Trends in the evolution of the elasmobranch melanocortin-2 receptor: Insights from structure/function studies on the activation of whale shark Mc2r.

To understand the mechanism for activation of the melanocortin-2 receptor (Mc2r) of the elasmobranch, Rhincodon typus (whale shark; ws), wsmc2r was co-expressed with wsmrap1 in CHO cells, and the transfected cells were stimulated with alanine-substituted analogs of ACTH(1-24) at the "message" motif (H6F7R8W9) and the "address" motif (K15K16R17R18P19). Complete alanine substitution of the H6F7R8W9 motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W9 > R8, and substitution at F7 and H6 had no effect on activation. The same analysis was done on a representative bony vertebrate Mc2r ortholog (Amia calva; bowfin; bf) and the order of position importance for activation was W9 > R8 = F7, (alanine substitution at H6 was negligible). Complete alanine substitution at the K15K16R17R18P19 motif resulted in distinct outcomes for wsMc2r and bfMc2r. For bfMc2r, this analog blocked activation-an outcome typical for bony vertebrate Mc2r orthologs. For wsMc2r, this analog resulted in a shift in sensitivity to stimulation of the analog as compared to ACTH(1-24) by two orders of magnitude, but the dose response curve did reach saturation. To evaluate whether the EC2 domain of wsMc2r plays a role in activation, a chimeric wsMc2r was made in which the EC2 domain was replaced with the EC2 domain from a melanocortin receptor that does not interact with Mrap1 (i.e., Xenopus tropicalis Mc1r). This substitution did not negatively impact the activation of the chimeric receptor. In addition, alanine substitution at a putative activation motif in the N-terminal of wsMrap1 did not affect the sensitivity of wsMc2r to stimulation by ACTH(1-24). Collectively, these observations suggest that wsMc2r may only have a HFRW binding site for melanocortin-related ligand which would explain how wsMc2r could be activated by either ACTH or MSH-sized ligands.

Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2.

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1-24) with an EC50 of 2.6 × 10-10 M ± 9.7 × 10-11. When the receptor was expressed alone, stimulation was only observed at [10-6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. Furthermore, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1-24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, whereas, co-expression with MRAP2 increases trafficking, but has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1-24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. In addition, co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

A basal actinopterygian melanocortin receptor: Molecular and functional characterization of an Mc2r ortholog from the Senegal bichir (Polypterus senegalus).

In bony vertebrates, melanocortin 2 receptor (Mc2r) specifically binds adrenocorticotropic hormone (ACTH) and is responsible for mediating anterior pituitary signaling that stimulates corticosteroid production in the adrenal gland/interrenal cells. In bony fishes Mc2r requires the chaperoning of an accessory protein (Mrap1) to traffic to the membrane surface and bind ACTH. Here, we evaluated the structure and pharmacological properties of Mc2r from the Senegal bichir (Polypterus senegalus), which represents the most basal bony fish from which an Mc2r has been pharmacologically studied to date. In our experiments, cDNA constructs of the Mc2r from the Senegal bichir (sbMc2r) and various vertebrate Mrap1s were heterologously co-expressed in Chinese hamster ovary (CHO) cells, stimulated by ACTH or melanocyte-stimulating hormone (α-MSH) ligands, and assessed using a luciferase reporter gene assay. When expressed without an Mrap1, sbMc2r was not activated by ACTH. When co-expressed with Mrap1 from either chicken (Gallus gallus) or bowfin (Amia calva), sbMc2r could be activated in a dose-dependent manner by ACTH, but not α-MSH. Co-expression of sbMrap2 with sbMc2r resulted in no detectable activation of the receptor. Collectively, these results demonstrate that sbMc2r has pharmacological properties similar to those of Mc2rs of later-evolved bony fishes, such as Mrap1 dependence and ACTH selectivity, indicating that these qualities of Mc2r function are ancestral to all bony fish Mc2rs.

Analyzing the Hypothalamus/Pituitary/Interrenal axis of the neopterygian fish, Lepisosteus oculatus: Co-localization of MC2R, MC5R, MRAP1, and MRAP2 in interrenal cells.

