1), goat Capra hircus the genome of lampreys (Vastermark & Schioth 2011). (NP_001272520.1), cattle Bos taurus (NP_776535.1), In zebrafish, mrap2 has two homologs: mrap2a and water buffalo Bubalus bubalis (AHN49803.1), domestic mrap2b. Both of MRAP2a and MRAP2b can interact with yak Bos grunniens (ADH51715.1), wild yak Bos mutus zebrafish MC4R: MRAP2a inhibit the binding of MC4R (ADZ24287.1), American alligator Alligator mississippiensis to its agaonist α-MSH, whereas MRAP2b stimulates (KYO35523.1), Ring-necked pheasant Phasianus colchicus MC4R activation by increase ligand-binding ability and (ACF35264.1), chicken Gallus gallus (NP_001026685.1), surface expression (Sebag et al. 2013). Josep Agulleiro hazel grouse Tetrastes bonasia (ACF35263.1), common carp et al. (2013) also found that MC4R could be activated Cyprinus carpio (CBX89936.1), goldfish Carassius auratus by ACTH when co-expressed with MRAP2a. MRAP2b can (CAD58853.1), Ya-fish Schizothorax prenanti (AGF80338.1), decrease the constitutive activity of MC4R during fasting zebrafish Danio rerio (NP_775387.1) and (NP_775386.1). which suggests that the deletion of MRAP2b may induce Phylogenetic tree based on the amino acid sequences growth changes or obesity. As in zebrafish, MRAP2 can was constructed by neighbor-joining (NJ) methods with enhance the sensitivity of MC4R for ACTH and block 1000 bootstrap resampling and Poisson correction model, the constitutive activity of MC4R in chickens (Zhang pairwise-deletion option using Mega 7.0 software. To et al. 2017). Unlike MC4R, the physiological functions determine whether mc5ra or mc5rb is more orthologous between MC5Rs and MRAP2 proteins are largely to mammals, synteny analysis was performed between unknown in zebrafish. zebrafish, medaka, spotted gar, human, mouse and rat In this study, we examined the interaction and with ensemble (http://www.ensembl.org/index.html). pharmacological modulation between two pairs of The adjacent genes of zebrafishmc5ra or mc5rb were as zebrafish proteins: MC5Ra, MC5Rb and MRAP2a, MRAP2b. follow: rnmt, mRNA cap guanine-N7 methyltransferase; MRAP2a prevents both MC5Ra and MC5Rb expression fam210aa, uncharacterized protein C18orf19 homolog

A; cthrc1a, collagen triple helix repeat containing 1a; and centrifuged, suspended in loading buffer and boiled slc25a32a, Slc25a32a protein; fam210ab, uncharacterized for 15 min. 10% gels were used for SDS–PAGE. Anti-HA protein C18orf19 homolog B; ldlrad4b, low-density (Abcam) and anti-Flag (abclonal) antibodies were used at lipoprotein receptor class A domain-containing protein a dilute of 1:4000 for immunoblotting. 4-like; mc2r, melanocortin 2 receptor; lmna, lamin.

