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Modulation of Canine Melanocortin-3 and -4 Receptors by Melanocortin-2 Receptor Accessory Protein 1 and 2

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have crucial roles in regulating energy homeostasis. The melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) have been shown to regulate neural MCRs in a species-specific manner. The potential effects of MRAP1 and MRAP2 on canine neural MCRs have not been investigated before. Herein, we cloned canine (c) MC3R and identified one canine MRAP2 splice variant, MRAP2b, with N-terminal extension of cMRAP2a. Canine MC3R showed higher maximal responses to five agonists than those of human MC3R. We further investigated the modulation of cMRAP1, cMRAP2a, and cMRAP2b, on cMC3R and cMC4R pharmacology. For the cMC3R, all MRAPs had no effect on trafficking; cMRAP1 significantly decreased Bmax whereas cMRAP2a and cMRAP2b significantly increased Bmax. Both MRAP1 and MRAP2a decreased Rmaxs in response to α-MSH and ACTH; MRAP2b only decreased α-MSH-stimulated cAMP generation. For the MC4R, MRAP1 and MRAP2a increased cell surface expression, and MRAP1 and MRAP2a increased Bmaxs. All MRAPs had increased affinities to α-MSH and ACTH. MRAP2a increased ACTH-induced cAMP levels, whereas MRAP2b decreased α-MSH- and ACTH-stimulated cAMP production. These findings may lead to a better understanding of the regulation of neural MCRs by MRAP1 and MRAP2s.

MC3R and MC4R are activated by the endogenous agonists, including α-, β-, γmelanocyte-stimulating hormones (MSHs) and adrenocorticotropin (ACTH) [26,27]. As members of the family of G protein-coupled receptors (GPCRs), the neural MCRs primarily couple to the stimulatory G protein (Gαs) to stimulate adenylyl cyclase activity, resulting in increased generation of the intracellular second messenger cyclic adenosine monophosphate (cAMP) to trigger downstream signaling.
From the studies on the regulation of MC3R and MC4R by MRAPs, it was shown that MRAPs modulate neural MCRs in a receptor-and species-dependent manner. The effects of MRAP1 and MRAP2 on canine (Canis lupus familiaris) (c) MC3R and cMC4R have not been studied before. Herein, we investigated the pharmacology of cMC3R as well as the pharmacological modulation of cMC3R and cMC4R by cMRAP1 and cMRAP2a/cMRAP2b. We have previously reported the pharmacology of cMC4R [54].

Flow Cytometry Assay
The regulation of cMRAP1 or cMRAP2s on the expression of cMC3R and cMC4R was carried out using flow cytometry (Accuri Cytometers, Ann Arbor, MI, USA) as described previously [59,60]. Cells were co-transfected with cMC3R or cMC4R and cMRAP1, cM-RAP2a, or cMRAP2b plasmids (1:5). Fluorescence of cells transfected with pcDNA3.1 was used for background staining. The expression of receptors was calculated as the percentage of the cell transfected with receptors in absence of MRAPs (set as 100%) [59].