RT-PCR analysis indicated that steroidogenic tissues are located along the length of the kidney of the neopterygian fish, Lepisosteus oculatus (spotted gar; g). However, RT-PCR analysis of the distribution of mc2r mRNA and mrap1 mRNA, critical components of the gar hypothalamus/pituitary/interrenal (HPI) axis, was only associated with the anterior and medial regions of the kidney. Steroidogenic cells were designated as interrenal cells that possess star mRNA (in situ hybridization) and lipid vesicles (histological analysis) within the kidney. RT-PCR also detected mc5r mRNA along the length of the tissues associated with the kidney. In situ hybridization analysis of the putative interrenal cells revealed co-expression of mc2r, and mc5r mRNAs in the same steroidogenic cells. Co-expression of gar Mc2r (gMc2r) and Mrap1 (gMrap1) in Chinese Hamster Ovary (CHO) cells stimulated with ACTH(1-24) resulted in activation with an EC50 value of 1.0 × 10-11M +/- 4.6 × 10-11); whereas stimulation of CHO cells co-expressed with gar Mc5r (gMc5r) and gMrap1 and stimulated with ACTH(1-24) resulted in an EC50 value that was 3 orders of magnitude lower (2.1 × 10-8 M +/- 3.5 × 10-9). Interesting, when CHO cells were co-transfected with gMc2r, gMc5r, and gMrap1 there was a decline in activation as measured by the Vmax values for CHO cells stimulated with either ACTH(1-24) or α-MSH. These results suggest that some interaction may occur between gMc2r and gMc5r when both receptors are expressed in the same cells. Phylogenetic and selection pressure analyses of vertebrate mc2r and mc5r genes concluded that the two genes are evolving at different rates after duplication from a proposed common ancestral gene.

Evaluating interactions between the melanocortin-5 receptor, MRAP1, and ACTH(1-24): A phylogenetic study.

The melanocortin-2 receptor (MC2R) and the melanocortin-5 receptor (MC5R) are found on the same chromosome in most vertebrate genomes, and for the species analyzed in this study, MC2R and MC5R are co-expressed in glucocorticoid-producing cells that also express the accessory protein MRAP1. Since MRAP1 affects the ligand sensitivity of MC2R orthologs, this study tested the hypothesis that co-expression of MC5R with MRAP1 would also affect the ligand sensitivity of MC5R. The hypothesis was confirmed for stingray, rainbow trout, and chicken, MC5R orthologs. However, elephant shark MC5R was not affected in the same way by co-expression of MRAP1. It appears that, for some MC5R orthologs (i.e., stingray, rainbow trout, and chicken), a docking site for the R/KKRRP motif of ACTH(1-24) may become exposed on the receptor following co-expression with MRAP1. However, for elephant shark MC5R co-expression with MRAP1 may not affect engagement ACTH(1-24). Hence during the radiation of the chordates, the interaction between MRAP1 and MC5R has diverged.

Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2.

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1-24) with an EC50 of 2.6 × 10-10 M ± 9.7 × 10-11. When the receptor was expressed alone, stimulation was only observed at [10-6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. In addition, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1-24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, and while co-expression with MRAP2 increases trafficking, it has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1-24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. Co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

Elephant shark melanocortin receptors: Novel interactions with MRAP1 and implication for the HPI axis.

The presence of Mrap1 and Mrap2 orthologs in the genome of the elephant shark (es), a cartilaginous fish, presented an opportunity to evaluate the potential interactions between these accessory proteins and melanocortin receptors of a cartilaginous fish. RT-PCR analysis indicated that Mrap1 mRNA was present in interrenal, brain, and pituitary tissue with mRNA for Mc2R, Mc3R, Mc4R, and Mc5r. Co-expression of esMrap1 cDNA with esMc2r cDNA or esMc5r cDNA in CHO cells increased sensitivity to stimulation with ACTH(1-24) 10 fold and 100 fold, respectfully, but had no effect on sensitivity to stimulation with DesAc-αMSH [i.e., ACTH(1-13)NH2] for either receptor, and had no effect on the ligand sensitivity of either esMc3r or esMc4r. Fluorescence image analysis indicated co-localization of esMrap1/esMc2r, and esMrap1/esMc5r on the plasma membrane; however, cell surface ELISA analysis indicated that co-expression with esMrap1 had no effect, positive or negative, on the trafficking of either esMc2r or esMc5r to the plasma membrane. RT-PCR analysis also indicated that Mrap2 mRNA, as well as, mRNAs for Mc2r, Mc3r, Mc4r, and Mc5r could be detected in brain tissue, however no Mrap2 mRNA was detected in interrenal tissue. Co-expression of esMrap2 in CHO cells with, respectively, esMc2r, esMc4r, or esMc5r had no effect on ligand sensitivity. However, co-expression of esMrap2 with esMc3r did lower sensitivity to stimulation by DesAc-αMSH 10 fold. These observations are discussed in the context of the parallel evolution of melanocortin receptors and their accessory proteins, and the hypothalamus/pituitary/adrenal axis and the hypothalamus/pituitary/interrenal axis in bony vertebrates and cartilaginous fishes.

