Divergent Pharmacology and Biased Signaling of the Four Melanocortin-4 Receptor Isoforms in Rainbow Trout (Oncorhynchus mykiss)

The melanocortin-4 receptor (MC4R) is essential for the modulation of energy balance and reproduction in both fish and mammals. Rainbow trout (Oncorhynchus mykiss) has been extensively studied in various fields and provides a unique opportunity to investigate divergent physiological roles of paralogues. Herein we identified four trout mc4r (mc4ra1, mc4ra2, mc4rb1, and mc4rb2) genes. Four trout Mc4rs (omMc4rs) were homologous to those of teleost and mammalian MC4Rs. Multiple sequence alignments, a phylogenetic tree, chromosomal synteny analyses, and pharmacological studies showed that trout mc4r genes may have undergone different evolutionary processes. All four trout Mc4rs bound to two peptide agonists and elevated intracellular cAMP levels dose-dependently. High basal cAMP levels were observed at two omMc4rs, which were decreased by Agouti-related peptide. Only omMc4rb2 was constitutively active in the ERK1/2 signaling pathway. Ipsen 5i, ML00253764, and MCL0020 were biased allosteric modulators of omMc4rb1 with selective activation upon ERK1/2 signaling. ML00253764 behaved as an allosteric agonist in Gs-cAMP signaling of omMc4rb2. This study will lay the foundation for future physiological studies of various mc4r paralogs and reveal the evolution of MC4R in vertebrates.


Introduction
Melanocortin-4 receptor (MC4R), a Family A rhodopsin-like G protein-coupled receptor (GPCR), primarily couples to the stimulatory heterotrimeric G protein and activates adenylyl cyclase, resulting in elevated intracellular cyclic adenosine monophosphate (cAMP) generation to regulate downstream signaling [1].α-Melanocyte-stimulating hormone (α-MSH) and other peptides derived from posttranslational processing of proopiomelanocortin (POMC) are endogenous agonists, and agouti-related peptide (AgRP) is an endogenous inverse agonist of MC4R.MC4R has high expression in the central nervous system and is essential for regulation of energy homeostasis, as well as reproduction and sexual function in mammals [1][2][3].Mc4r-deficient mice show an obese phenotype with increased food intake and decreased energy expenditure [4,5].More than 300 mutations in human MC4R have been identified from patients, and MC4R mutations are considered to be the leading cause of monogenic obesity [3,[6][7][8][9].
Mc4r has also been studied in teleosts.Fish mc4r is widely distributed in both the central nervous system and the periphery [10][11][12][13][14][15][16][17][18][19].Activation of Mc4r with agonists decreases food intake in rainbow trout and goldfish, while injection of antagonists results in increased food intake [11,[20][21][22].Aspiras et al. reported that Mexican cavefish mc4r mutations contribute to elevated appetite, starvation resistance, and growth, playing crucial roles in adaptation to a nutrient-poor environment [23].Furthermore, Mc4r is shown to play key roles in sexual behavior and reproduction in teleosts [24][25][26][27] (reviewed in [9]).These studies indicate that teleost Mc4r is also an important regulator in energy balance and reproduction.Hence, a better understanding of physiological functions of teleost Mc4r will be beneficial for growth and artificial breeding of cultured fish.
Rainbow trout (Oncorhynchus mykiss), cultured worldwide, belongs to the salmonid family and has been extensively studied in various fields, such as toxicology, carcinogenesis, ecology, immunology, physiology, and nutrition [52,53].The presence of whole-genome duplication of rainbow trout also provides a unique opportunity to explore the early evolutionary feature of a duplicated vertebrate genome and contributes to investigating the divergent physiological roles of the paralogues [54,55].
In this study, we identified four paralogs of mc4r genes in rainbow trout, and we further investigated the pharmacological characteristics of the four trout Mc4r isoforms (omMc4rs) encoded by these duplicated genes.Inverse agonists have been extensively used in mammalian MC4Rs.However, the effects of mammalian MC4R inverse agonists on teleost Mc4r are not well understood [51].Especially, small molecule inverse agonists, which could potentially be added into the feed, will contribute to developing novel feeding strategies in aquaculture.Thus, several MC4R inverse agonists, such as AgRP, MCL0020 [56], ML00253764 [57], and Ipsen 5i [58], were used to explore their effects on two signaling properties (Gαs-cAMP and ERK1/2 signaling) of the four trout Mc4rs.This study will facilitate the investigation of potential physiological functions of different mc4r paralogs, as well as contribute to revealing the evolution of MC4R in vertebrates.