Statistical Analysis
All data were presented as mean ± S.E.M. GraphPad Prism 8.3 software (GraphPad, San Diego, CA, USA) was used to calculate the parameters of ligand binding, cAMP signaling, and flow cytometry assay. The significance in binding and signaling parameters between cMC3R and hMC3R, as well as vehicle and ligand-treated groups, were all determined by Student's t-test. One-way ANOVA was used to analyze the significant differences in binding, cAMP, and flow cytometry between multiple groups.
The canine MRAP1 had 336 bp ORF that encoded a putative protein of 111 amino acids with 12.90 kDa molecular mass ( Figure 2A). cMRAP1 had the classical characteristic of other MRAP orthologs, including two potential N-linked glycosylation sites (Asn 3 and Asn 6 ), YEYY motif, LDYL motif, LKANKYL motif, and a single TMD (Figure 2A). cMRAP1 shared high identities with cat MRAP1 (90%), and lower identities with other MRAP1 (44-79%). The phylogenetic tree showed that cMRAP1 clustered with mammalian MRAP1s and was evolutionarily closer to red fox MRAP1 ( Figure 2B). The canine MRAP1 had 336 bp ORF that encoded a putative protein of 111 amino acids with 12.90 kDa molecular mass ( Figure 2A). cMRAP1 had the classical characteristic of other MRAP orthologs, including two potential N-linked glycosylation sites (Asn 3 and Asn 6 ), YEYY motif, LDYL motif, LKANKYL motif, and a single TMD (Figure 2A). cMRAP1 shared high identities with cat MRAP1 (90%), and lower identities with other MRAP1 (44-79%). The phylogenetic tree showed that cMRAP1 clustered with mammalian MRAP1s and was evolutionarily closer to red fox MRAP1 ( Figure 2B). The canine MRAP2 consists of 11 exons. Two MRAP2 splice variants were identified: MRAP2a (XM_038682814.1) derived from four exons (2, 6, 7, and 11) that had 621 bp ORF, encoding a putative protein of 206 amino acids with 23.62 kDa molecular mass ( Figure 3A); MRAP2b (XM_038682813.1) derived from five exons (3, 4, 6, 7, and 11) that had 684 bp ORF encoding a putative protein of 232 amino acids with 26.71 kDa molecular mass ( Figure 3A). Canine MRAP2b had an extended N-terminus (26 amino acids) compared with cMRAP2a, and they shared the common structure with other MRAP2s, such as one potential N-linked glycosylation sites (Asn 9 in MRAP2a and Asn 35 in MRAP2b), YEYY motif, LKAHKYS motif, and a single TMD ( Figure 3B,C). Multiple sequence alignment analysis showed that dog MRAP2a and MRAP2b shared high identities with mammalian MRAP2s (>81%) and lower identities with MRAP2s from other species (<78%). The two isoforms of cMRAP2 were clustered with different MRAP2s, in which MRAP2a was evolutionarily closer to cat MRAP2, and MRAP2b was nested with Nile tilapia MRAP2 ( Figure 3D).  evolutionarily closer to cat MRAP2, and MRAP2b was nested with Nile tilapia MRAP2 ( Figure 3D).

Ligand Binding Properties of cMC3R
The binding assay was performed using multiple MC3R ligands, including NDP-MSH, α-MSH, β-MSH, ACTH, and D-Trp 8 -γ-MSH. We included hMC3R for comparison in the same experiments to explore whether cMC3R shows any unique pharmacological characteristics. The maximal binding value (Bmax) of cMC3R was 247.97 ± 13.44% of that of hMC3R ( Figure 4 and Table 1). Canine MC3R had significantly lower affinities to NDP-MSH and D-Trp 8 -γ-MSH than that of hMC3R ( Figure 4 and Table 1). The two MC3Rs showed similar IC50s when α-MSH, β-MSH, and ACTH, were used ( Figure 4 and Table 1).

Ligand Binding Properties of cMC3R
The binding assay was performed using multiple MC3R ligands, including NDP-MSH, α-MSH, β-MSH, ACTH, and D-Trp 8 -γ-MSH. We included hMC3R for comparison in the same experiments to explore whether cMC3R shows any unique pharmacological characteristics. The maximal binding value (B max ) of cMC3R was 247.97 ± 13.44% of that of hMC3R ( Figure 4 and Table 1). Canine MC3R had significantly lower affinities to NDP-MSH and D-Trp 8 -γ-MSH than that of hMC3R ( Figure 4 and Table 1). The two MC3Rs showed similar IC 50 s when α-MSH, β-MSH, and ACTH, were used ( Figure 4 and Table 1).   Values are expressed as the mean ± SEM of at least three independent experiments. a Significant difference from the parameter of hMC3R, p < 0.05. b Significant difference from the parameter of hMC3R, p < 0.001.

cAMP Signaling Properties of cMC3R
Intracellular cAMP levels were determined to explore whether cMC3R could respond to these agonists. All agonists could dose-dependently stimulate cMC3R and increase cAMP production ( Figure 5 and Table 2). Canine MC3R showed higher maximal responses (Rmax) in response to four agonists (NDP-MSH, β-MSH, ACTH, and D-Trp 8 -γ-MSH) compared to hMC3R ( Figure 5 and Table 2). Similar EC50s between the two MC3Rs were observed in response to five agonists ( Figure 5 and Table 2). Additionally, cMC3R showed similar basal activity as hMC3R ( Table 2).