The melanocortin-2 receptor of the rainbow trout: Identifying a role for critical positions in transmembrane domain 4, extracellular loop 2, and transmembrane domain 5 in the activation of rainbow trout MC2R.

The activation of either teleost or tetrapod melanocortin-2 receptor (MC2R) orthologs requires interaction between the HFRW motif and R/KKRRP motif in the primary sequence of ACTH, and two corresponding sites on the melanocortin 2 receptor. While the HFRW contact site on MC2R appears to involve residues in TM2, TM3, and TM6, several studies on human MC2R point to the EC2/TM5 region of MC2R as a possible location for the R/KKRRP contact site. In this study nineteen single-alanine mutants of rainbow trout (rt) MC2R were made beginning at V153 in TM4, at all positions in EC2 (extracellular loop 2), to F175 in TM5. For twelve of these alanine mutants (i.e., V153, G155, C162, D163, T165, V166, I167, H169, F170, H172, V173, L174), alanine substitution did not have a statistically significant effect on activation of the receptor. For four of these alanine mutations (i.e., V157, M158, F161, K168), while the negative shift in ligand sensitivity was statistically significant, the magnitude of the negative shift in activation was fivefold or less. However, for substitution at V159 in TM4 (negative shift in activation: 110 fold), F171 in TM5 (negative shift in activation: 48-fold), and F175 in TM5 (negative shift in activation: 100 fold), the effect on activation was both statistically significant and may be physiologically relevant. To support this conclusion, a triple alanine mutant of rtMC2R (V159/A, F171/A, F175/A), and this mutant receptor could not be activated by ACTH at concentrations as high as 10-6M. A Cell Surface ELISA analysis indicated that the trafficking of the triple alanine mutant rtMC2R to the plasma membrane was not impaired by the alanine substitutions. Collectively, these observations point to a critical role for TM4 and TM5 in the activation of the rainbow trout melanocortin-2 receptor.

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1-24) or ACTH(1-13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10-13M to 10-6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

Evaluating the interactions between red stingray (Dasyatis akajei) melanocortin receptors and elephant shark (Callorhinchus milii) MRAP1 and MRAP2 following stimulation with either stingray ACTH(1-24) or stingray Des-Acetyl-αMSH: A pharmacological study in Chinese Hamster Ovary cells.

Previous studies on bony vertebrate MC2R orthologs (i.e., ray finned fishes, amphibians, reptiles, birds, and mammals) have shown that these MC2R orthologs have an obligatory requirement for interaction with bony vertebrate MRAP1 orthologs to a) allow for the trafficking of the MC2R ortholog to the plasma membrane; and b) to allow activation by ACTH, but not by any MSH-sized ligand. In addition, previous studies have found that co-expression of teleost and mammalian MC4R orthologs with corresponding MRAP2 has positive effects on sensitivity to stimulation by αMSH or ACTH. MRAP1 and MRAP2 paralogs have been detected in the genome of a cartilaginous fish (elephant shark), yet two cartilaginous fish MC2R orthologs (elephant shark and red stingray) do not apparently require MRAP1 for trafficking to the plasma membrane when expressed in Chinese Hamster Ovary (CHO) cells, and both orthologs can be activated by either ACTH or MSH-sized ligands. This study was done to determine whether sensitivity to stimulation by ACTH(1-24) or Des-Acetyl-αMSH is affected when stingray (sr) MC1R, MC2R, MC3R, MC4R or MC5R were co-expressed in CHO cells with either elephant shark (es) MRAP1 or esMRAP2. The results indicated that co-expression with heterologous MRAP1 increased the sensitivity of all five stingray melanocortin receptors for srACTH(1-24), but had not statistically significant effect on stimulation by srDes-Acetyl-αMSH for any of the stingray melanocortin receptors. Conversely, co-expression with esMRAP2 only enhanced sensitivity for srDes-Acetyl-αMSH for srMC4R, but had no effect on the other stingray orthologs, and there was no increase in sensitivity for srACTH(1-24) for any of the stingray melanocortin receptors. It appears then that some stingray melanocortin receptors have retained the ability to interact with a cartilaginous MRAP1 paralog. These results are discussed with reference to radiation of MRAP-related accessory proteins in cartilaginous fishes.

Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs.

The Multifaceted Melanocortin Receptors.

The 5 known melanocortin receptors (MCs) have established physiological roles. With the exception of MC2, these receptors can behave unpredictably, and since they are more widely expressed than their established roles would suggest, it is likely that they have other poorly characterized functions. The aim of this review is to discuss some of the less well-explored aspects of the 4 enigmatic members of this receptor family (MC1,3-5) and describe how these are multifaceted G protein-coupled receptors (GPCRs). These receptors appear to be promiscuous in that they bind several endogenous agonists (products of the proopiomelanocortin [POMC] gene) and antagonists but with inconsistent relative affinities and effects. We propose that this is a result of posttranslational modifications that determine receptor localization within nanodomains. Within each nanodomain there will be a variety of proteins, including ion channels, modifying proteins, and other GPCRs, that can interact with the MCs to alter the availability of receptor at the cell surface as well as the intracellular signaling resulting from receptor activation. Different combinations of interacting proteins and MCs may therefore give rise to the complex and inconsistent functional profiles reported for the MCs. For further progress in understanding this family, improved characterization of tissue-specific functions is required. Current evidence for interactions of these receptors with a range of partners, resulting in modulation of cell signaling, suggests that each should be studied within the full context of their interacting partners. The role of physiological status in determining this context also remains to be characterized.

Structure/Function Studies on the Activation Motif of Two Non-Mammalian Mrap1 Orthologs, and Observations on the Phylogeny of Mrap1, Including a Novel Characterization of an Mrap1 from the Chondrostean Fish, Polyodon spathula.

In derived bony vertebrates, activation of the melanocortin-2 receptor (Mc2r) by its ACTH ligand requires chaperoning by the Mc2r accessory protein (Mrap1). The N-terminal domain of the non-mammalian tetrapod MRAP1 from chicken (c; Gallus gallus) has the putative activation motif, W18D19Y20I21, and the N-terminal domain in the neopterygian ray-finned fish Mrap1 from bowfin (bf; Amia calva) has the putative activation motif, Y18D19Y20I21. The current study used an alanine-substitution paradigm to test the hypothesis that only the Y20 position in the Mrap1 ortholog of these non-mammalian vertebrates is required for activation of the respective Mc2r ortholog. Instead, we found that for cMRAP1, single alanine-substitution resulted in a gradient of inhibition of activation (Y20 >> D19 = W18 > I21). For bfMrap1, single alanine-substitution also resulted in a gradient of inhibition of activation (Y20 >> D19 > I21 > Y18). This study also included an analysis of Mc2r activation in an older lineage of ray-finned fish, the paddlefish (p), Polyodon spathula (subclass Chondronstei). Currently no mrap1 gene has been found in the paddlefish genome. When pmc2r was expressed alone in our CHO cell/cAMP reporter gene assay, no activation was observed following stimulation with ACTH. However, when pmc2r was co-expressed with either cmrap1 or bfmrap1 robust dose response curves were generated. These results indicate that the formation of an Mc2r/Mrap1 heterodimer emerged early in the radiation of the bony vertebrates.

Human Melanocortin-2 Receptor: Identifying a Role for Residues in the TM4, EC2, and TM5 Domains in Activation and Trafficking as a Result of Co-Expression with the Accessory Protein, Mrap1 in Chinese Hamster Ovary Cells.