cAMP assays Animal care HEK 293T cells were plated into 24-well plates, and WT zebrafish (TU) were raised at 26–28°C, with 14-h light transfected with indicated plasmids next day. After 24 h of and 10 h night cycle. Fish aged from 5 to 10 dpf were fed transfection, cells were treated with α-MSH or SHU9119 in twice a day with paramecia, fish aged from 10 to 15 dpf DMEM medium supplemented with 0.1% bovine serum were fed with paramecia and brine shrimp, adult fish albumin for 4 h at 37°C, concentration of α-MSH ranged were fed with brine shrimp in system water. Zebrafish from 10−12 M to 10−6 M, concentration of SHU9119 ranged care and experimental procedures were approved by the from 10−13 M to 10−7 M signaling assay was tested in HEK Institutional Animal Care and Use Committee (IACUC) of 293T cells stimulated by 2 × 10-9 M α-MSH and AgRP with Tongji University. 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 Plasmids and peptides Spectramax M5 plate reader. All data were normalized to 3HA-zMC5Ra, 3HA-zMC5Rb, 2Flag-zMRAP2a and 2Flag- Renilla luminescence. zMRAP2b, cloned from WT zebrafish, were constructed into vector pcDNA3.1 (+) (Invitrogen). α-MSH and Cell-surface ELISA SHU9119 were purchased from GenScrpipt Corporation Ltd. (China). AgRP (83–132) were synthesized by Chinese Twelve-well plates were treated with Poly-l-lysine Peptide Company (Hangzhou, China). 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% Cell culture and transfection poly-formaldehyde for 20 min at room temperature. After Human embryonic kidney (HEK) 293T cells were cultured fixation, cells were washed, blocked for 30 min in 5% milk in Dulbecco’s Modified Eagle’s Medium (DMEM) medium in D-PBS for surface groups or in 5% milk in lysis buffer contain 10% fetal bovine serum and 1% penicillin/ (Beyotime) for whole-cell groups and incubated with streptomycin. Chinese hamster ovary (CHO) cells anti-HA antibody (Abcam) for 2 h at room temperature. were cultured in DMEM/F12 medium contain 5% fetal After washed three times with D-PBS, cells were incubated bovine serum and 1% penicillin/streptomycin. Cells with secondary antibody for 1 h. Then, cells were washed were maintained in a humidified atmosphere consisting three times with D-PBS and incubated with TMB solution of 5% CO2 at 37°C. For transfection, when cells growth for 15–30 min, the reaction was stopped by adding 2 M to 70–80% confluence, plasmids were transfected using H2SO4. Spectramax M5 plate reader was used to record OD ViaFect Transfection Reagent (Promega). 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 Co-immunoprecipitation and Western blotting water. After removal of all the water, added 200 μL 0.5 M 1 × 106 HEK 293T cells were seeded into 60 mm dishes HCl to shake for 10 min, record OD at 595 nm. All data and transfected next day. After 48 h of transfection, were normalized to OD 595 nm before used. Statistical cells were washed with PBS and lysed with lysis buffer analyses were performed using Graph Pad Prism, version (0.75% Triton-X, 50 mM Tris–HCl pH 7.9, 150 mM NaCl 6 (GraphPad Software). and proteinase inhibitor cocktail from Roche) for 1 h at 4°C and centrifuged. Supernatants were incubated with Immunofluorescence anti-HA (Abcam) overnight at 4°C. Protein A/G agarose beads were added to cell lysate next day, and rotated for YFP fragments YFP-F1 and YFP-F2 (Sebag & Hinkle 2009) 4 h at 4°C, beads were washed three times using lysis buffer were constructed into C-terminal of GPCRs respectively