Cell Culture and Transfection
Human Embryonic Kidney (HEK) 293T cells, provided by American Type Culture Collection (Manassas, VA, USA), were cultured as described previously [18,62].Cells were plated into 24-well or 6-well plates pre-coated with 0.1% gelatin and transfected with plasmids by the calcium phosphate precipitation method [63].

Flow Cytometry Assay
Flow cytometry was used to determine the cell-surface and total expression levels of trout and human MC4Rs as described earlier [34,64].Cells were plated into 6-well plates and transiently transfected with empty vector and trout and human MC4R plasmids.Forty-eight hours after transfection, cells were washed twice with cold PBS.Cells were detached with 1 mL PBS by pipetting up and down.Two wells were collected for nonpermeabilized cells and four wells for permeabilized cells.For permeabilized cells, cells were fixed with 200 µL of 4% paraformaldehyde and were permeabilized with 200 µL of 0.1% Triton 100.Non-permeabilized and permeabilized cells were incubated with primary antibody (mouse anti-myc 9E10 antibody) and secondary antibody (Alexa Fluro@488 goat anti-mouse antibody).The CytoFLEX LX flow cytometer (Beckman Coulter, Indianapolis, IN, USA) was used for analysis.Empty vector (pcDNA3.1)fluorescence was used for correction of background staining.The expression of trout Mc4r was calculated as the percentage of hMC4R, after subtraction of background staining [65].

Statistical Analysis
Results were represented as mean ± SEM.GraphPad Prism 8.3 software (San Diego, CA, USA) was used to analyze data.The significance of differences was determined by one-way ANOVA.
Phylogenetic tree analysis between omMc4rs and other MC4Rs showed that four omMc4rs were clustered into four different groups (Figure 4

Cell-Surface and Total Expression of omMc4rs
Flow cytometry was used to determine cell-surface and total expression of omMc4rs (Figure 5).Results showed that all four trout Mc4rs had higher cell-surface expression than hMC4R (Figure 5A).Trout Mc4rb1 also showed higher total expression compared to hMC4R (Figure 5B).Additionally, three trout Mc4rs, including omMc4ra1, omMc4ra2, and omMc4rb1, showed higher surface ratios (surface expression/total expression) at greater than 70%, and hMC4R only had around a 40% surface ratio (Figure 5C).
For the antagonists, only AgRP (83-132) could dose-dependently displace the 125 I-NDP-MSH binding to the four omMc4rs with lower IC 50 values than that of hMC4R (Figure 6 and Table 1).MCL0020 only displaced 125 I-NDP-MSH binding to hMC4R and omMc4rb1, and omMc4rb1 showed a higher IC 50 than that of hMC4R (Figure 6 and Table 1).ML00253764 and Ipsen 5i displaced 125 I-NDP-MSH binding to hMC4R, with IC 50 values of 1644.57± 215.24 nM and 399.86 ± 122.11 nM, respectively, and these two compounds could not displace 125 I-NDP-MSH binding to the four trout Mc4rs (Figure 6 and Table 1).Values are expressed as the mean ± SEM of at least three independent experiments.a Significant difference from the parameter of hMC4R, p < 0.05.b Significant difference from the parameter of hMC4R, p < 0.01.c Significant difference from the parameter of hMC4R, p < 0.001.N/A: could not be determined.
In this study, the basal signaling of omMc4ra1, omMc4ra2, omMc4rb1, and omMc4rb2, was 1.16-, 2.53-, 6.48-, and 0.83-fold that of hMC4R, respectively, indicating that trout Mc4rs might be constitutively active, especially omMc4ra2 and omMc4rb1 (Figure 8A).Values are expressed as the mean ± SEM of at least three independent experiments.a Significant difference from the parameter of hMC4R, p < 0.05.b Significant difference from the parameter of hMC4R, p < 0.01.c Significant difference from the parameter of hMC4R, p < 0.001.

Constitutive Activities of omMc4rs in Response to Four Antagonists
The basal activity of hMC4R and constitutively active mutant hMC4Rs were shown to be reduced by inverse agonists, such as AgRP (83-132), ML00253764, MCL0020, and Ipsen 5i [1,28,48,51].To investigate their effects on the basal activities of trout Mc4rs, cells transfected with the four trout Mc4rs were treated with 10 nM AgRP (83-132), 1 µM MCL0020, 1 µM ML00253764, or 1 µM Ipsen 5i, respectively.Results showed that AgRP (83-132) acted as an inverse agonist for the four trout Mc4rs (Figure 8).ML00253764 acted as an agonist for omMcrb2 and significantly increasing the cAMP levels (Figure 8).Ipsen 5i and MCL0020 had no significant effect on the cAMP levels of the four trout Mc4rs (Figure 8).