Human melanocortin-2 receptor (hMC2R) co-expressed with the accessory protein mouse (m)MRAP1 in Chinese Hamster Ovary (CHO) cells has been used as a model system to investigate the activation and trafficking of hMC2R. A previous study had shown that the N-terminal domain of mMRAP1 makes contact with one of the extracellular domains of hMC2R to facilitate activation of hMC2R. A chimeric receptor paradigm was used in which the extracellular domains of hMC2R were replaced with the corresponding domains from Xenopus tropicalis MC1R, a receptor that does not interact with MRAP1, to reveal that EC2 (Extracellular domain 2) is the most likely contact site for hMC2R and mMRAP1 to facilitate activation of the receptor following an ACTH binding event. Prior to activation, mMRAP1 facilitates the trafficking of hMC2R from the ER to the plasma membrane. This process is dependent on the transmembrane domain (TM) of mMRAP1 making contact with one or more TMs of hMC2R. A single alanine substitution paradigm was used to identify residues in TM4 (i.e., I163, M165), EC2 (F167), and TM5 (F178) that play a role in the trafficking of hMC2R to the plasma membrane. These results provide further clarification of the activation mechanism for hMC2R.

Pharmacological Modulation of Melanocortin 1 Receptor Signaling by Mrap Proteins in Xenopus tropicalis.

The melanocortin system consists of five G protein-coupled receptors (MC1R-MC5R), the bidirectional endogenous ligands (MSH and Agouti families), and accessory proteins (MRAP1 and MRAP2). Accumulative studies of vertebrate species find high expression level of melanocortin 1 receptor (MC1R) in the dermal melanocyte and elucidate the essential roles in the skin and fur pigmentation, morphological background adaptation, and stress response. The diploid amphibian Xenopus tropicalis (xt) has been utilized as a fantastic animal model for embryonic development and studies of physiological cryptic colouring and environmental adaptiveness. However, the interaction of xtMc1r signaling with xtMrap proteins has not been assessed yet. In this study, we carried out in silico evolutionary analysis of protein alignment and genetic phylogenetic and genomic synteny of mc1r among various vertebrates. Ubiquitous expression of mrap1 and mrap2 and the co-expression with mc1r transcripts in the skin were clearly observed. Co-immunoprecipitation (ip) and fluorescent complementary approach validated the direct functional interaction of xtMc1r with xtMrap1 or xtMrap2 proteins on the plasma membrane. Pharmacological assay showed the improvement of the constitutive activity and alpha melanocyte-stimulating hormone (α-MSH) stimulated plateau without dramatic alteration of the cell surface translocation of xtMc1r in the presence of xtMrap proteins. Overall, the pharmacological modulation of xtMc1r by dual xtMrap2 proteins elucidated the potential role of this protein complex in the regulation of proper dermal function in amphibian species.

Characterization of the chicken melanocortin 5 receptor and its potential role in regulating hepatic glucolipid metabolism.

Melanocortin receptors (MC1R-MC5R) and their accessory proteins (MRAPs) are involved in a variety of physiological processes, including pigmentation, lipolysis, adrenal steroidogenesis, and immunology. However, the physiological roles of MC5R are rarely characterized in vertebrates, particularly in birds. In this work, we cloned the full-length cDNA of chicken MC5R and identified its core promoter region. Functional studies revealed that cMC5R was more sensitive to ACTH/α-MSH than ß-MSH/γ-MSH, and was coupled to the cAMP/PKA signaling pathway. We demonstrated that MRAP2 decreased MC5R sensitivity to α-MSH, whereas MRAP1 did not have a similar effect, and that both MRAPs significantly reduced MC5R expression on the cell membrane surface. Transcriptome and qPCR data showed that both MRAP1 and MC5R were highly expressed in chicken liver. Additionally, we observed that ACTH might increase hepatic glucose production and decrease lipogenesis in primary hepatocytes, and dose-dependently downregulated the expression levels of ELOVL6 and THRSPA genes. These findings indicated that ACTH may act directly on hepatocytes to regulate glucolipid metabolism, which will help to understand the function of MC5R in avian.

Identification of MRAP protein family as broad-spectrum GPCR modulators.

BACKGROUND: The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. METHODS: Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. RESULTS: 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. CONCLUSIONS: Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.

Hypothesis and Theory: Evaluating the Co-Evolution of the Melanocortin-2 Receptor and the Accessory Protein MRAP1.