CHO cells were seed into a 12-well plates with slides and extracellular and intracellular loops with an extracellular transfected next day. After 24 h of transfection, cells were N-terminus and an intracellular C-terminus (Supplementary fixed with paraformaldehyde for 20 min, and then washed Fig. 1, see section on supplementary data given at the end 3 times with PBS. To detect surface expression of MRAP2s, of this article). Alignment of the amino acid sequences cells were incubated with anti-V5 antibody (Abcam) of MC5R with that of other species demonstrated that at 1:1000 for 2 h at 37°C. Then washed 3 times and zebrafish MC5Ra and MC5Rb were homologs to tetraodon incubated with 1:1000 secondary antibody Alexa Fluor594 (78.53 and 78.85%, respectively), and fugu (78.53 and (Abcam) for 2 h at 37°C. For FACS analysis, MRAP2:MC5R- 75.00%, respectively) MC5Rs, whereas the homology of F1:MC5R-F2 were transfected at ratio 2:1:1, after 36 h of zebrafish MC5R to mammalian MC5Rs, such as human transfection, cells were digested into single cell using and mouse MC5Rs, was lower (61.83–75%). As shown in 0.02% EDTA and centrifuged, 5 × 105 cells were dispensed Supplementary Fig. 1, the amino acid sequences in the into 500 μL DMEM/F12 medium, filtered through a 40 μm transmembrane domains and intracellular loops of MC5Ra mesh sieve into a 5 mL Polystyrene Round-Bottom Tube and MC5Rb were found to be highly identical to that of (FALCON), and then analyzed by BD FACSVerse. other species while lower homology was identified at the N-termini and extracellular loops. A NJ phylogenetic tree Fasting and real-time quantitative PCR was analyzed based on amino acid sequences of MC5Rs from different species and our results indicated that MC5Ra Male WT zebrafish (TU) were fasted 0, 2, 10, 15 days of zebrafish was more evolutionarily related to tetraodon respectively, keeping water change every day. On indicated and fugu MC5Rs. However, the zebrafish MC5Rb out- day, the brain RNA of adult zebrafish was extracted using groups to all bony vertebrate MC5R orthologs (Fig. 1A). RNAiso Plus (TaKaRa), according to the manufacturer’s To determine which mc5r is more orthologous to instructions. 1 μg RNA was reverse transcribed using a mammals, synteny analysis was performed between TIANGEN FastKing RT kit (Beijing, China). cDNA reaction zebrafish, spotted gar, human, mouse and rat (Fig. 1B). Our was diluted 1:10 as a template for qPCR. Real-time PCR results showed that adjacent genes of mc5ra were highly was performed using LightCycler 96 (Roche), the PCR conserved between zebrafish and human, mouse and conditions were: preincubation at 95°C for 600 s; 45 cycles rat MC5Rs. The adjacent genes of mc5ra, including rnmt, of 95°C 10 s, 60°C for 10 s, and 72°C for 10 s; melting at 95°C fam210aa, ldlrad4a, sehil, ptpn2b and psmg2 were identical for 10 s, 65°C for 60 s and 97°C for 1 s. Primers for qPCR: with those of spotted gar, human, mouse and rat MC5Rs. mc5ra, forward 5′-TCCTCATCCTTGGCATCGTCAGT- Interestingly, the adjacent genes of mc5rb: ldlrad4b, 3′,reverse 5′-TGGCGATTGGTGAGCAGGTAGAT-3′; mc5rb, fam210ab and especially mc2r were conserved between forward 5′-AGAACAAGAACCTCCACTCACCAATG-3′, zebrafishmc5rb and mouse, rat and human MC5Rs. reverse 5′-TTGCCAGCAGATGAATGACGATTGT-3′; ef1α, forward 5′-CCTGCCAGTGTTGCCTTCGT-3′, reverse Interaction of zebrafish MRAP2s with MC5Rs 5′-CCTCCTTGCGCTCAATCTTCC-3′. All reactions were run in triplicate and repeated three times. To verify the protein–protein interactions between two MRAP2s and two MC5Rs, four groups of HEK 293T cells Statistical analyses were transfected with 2Flag-zMRAP2a and 3HA-zMC5Ra, 2Flag-zMRAP2b and 3HA-zMC5Ra, 2Flag-zMRAP2a and All experiments were performed at least three times. 3HA-zMC5Rb, 2Flag-zMRAP2b and 3HA-zMC5Rb, Data were analyzed using GraphPad Prism 6 (GraphPad respectively. Co-immunoprecipitation results showed Software, Inc.). Results of cAMP assays were analyzed by that zebrafish MRAP2a (Fig. 2A and B) and MRAP2b two-way ANOVA with Tukey post-test. Results of surface (Fig. 2C and D) could interact with MC5Ra and MC5Rb, ELISA and qPCR were analyzed by two-tailed t test. All suggesting that MRAP2a and MRAP2b could modulate the Results were shown as mean ± s.e.m. signaling of MC5Ra and MC5Rb in zebrafish.

Results Surface expression of MC5Rs was influenced Amino acid sequences of MC5R by MRAP2s

The amino acid sequences of MC5R consisted of To investigate the influences of MRAP2s on the MC5R seven transmembrane domains connected by alternating trafficking, we next measured the surface expression

A MRAP2a and MRAP2b modulate signaling of MC5Ra 100 SheepMC5R 97 Goat MC5R and MC5Rb in different ways 99 CowMC5R 98 PigMC5R To determine how MRAP2a and MRAP2b affect MC5Rs HumanMC5R 100 93 Chimpanzee MC5R signaling, CRE-luciferase reporter assay was performed 100 RabbitMC5R to detect the cAMP level stimulated by α-MSH through HousemouseMC5R 99 MC5Rs. We found that MRAP2a dose-dependently 100 Norway ratMC5R

96 ChickenMC5R inhibited the efficacy of both MC5Ra and MC5Rb, 98 Xenopus MC5R MRAP2b inhibited MC5Ra efficacy but increased the Elephant sharkMC5R 99 Mudshark MC5R ZebrafishMC5Ra