cAMP Signaling Properties of cMC3R
Intracellular cAMP levels were determined to explore whether cMC3R could respond to these agonists. All agonists could dose-dependently stimulate cMC3R and increase cAMP production ( Figure 5 and Table 2). Canine MC3R showed higher maximal responses (R max ) in response to four agonists (NDP-MSH, β-MSH, ACTH, and D-Trp 8 -γ-MSH) compared to hMC3R ( Figure 5 and Table 2). Similar EC 50 s between the two MC3Rs were observed in response to five agonists ( Figure 5 and Table 2). Additionally, cMC3R showed similar basal activity as hMC3R (Table 2).   Values are expressed as the mean ± SEM of at least three independent experiments. a Significant difference from the parameter of hMC3R, p < 0.05.

Modulation of cMC3R and cMC4R Expression by MRAPs
Canine MC3R or MC4R expression regulated by MRAPs was measured using flow cytometry. Results showed that cMRAP1, cMRAP2a, and cMRAP2b had no effect on the cell surface and total expression of cMC3R ( Figure 6A,B). For cMC4R, cMRAP1 and cMRAP2a increased cell surface and total expression, and cMMRAP2b only increased total expression of cMC4R ( Figure 6C,D).

Modulation of cMC3R and cMC4R Expression by MRAPs
Canine MC3R or MC4R expression regulated by MRAPs was measured using flow cytometry. Results showed that cMRAP1, cMRAP2a, and cMRAP2b had no effect on the cell surface and total expression of cMC3R ( Figure 6A,B). For cMC4R, cMRAP1 and cMRAP2a increased cell surface and total expression, and cMMRAP2b only increased total expression of cMC4R ( Figure 6C,D).

Modulation of cMC3R Pharmacology by MRAPs
Ligand binding assays with α-MSH and ACTH showed that cMRAP1 decreased the B max , whereas cMRAP2a and cMRAP2b increased B max s of cMC3R ( Figure 7A,B and Table 3). All MRAPs had no significant effect on IC 50 s of cMC3R to α-MSH and ACTH ( Figure 7A,B and Table 3). Modulation of cMRAPs on cMC3R signaling was also studied. Results showed that all MRAPs did not alter α-MSH and ACTH potencies of cMC3R (Table 4). Both MRAP1 and MRAP2a decreased R max s in response to α-MSH and ACTH; MRAP2b significantly decreased α-MSH-stimulated cAMP generation but showed similar ACTH-induced cAMP production of cMC3R ( Figure 7C,D and Table 4). Only MRAP2b decreased the basal activity, and the other MRAPs had no effect on the basal cAMP levels of cMC3R (Table 4).

Modulation of cMC3R Pharmacology by MRAPs
Ligand binding assays with α-MSH and ACTH showed that cMRAP1 decreased the Bmax, whereas cMRAP2a and cMRAP2b increased Bmaxs of cMC3R ( Figure 7A,B and Table  3). All MRAPs had no significant effect on IC50s of cMC3R to α-MSH and ACTH ( Figure  7A,B and Table 3). Modulation of cMRAPs on cMC3R signaling was also studied. Results showed that all MRAPs did not alter α-MSH and ACTH potencies of cMC3R (Table 4). Both MRAP1 and MRAP2a decreased Rmaxs in response to α-MSH and ACTH; MRAP2b significantly decreased α-MSH-stimulated cAMP generation but showed similar ACTHinduced cAMP production of cMC3R ( Figure 7C,D and Table 4). Only MRAP2b decreased the basal activity, and the other MRAPs had no effect on the basal cAMP levels of cMC3R (Table 4).     Values are expressed as the mean ± SEM of at least three independent experiments. a Significant difference from the parameter of cMC3R, p < 0.05. Values are expressed as the mean ± SEM of at least three independent experiments. a Significant difference from the parameter of cMC3R, p < 0.05. b Significant difference from the parameter of cMC3R, p < 0.001.