Discussion
In the present study, we identified four rainbow trout mc4r genes (mc4ra1, mc4ra2, mc4rb1, and mc4rb2).The four trout Mc4rs had similar structural features as the MC4Rs of other species (Figure 1).These conserved motifs and residues have been shown to play crucial roles in maintaining receptor structure and function [1].Multiple sequence alignment, a phylogenetic tree, and chromosomal synteny analysis indicated that mc4ra1 and mc4ra2 might be two copies of mc4ra, and mc4rb may have two copies: mc4rb1 and mc4rb2 (Figures 1-4).These results were possibly due to whole-genome duplication, similar to other salmonids, indicating that mc4r may have undergone different evolutionary processes.In Atlantic salmon, four mc4r (a1, a2, b1, and b2) were identified and have high expression in the different regions of brain [68].Administration of MC4R agonist decreases food intake, and antagonists result in increased food intake in rainbow trout [22].These indicated that four different Mc4rs of trout and salmon might have various roles in appetite regulation and energy balance.
The spontaneously active conformation of GPCRs in the absence of the agonist results in the constitutive activation of receptors [72].High basal activities are observed in numerous fish Mc4rs [13][14][15][16]18,19,33,34].In the present study, of the four omMc4rs, two (omMc4ra2 and omMc4rb1) had high basal cAMP levels, whereas the other two had modest basal activities (Figure 8A).The differences in constitutive activity between these omMc4rs and hMC4R might be attributed to their evolutionarily distinct structures.Although TMDs of MC4Rs are conserved from teleosts to mammals, less homology is present at N-termini and extracellular loops of the four omMc4rs and other teleost Mc4rs compared to hMC4R.N-terminus and extracellular loops play key roles in the modulation of constitutive activities in hMC4R [30,73], as well as luteinizing hormone receptor [74] and thyroid-stimulating hormone receptor [75,76].However, more research is needed to determine whether these domains contribute to the constitutive activation of the omMc4rs and whether high basal activity of Mc4r is more common among teleosts.
Numerous constitutively active mutants have been reported in several GPCRs, resulting in various human diseases, and inverse agonism will be vital for correcting the diseases caused by activating mutations in GPCRs [32,77].The constitutive activity of teleost Mc4r may have important physiological functions.Our data showed that AgRP (83-132), an inverse agonist, reduced the basal cAMP levels of four omMc4rs (Figure 8), similar to results on spotted scat, grass carp, and Nile tilapia Mc4rs [17,51].Although fish have four endogenous antagonists, including agouti signaling peptide 1 (Asip1), Asip2, Agrp1, and Agrp2, Agrp1 shows high identity with tetrapod AgRP at the C-terminus with ten conserved cysteine residues (forming five disulfide bridges) and a highly conserved Arg-Phe-Phe (RFF) motif, which is essential for maintaining the structure and biological function of AgRP [20,78].Thus, the active fragment in the C-terminal human AgRP behaves as an inverse agonist in fish Mc4rs, indicating that AgRP-induced inverse agonism on MC4R is conserved in vertebrates, and inverse agonism and antagonism of teleost Mc4r might provide new insight for promoting feed intake and growth performance in aquaculture.
Three small molecule inverse agonists of hMC4R were used in the current study to determine their potential modulation on cAMP and ERK1/2 signaling pathways of four omMc4rs (Figure 8).MCL0020 is a neutral antagonist of hMC4R [51,56,79].MCL0020 replaces NDP-MSH binding to human, grass carp (ciMc4r), and spotted scat (saMc4r) MC4Rs, it and antagonizes NDP-MSH-induced cAMP generation and decreases the basal cAMP production of ciMc4r, whereas it has no effect on basal and ligand-induced cAMP levels on saMc4r [51].Our results indicated that MCL0020 only bound to omMc4rb1 and hMC4R by displacing NDP-MSH, suggesting that binding sites for this compound and NDP-MSH were overlapping in hMC4R and omMc4rb1, which is different than omMc4ra1, omMc4ra2, and omMc4rb2 (Figure 6).For signaling, MCL0020 had no significant effects on the cAMP level of four trout Mc4rs (Figure 8).
MC4R-mediated ERK1/2 signaling has a crucial role in the modulation of energy homeostasis [46].Although teleost Mc4rs have much higher basal activities in Gs-cAMP signaling, constitutive activation of ERK1/2 signaling is only observed in topmouth culter Mc4r but not in saMc4r and ciMc4r [51].Our results showed that only omMc4rb2 was constitutively active in ERK1/2 signaling, and the other three omMc4rs had similar pERK1/2 levels as the empty vector (Figure 9).For ligand-induced ERK1/2 signaling, α-MSH was shown to stimulate pERK1/2 of four omMc4rs (Figure 9).AgRP, as a biased ligand, was shown to decrease Gs-cAMP signaling and activate ERK1/2 signaling on hMC4R and fish Mc4rs (saMc4r and ciMc4r) [48,51,81].Our data indicated that AgRP could decrease cAMP levels of four omMc4rs, whereas it significantly stimulated ERK1/2 signaling at omMc4ra1 and omMc4rb1 but not at omMc4ra2 and omMc4rb2 (Figure 9), suggesting that AgRP is a biased ligand for omMc4ra1 and omMc4rb1.Although the physiological roles of fish Mc4r-regulated ERK1/2 signaling are not well understood, our findings presented vital new knowledge on the complexity of fish Mc4r pharmacology.
Three small molecules (Ipsen 5i, ML00253764, and MCL0020) were suggested to act as biased ligands for hMC4R and fish Mc4rs (spotted scat and grass carp Mc4r) with preferred activation on ERK1/2 signaling [48,51,81].Our data documented that these three compounds only activated ERK1/2 signaling of omMc4rb1 but not the other three omMc4rs (Figure 9).Furthermore, ML00253764 could stimulate cAMP signaling but had no effect on ERK1/2 signaling of omMc4rb2 (Figures 8 and 9).Collectively, these findings suggested that Ipsen 5i, ML00253764, and MCL0020 were biased allosteric modulators for omMc4rb1 with selective activation on ERK1/2 signaling, while ML00253764 behaved as an allosteric agonist for omMc4rb2, preferentially activating Gs-cAMP signaling.Thus, small molecules developed for hMC4R had various effects on the Mc4r in different fishes.We need to investigate the pharmacological characteristics of Mc4r in the intended fish before field trials.