84 Fugu MC5R IP: HA Lysate Lysate 100 Tetraodon MC5R A B IP: HA 3HA-zMC5Ra 3HA-zMC5Ra ZebrafishMC5Rb 2Flag-zMRAP2a 2Flag-zMRAP2b B

cep76 PSMG2 Cep76 Cep76 gstk2 ncaldb zMC5Ra dgat1b psmg2 psmg2 CEP76 Psmg2 Psmg2 zMC5Ra gpr20 ptpn2b ptpn2a PTPN2 Ptpn2 Ptpn2 { { slc45a4 ftr56 seh1l SEH1L Seh1l Cep192 dennd3b seh1l cep192 CEP192 Cep192 RGD1562758 ptk2ab ldlrad4a ldlrad4a LDLRAD4 Ldlrad4 Ldlrad4 zMRAP2a zMRAP2b ldlrad4b fam210aa fam210aa FAM210A Fam210a Fam210a IP: HA Lysate fam210ab rnmt rnmt RNMT Rnmt Rnmt C IP: HA Lysate D mc5rb mc5ra mc5ra MC5R Mc5r Mc5r b 3HA-zMC5Rb 3HA-zMC5R 2Flag-zMRAP2b mc2r cthrc1a mc2r MC2R Mc2r Mc2r 2Flag-zMRAP2a lmna slc25a32a fzd6 ZNF519 4930546C10Rik Tcf4 pear1 rims2b cthrc1a POTEC Tcf4 Poli zMC5Rb zMC5Rb zgc:77086 zfpm2b slc25a32a ANKRD30B Ccdc68 Mbd2 cct3 lrp12 dcaf13 ROCK1 Rab27b Dcc { { tmem79b srsf4 rims2 GREB1L Dynap Ccdc68 CABZ01063757.1 epb41a dcstamp ESCO1 Stard6 Rab27b sema6e ctgfb dpys SNRPD1 Poli Dynap zMRAP2a zMRAP2b

Zebrafish Zebrafish Spotted gar Human Mouse Rat Chr. 16 Chr. 19 LG.11 Chr. 18 Chr. 18 Chr. 18 EF G ) ) ) 150 150 150 ne ne ne lo lo lo C5Ra C5Rb C5Ra 100 100 100 ba aa Figure 1 aa eM eM eM 5R 5R 50 5R 50 50 ac Phylogenetic and synteny analysis of zebrafish MC5R receptors. (A) ac MC MC MC Surf Surf Phylogenetic tree of MC5Rs constructed by NJ method with Mega 6.0 Surfac (% (% 0 (% 0 0 software. Jones–Taylor–Thornton (JTT) model was used. The strength of k :0 :3 :6 k :0 :3 :6 k :0 oc oc 1 oc a 1 a 1 a 1 b 1 b 1 a 1 a1:3 a1:6 branch relationships was assessed by bootstrap replication (N = 1000 m m 2b m 2 P2 P2 P2 P2 P2 P P P2 P2 A replicates). Asterisk (*) indicates zebrafishmc5ra and mc5rb. (B) Synteny RA RA RA RA RA RA zM zM zM zM zM zM zMR zMRAzMRA analysis of MC5Rs. Genes in solid black square are conserved among a + a + a + a + a + a + b + b + b + R 5R 5R 5R 5R 5R 5 5R 5R 5R zebrafish, spotted agar, human, mouse, and rat. MC MC zMC zMC zMC zMC zMC zMC zMC z z

H ) 150 I 150 J 150 of MC5Ra and MC5Rb in the absence or presence of e) ne ) R on l lone lo C5 R C5 MRAP2a or MRAP2b. 293T cells were transfected with C5Rb 100 100 100 ba Ra Ra eM eM eM 5R 50 C5 50 MC5R alone (1:0) and MRAP2 at progressive ratio (1:3, ac 50 MC mM hMC5 Surfac Surf Surfac