The melanocortin receptors (MCRs) and the MRAP accessory proteins belong to distinct gene families that are unique to the chordates. During the radiation of the chordates, the melancortin-2 receptor paralog (MC2R) and the MRAP1 paralog (melanocortin-2 receptor accessory protein 1) have co-evolved to form a heterodimer interaction that can influence the ligand selectivity and trafficking properties of MC2R. This apparently spontaneous interaction may have begun with the ancestral gnathostomes and has persisted in both the cartilaginous fishes and the bony vertebrates. The ramifications of this interaction had profound effects on the hypothalamus/anterior pituitary/adrenal-interrenal axis of bony vertebrates resulting in MC2R orthologs that are exclusively selective for the anterior pituitary hormone, ACTH, and that are dependent on MRAP1 for trafficking to the plasma membrane. The functional motifs within the MRAP1 sequence and their potential contact sites with MC2R are discussed. The ramifications of the MC2R/MRAP1 interaction for cartilaginous fishes are also discussed, but currently the effects of this interaction on the hypothalamus/pituitary/interrenal axis is less clear. The cartilaginous fish MC2R orthologs have apparently retained the ability to be activated by either ACTH or MSH-sized ligands, and the effect of MRAP1 on trafficking varies by species. In this regard, the possible origin of the dichotomy between cartilaginous fish and bony vertebrate MC2R orthologs with respect to ligand selectivity and trafficking properties is discussed in light of the evolution of functional amino acid motifs within MRAP1.

Evidence for diversity in the activation of the melanocortin 2 receptor: A study on gar, elephant shark and stingray MC2Rs.

The melanocortin-2 receptor (MC2R) is a critical component of the HPI and HPA axes of cartilaginous fishes, teleosts and tetrapods. Studies on teleost and tetrapod orthologs suggest two contact sites between ACTH and the receptor involving the following motifs on ACTH: H6F7R8W9 and K15K16R17R18P19. Using spotted gar (g) MC2R as a representative bony fish MC2R ortholog, we found that activation of gMC2R in Chinese Hamster Ovary (CHO) cells was diminished following stimulation of the transfected cells with hACTH(1-24) analogs substituted with alanine at either the H6F7R8W9 or K15K16R17R18P19 motifs compared to stimulation with hACTH(1-24). This observation suggests two ligand contact sites necessary for activation of the gMC2R. The same experiments were done with elephant shark (es) MC2R, however only the H6F7R8W9 analogs blocked activation, pointing to a single contact on esMC2R. Conversely, the red stingray (sr) MC2R activation was blocked by both the H6F7R8W9 and K15K16R17R18P19 alanine-substituted analogs. Together these results build a picture of the evolution of the ligand and receptor interaction between ACTH and MC2R orthologs of different taxa. These results will be discussed in light of the parallel evolution of MC2R orthologs in cartilaginous fishes and bony vertebrates.

Cadmium disrupts melanocortin 2 receptor signaling in rainbow trout.

Cadmium is an endocrine disruptor and inhibits corticosteroid production, but the mechanisms are far from clear. We tested the hypothesis that sublethal exposure to environmentally realistic levels of cadmium impairs cortisol production by disrupting the melanocortin 2 receptor (MC2R) signaling in rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of cadmium (0.75 or 2.0 µg/L) in a flow-through system for 7 d and subjected to an acute secondary stressor to evoke a cortisol response. Cadmium exposure for 7 d did not affect plasma cortisol concentrations, but head kidney mc2r mRNA levels were higher than in control fish. The cortisol stress performance to a secondary-stressor was attenuated in the cadmium groups, and this corresponded with transient reduction in transcript abundance of mc2r and the gene encoding its accessory protein MRAP1 but not MRAP2 in the head kidney. Furthermore, in vivo cadmium exposure attenuated the adrenocorticotropic hormone (ACTH)-, but not 8-br-cAMP-stimulated cortisol production in head kidney slices ex vivo. This corresponded with reduced transcript abundance of mc2r and mrap1, but not mrap2 in these tissue slices. Also, reporter assays with CHO cells transiently transfected with rainbow trout mc2r and zebrafish mrap1 revealed a dose-independent inhibition in ACTH-stimulated luciferase activity by cadmium. Collectively, waterborne exposure to environmentally realistic concentration of cadmium compromises the stressor-induced cortisol response, and a mode of action involves the disruption of MC2R signaling in rainbow trout.