Conclusions
In summary, four trout mc4r genes were located at different chromosomes, and these four Mc4r isoforms showed different pharmacological characteristics, indicating that trout mc4r may have undergone different evolutionary processes and have various physiological functions.Trout Mc4rs had constitutive activities in cAMP signaling that were decreased by AgRP.Several MC4R ligands possessed divergent effects on trout Mc4rs, resulting in biased cAMP and ERK1/2 signaling.These findings contributed to a better understanding of pharmacology in Mc4r isoforms encoded by different mc4r paralogs.A very recent report investigated the modulation of trout Mc4rb1 by Mrap2a [82].Taken together, these results will aid further investigations of the physiological role of fish Mc4r in energy homeostasis and of the evolution of MC4R in vertebrates.
). Trout Mc4ra1 was nested with other Mc4ra1s of salmonids and trout, and similar results were observed in Mc4ra2, Mc4rb1, and Mc4rb2.In addition, omMc4ra1 was close to omMc4ra2 (Figure 4).

Figure 2 .
Figure 2. Amino acid sequence identities of omMc4rs and other MC4Rs.

Figure 3 .
Figure 3. Synteny analyses of MC4Rs.The syntenic genes are displayed as dark boxes linked by lines.MC4R genes are boxed.

Figure 4 .
Figure 4. Phylogenetic tree of MC4Rs.The tree was constructed using the neighbor-joining (NJ) method.Numbers at nodes indicate the bootstrap values, as percentages, obtained for 1000 replicates.Shapes indicate trout Mc4r isoforms.

Figure 7 .
Figure 7. Signaling properties of omMc4rs.HEK293T cells were transiently transfected with omMc4r or hMC4R plasmids, and intracellular cAMP levels were measured by RIA after stimulation with different concentrations of α-MSH (A) or ACTH (1-24) (B).Data are mean ± SEM from triplicate measurements within one experiment.All experiments were performed at least three times independently.

Table 1 .
The ligand-binding properties of omMc4rs to different ligands.

Table 2 .
The signaling properties of omMc4rs.