(% 0 0 1:6) as used in previous studies (Liang et al. 2018). (% 0 (% k k c :0 :3 :6 :0 :3 :6 ck :0 :3 :6 o oc o 1 1 1 The results showed that MRAP2a decreased the surface m b 1 b 1 b 1 M 2 1 2 1 2 1 P P M P2 P2 P2 A AP A R RAP2RAP2RAP2 expression of both MC5Ra and MC5Rb, whereas RA RA RA MR MR zM zM zM mM hM hM hM + m + m + + + b + b + b + R R + R R R MRAP2b had no significant effect (Fig. 2E, F, G and H). R 5 5 5 5 5 5R 5R C5 C C C MC MC5RM M M M h h h Since the results were different from previous studies in zMC zMC zMC m m m human (Chan et al. 2009, Sebag & Hinkle 2009), then Figure 2 we validated the surface expression of mammalian MRAP2s interact and regulate surface expressions of MC5Rs. MC5Rs in the absence or presence of mammalian Co-immunoprecipitation assay exhibits the interactions between zMC5Ra MRAP2s. These results showed that human MRAP2 and zMRAP2a (A), zMRAP2b (B), also between zMC5Rb and zMRAP2a (C), zMRAP2b (D). *IgG heavy chain. Surface expression of zMC5Ra and decreased the surface expression of human MC5R zMC5Rb measured by whole-cell ELISA in 293T cells transfected with like MRAP2a, but mouse MRAP2 had no significant zMC5Ra or zMC5Rb and different amounts of zMRAP2a or zMRAP2b (E, F, influence on the surface expression of mouse MC5R, 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 which was similar to the results of MRAP2b in zebrafish measured by whole-cell ELISA in 293T cells transfected with mammalian (Fig. 2I and J). MC5R and different amounts of mammalian MRAP2 (I and J).

https://jme.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JME-18-0104 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 08:17:44PM via free access Journal of Molecular M Zhu, M Wang et al. Pharmacological modulation of 62:1 32 Endocrinology MC5R by MRAP2 maximal stimulated efficacy of MC5Rb Fig. ( 3A, B, C Unexpectedly, zMC5Ra and zMC5Rb could also form and D). To test whether this effect was stable with other heterodimers (Fig. 4C). However, when co-expressed with agonist, similar results were seen when stimulated with MRAP2a or MRAP2b, the YFP fluorescence could barely be SHU9119, another reported MC5R agonist (Fig. 3E, F, G detected (Fig. 4D, E, F, G, H and I). Thus, we hypothesized and H). We also tested the effect of mouse MRAP2 on MC5R that MRAP2a and MRAP2b could disrupt dimerization of and found that MRAP2 dose-dependently inhibited the zMC5Rs just like the effect of MRAP on human MC5R. To efficacy of mouse MC5R Fig. 3I( and J), which was similar validate our hypothesis, we counted the fluorescent cells to zebrafish MC5Ra. However, both MRAP2a and MRAP2b by FACS (Fluorescence Activated Cell Sorter). Compared did not affect the agonists and antagonists signaling to control groups that co-expressed with RAMP3, another competing affinity (Fig. 3K, L, M and N). LogEC50 values reported transmembrane protein that did not regulate of each curve were shown in Table 1. melanocortin receptor trafficking Sebag( & Hinkle 2009), zMRAP2a and zMRAP2b significantly decreased the percentage of YFP fluorescent cells (Supplementary Fig. 2). Dimerization of zebrafish MC5Rs Thus, zMRAP2a and zMRAP2b could disrupt the dimerization Human MC5R has been reported to form homodimers, of both zMC5Rs homodimers and heterodimers, similar to which could be disrupted by MRAP (Sebag & Hinkle the effect of MRAP on human MC5R. 2009). To assess the ability of dimerization of two zebrafish MC5Rs, YFP fragments were fused to C-terminal Fasting increased the expression of mc5ra in of zMC5Ra and zMC5Rb. zMC5R-YFP-F1 and zMC5R- zebrafish brain YFP-F2 were co-expressed in CHO cells and the YFP fluorescence indicated the presence of zMC5Ra and The melanocortin system may be involved in regulating zMC5Rb homodimers on cell surface (Fig. 4A and B). food intake of fish, but no experiments had been carried

ABCD zMC5Ra : zMRAP2a zMC5Ra : zMRAP2b zMC5Rb : zMRAP2a zMC5Rb : zMRAP2b 1:0 1:0 1:0 1:0 100 1:3 100 1:3 100 100 1:3 1:3

1:6 1:6 ) 1:6 ) x) 1:6

75 75 ax 75 ax 75 M M (% (% (%Ma (%Max) 50 50 50 50 MP MP MP MP cA cA cA cA 25 25 25 25

0 0 0 0 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 a-MSH a-MSH a-MSH a-MSH