Modulation of cMC4R Pharmacology by MRAPs
Ligand binding assays showed that MRAP1 and MRAP2a increased B max s of cMC4R and MRAP2b did not affect the B max ( Figure 8A,B and Table 5). All MRAPs increased the affinities of cMC4R to α-MSH and ACTH ( Figure 8A,B and Table 5). induced cAMP signaling, whereas MRAP2b increased ACTH-stimulated signaling and did not affect α-MSH-stimulated cAMP signaling ( Figure 8C,D and Table 6). Canine MC4R showed higher basal cAMP production than that of hMC4R (2.38 times that of hMC4R), indicating that cMC4R might be constitutively active. In this study, all MRAPs decreased the basal cAMP levels of cMC4R (Table 6).    Signaling results showed that all MRAPs did not affect EC 50 s of cMC4R in response to α-MSH and ACTH (Table 6). MRAP1 had no effect on α-MSHand ACTH-stimulated cAMP level of cMC4R ( Figure 8C,D and Table 6). MRAP2a decreased α-MSHand ACTHinduced cAMP signaling, whereas MRAP2b increased ACTH-stimulated signaling and did not affect α-MSH-stimulated cAMP signaling ( Figure 8C,D and Table 6). Canine MC4R showed higher basal cAMP production than that of hMC4R (2.38 times that of hMC4R), indicating that cMC4R might be constitutively active. In this study, all MRAPs decreased the basal cAMP levels of cMC4R (Table 6). Values are expressed as the mean ± SEM of at least three independent experiments. a Significant difference from the parameter of cMC4R, p < 0.05. b Significant difference from the parameter of cMC3R, p < 0.001.