EFzMC5Ra : zMRAP2a zMC5Ra : zMRAP2b G zMC5Rb : zMRAP2a H zMC5Rb : zMRAP2b 1:0 1:0 1:0 1:0 100 100 100 1:3 100 1:3 1:3 1:6 1:3 ) )

1:6 x 1:6 x 1:6 75 75 75 75 Max) Ma Max) Ma %

(% 50 (% 50 (% 50 ( 50 MP MP MP MP 25 25 25 25 cA cA cA cA

0 0 0 0 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 SHU9119 SHU9119 SHU9119 SHU9119

I mMC5R : mMRAP2 J mMC5R : mMRAP2 Figure 3 1:0 100 1:3 100 1:0 Pharmacological modulation of MC5Rs by 1:6 1:3 75 75 1:6 MRAP2s. Dose response curves of -MSH induced

Max) α Max) (% 50 (% 50 cAMP production in 293T cells upon transfection MP MP with zMC5Ra (A and B) or zMC5Rb (C and D) in 25 cA 25 cA presence of different amount of zMRAP2a or 0 0 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 zMRAP2b. Dose response curves of SHU9119 a-MSH SHU9119 induced cAMP production in 293T cells transfected with zMC5Ra (E and F) or zMC5Rb (G KLzMC5Ra : zMRAP2a zMC5Ra : zMRAP2b MNzMC5Rb : zMRAP2a zMC5Rb : zMRAP2b and H) in presence of different amount of 1:0 100 1:0 100 1:0 100 1:0 100 1:3 1:3 1:3 zMRAP2a or zMRAP2b. Dose response curves of

) 1:3 x) x 1:6 1:6 x) 1:6 75 a 75 75 1:6 75 -MSH (I) and SHU9119 (J) induced cAMP Max) Ma Ma α %M (% ( (% 50 50 50 (% 50 production in 293T cells transfected with mouse MP MP MP MP MC5R and the indicated amount of mouse 25 25 25 25 cA cA cA cA MRAP2. Binding competition of agonist (α-MSH) 0 0 0 0 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 10 -12 10 -10 10 -8 10 -6 and antagonist (AgRP) of MC5Rs modulated by AgRP AgRP AgRP AgRP MRAP2s (K, L, M and N).

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 0.0001 0.0085 0.0584 0.005 0.9981 0.3975 0.3396 0.4559 0.3085 0.0371 0.1425 <0.0001 <0.0001 <0.0001 1:3 vs 1:6 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( ). comparison max 0.0005 0.0005 0.275 0.292 V <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 1:0 vs 1:6 Discussion

The fish-specific genome duplication (FSGD or 3R) occurred value for

P about 350 million years ago; thus, zebrafish experienced an

0.0006 0.0009 0.1858 0.038 0.0278 0.0116 0.0039 additional genome duplication than human (Meyer & Van <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 1:0 vs 1:3 de Peer 2005), which caused several gene duplications in zebrafish genome, such asmrap2a 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 1:6

9.32 ± 0.20 8.73 ± 0.17 8.81 ± 0.16 9.54 ± 0.14 8.91 ± 0.20 8.79 ± 0.20 8.33 ± 0.12 7.99 ± 0.09 7.85 ± 0.15 7.42 ± 0.31 (Sebag et al. 2013). Following the three whole genome 10.69 ± 0.26 10.66 ± 0.24 10.17 ± 0.14 10.41 ± 0.30 − − − − − − − − − − − − − − duplication event, chromosome reduction and gene loss, the only duplicated melanocortin receptor genes left in the zebrafish genome are themc5ra and the mc5rb, and all the other zebrafish melanocortin receptor genes are present in single copy. Actually, the function of MC5Rs has not

1:3 been elucidated in fish. Results of amino acid sequences 9.58 ± 0.20 9.09 ± 0.18 8.91 ± 0.09 9.28 ± 0.22 9.03 ± 0.19 9.01 ± 0.09 8.31 ± 0.09 8.03 ± 0.12 7.87 ± 0.14 7.30 ± 0.42 LogEC50 10.88 ± 0.39 10.65 ± 0.30 10.54 ± 0.11 10.59 ± 0.30 − − − − − − − − − − 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

1:0 hydrophobic transmembrane domains. Phylogenetic 9.84 ± 0.16 9.87 ± 0.21 9.53 ± 0.12 9.11 ± 0.16 10.8 ± 0.32 9.82 ± 0.14 8.02 ± 0.17 8.15 ± 0.15 7.50 ± 0.29 7.73 ± 0.44 10.49 ± 0.27 11.09 ± 0.12 10.82 ± 0.18 10.12 ± 0.41 − − − − − − − − − − 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. zMC5Ra:zMRAP2a zMC5Ra:zMRAP2b zMC5Rb:zMRAP2a zMC5Rb:zMRAP2b zMC5Ra:zMRAP2a zMC5Ra:zMRAP2b zMC5Rb:zMRAP2a zMC5Rb:zMRAP2b mMC5R:mMRAP2(a-MSH) mMC5R:mMRAP2(SHU9119) zMC5Ra:zMRAP2a zMC5Ra:zMRAP2b zMC5Rb:zMRAP2a zMC5Rb:zMRAP2b observed that hMRAP2 decreased hMC5R trafficking while we observed that mMRAP2 did not effect mMC5R

Analyze data in Fig. 3. trafficking. These data suggest that there appears to be two types of MRAP2 in mammals, and the alternative effects

Table 1 Analyze data in Fig. 3 A B C D E F G H I J K L M N Statistics were measured using a two-way ANOVA with Tukey post-test. are species specific. https://jme.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JME-18-0104 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 08:17:44PM via free access Journal of Molecular M Zhu, M Wang et al. Pharmacological modulation of 62:1 34 Endocrinology MC5R by MRAP2

A B C

MC5Ra- MC5Ra- MC5Rb- MC5Rb- MC5Ra- MC5Rb- YFP-F1 YFP-F2 YFP-F1 YFP-F2 YFP-F1 YFP-F2

D V5-MRAP2a YFPV5 E V5-MRAP2b YFP V5

MC5Ra- MC5Ra- MC5Ra- MC5Ra- YFP-F1 YFP-F2 YFP-F1 YFP-F2

F V5-MRAP2a YFPV5 G V5-MRAP2b YFP V5

MC5Rb- MC5Rb- MC5Rb- MC5Rb- YFP-F1 YFP-F2 YFP-F1 YFP-F2

H V5-MRAP2a YFPV5 I V5-MRAP2b YFP V5

MC5Ra- MC5Rb- MC5Ra- MC5Rb- YFP-F1 YFP-F2 YFP-F1 YFP-F2

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.

Our results suggested that the interactions between was considered as the zebrafish ortholog of MRAP2 (Asai MRAP2s and MC5Rs were conserved in zebrafish. et al. 2013). Although both MRAP2a and MRAP2b could interact with We further verified that MRAP2a and MRAP2b melanocortin receptors in zebrafish Agulleiro( et al. 2010, could modulate the signaling of MC5Ra and MC5Rb in Sebag et al. 2013, Cortes et al. 2014), they had distinct different ways. MRAP2a could inhibit maximal activity of functions on modulating the signaling of receptors. As both MC5Ra and MC5Rb, which might be explained by same as mouse MRAP2, MRAP2b had no influence on decreased cell surface expression. To our surprise, MRAP2b the surface expression of MC5R. Previous studies showed also inhibited MC5Ra activity in zebrafish without that MRAP2b could inhibit the adult zebrafish MC4R changing its surface expression, and unexpectedly constitutively activity but increase ligand-dependent increased MC5Rb activity. MC5R was reported to form activity (Sebag et al. 2013). Like MRAP2b, mouse MRAP2 homodimers and heterodimers with other GPCRs (Sebag could increase the signaling of mouse MC4R, so MRAP2b & Hinkle 2009, Kobayashi et al. 2016). To elucidate the

6 mc5ra Declaration of interest *** The authors declare that there is no conflict of interest that could be mc5rb perceived as prejudicing the impartiality of the research reported.

0 *** ay

d 4 o t ** Funding ns The work was supported by grants from National Key Research and Development Program of China (Grant No. 2017YFA0103902 and hange

c 2 2016YFA0102200); The National Natural Science Foundation of China (Grant

ld No. 81570760 and 31771283); One Thousand Youth Talents Program of

Fo 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 0 Funds for the Central Universities of Tongji University. 0210 15 0210 15 Day References

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Received in final form 17 October 2018 Accepted 25 October 2018 Accepted Preprint published online 25 October 2018