Discussion
In this study, we cloned canine MC3R and investigated its pharmacological properties. We have reported cMC4R pharmacology previously [54,61]. In the current study, we also identified a MRAP2 variant, MRAP2b. The potential regulation of cMRAP1 and two cMRAP2 isoforms on cMC3R/cMC4R pharmacology were further studied.
To investigate the pharmacology of cMC3R, ligand binding and signaling assays were performed. cMC3Rs showed a higher binding capacity than that of hMC3R, consistent with the results of other MC3Rs, including channel catfish [49], topmouth culter [52], and giant panda [62]. For signaling, different from the results of giant panda and pig MC3Rs [62,63], cMC3R had a higher ligand-induced cAMP level than that of hMC3R. In addition, our current results are consistent with mammalian MC3Rs in that MC3R has little or no basal cAMP signaling [62][63][64][65]. Of interest, high constitutive activities were present in several non-mammalian MC3Rs, including teleosts [49,52,66], amphibians (Mexican axolotl) [67], and avian (chicken) [38,68]. The amino acids accounting for the differences in the constitutive activity between these MC3Rs are not clear. N-termini and extracellular loops are essential for the modulation of constitutive activities in hMC4R [69,70], luteinizing hormone receptor [71], and thyroid-stimulating hormone receptor [72,73]. Lower homology is observed in the N-termini and extracellular loops of mammalian and non-mammalian MC3Rs. Further studies are needed to determine the exact molecular determinants.
The potential roles of cMRAPs on cMC3R/cMC4R trafficking were studied. Human MRAP1a decreased [30,74] or increased [43], and MRAP2a decreased [30,74] or had no effect [43] on the cell surface expression of hMC3R. The current study showed that all cMRAPs did not affect the cell surface expression of cMC3R. In other species, frog MRAP1 increased and chicken MRAP1 did not alter the surface expression of MC3Rs [38,39]. MRAP2 decreased the surface expression of clawed frog MC3R [39], increased the surface expression of topmouth culter Mc3r [52], and had no effect on the surface expression of Mexican axolotl and chicken MC3Rs [38,67]. For MC4R, hMRAP1a and hMRAP2a decreased [30,74] or increased [43] the cell surface expression of hMC4R. In this study, cMRAP1 and cMRAP2a increased cell surface expression of cMC4R. MRAP1 had no effect on the surface expression of chicken MC3R [38] and increased frog MC3R expression [42]. MRAP2 has been reported to decrease the surface expression of tilapia and Mexican axolotl MC4Rs [67,75], increase the membrane expression of zebrafish (Mrap2b) [31], topmouth culter (Mrap2a and Mrap2b) [51], and Xenopus MC4Rs [39], or have no effect on the surface expression of chicken and snakehead MC4Rs [38,53]. Collectively, MRAP1 and MRAP2 modulate the MC3R/MC4R trafficking to the plasma membrane in a species-and receptorspecific manner.
The potential modulation of cMRAPs on cMC4R pharmacology was also studied. Conflicting results were reported previously on hMRAP1a-and hMRAP2a-regulated signaling of hMC4R, where hMRAP1a decreased α-MSH-induced or did not affect α-MSHand ACTH-stimulated hMC4R signaling [37,43]; MRAP2a had no effect [76], decreased [43] or increased [41,74] α-MSH-stimulated and did not affect ACTH-induced [43,76] signaling of hMC4R. Chicken MRAP1 was shown to decrease α-MSH-stimulated and have no effect on ACTH-induced signaling of MC4R [38]. MRAP1 increased α-MSHand ACTH-stimulated signaling of Xenopus MC4R [39]. MRAP2-suppressed α-MSHand/or ACTH-stimulated signaling of MC4Rs were present in several teleosts [31,51,53,75,77]. Our results indicated that MRAP1 had no effect on the efficacy of cMC4R in response to α-MSH and ACTH, MRAP2a increased ACTH-stimulated but had no effect on α-MSH-induced cMC4R signaling, and MRAP2b decreased α-MSHand ACTH-induced signaling, suggesting that MRAP1 modulated cMC4R trafficking and ligand binding, but did not affect signaling; the interaction of MRAP2a and cMC4R might change MC4R ligand selectivity and sensitivity; MRAP2b might inhibit receptor coupling to G protein, resulting in decreased signaling. Hence, MRAPs might be involved in regulating receptor ligand selectivity and sensitivity in a species-dependent manner. The potential mechanisms of the MRAP regulation of receptor ligand selectivity and sensitivity need further study.
Human MC4R shows modest basal cAMP signaling [65]. The defect in basal activities of MC4R mutations can cause obesity [69,78]. Mrap2-and Agrp-suppressed basal activity of Mc4r play an important role in promoting the growth of zebrafish and culter [31,52,79]. These studies indicate that the basal activity of MC4R plays a pivotal role in the modulation of energy homeostasis [80]. Human MRAP1s increased [37,41,43] or did not affect [30,36] the basal activities of hMC4R, and MRAP2s decreased [43] or had no effect [30,36,41,81] on the basal activities of hMC4R. Our studies showed that cMRAP1 and cMRAP2s decreased the basal cAMP signaling of cMC4R. Decreased MC4R basal activities by MRAP2(s) were also reported in other species [31,51,53,75,77].
Alternative splicing is prevalent in eukaryotes and isoforms generated by alternative splicing might have different functions [82][83][84][85]. Splicing variants provide a nature-made chance to investigate the roles of specific domains. Human MRAP and MRAP2 have two and three alternatively spliced forms, respectively, and they show different effects on the hMC3R/hMC4R pharmacology [28,43]. Canine MRAP2 also had two alternatively spliced variants, MRAP2a and MRAP2b (MRAP2b with extension at N-terminus compared with MRAP2a). This extension sequence at the N-terminus of MRAP2b is not found in other MRAP2s. Our results showed that MRAP2a and MRAP2b had different effects on cMC3R/cMC4R pharmacology. The N-termini of MRAP1 and MRAP2, with several conserved motifs, have important roles in modulating GPCR pharmacology [43,[86][87][88]. We speculate that the extension sequences at the N-termini of MRAP2 might have important roles in MC3R/MC4R pharmacology.

Conclusions
In summary, we cloned canine MC3R and investigated its pharmacology, as well as modulation of MC3R and MC4R pharmacology by MRAPs. MRAP1 did not affect the MC3R trafficking and decreased α-MSHand ACTH-induced signaling, whereas MRAP1 increased the cell surface expression and decreased the basal activity of cMC4R. The two MRAP2 isoforms exerted different effects on cMC3R or MC4R pharmacology. MRAP2a decreased α-MSHand ACTH-induced signaling, whereas MRAP2b only the decreased α-MSH-stimulated signaling of cMC3R. MRAP2a increased the cell surface expression and ACTH-induced signaling, decreased the basal activity of cMC4R, whereas MRAP2b had no effect on trafficking, and decreased basal and α-MSHand ACTH-induced signaling. This study contributes to a better understanding of cMC3R/cMC4R.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors upon request, without undue reservation.