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. 2024 Feb 15;7(3):630–640. doi: 10.1021/acsptsci.3c00169

Evolutionary Identification of the Requirement of the Second Intracellular Loop for the Constitutive Activity of Melanocortin-4 Receptors

Bingxin Xu †, Jindong Yao †, Wenqi Song †, Xinyi Yan †, Ming Zhu †, Jiangtao Li ‡, Zhonglin Ma ‡, Yanchuan Li §, Yihao Li §, Yanbin Fu ∥, Liu Liu ⊥, Lei Li #,*, Jianjun Lyu §,*, Chao Zhang †,*
PMCID: PMC10928900  PMID: 38481681

Abstract

graphic file with name pt3c00169_0006.jpg

Melanocortin-4 receptor (MC4R) functions as a crucial neuroendocrine G protein-coupled receptor (GPCR) in the central nervous system of mammals, displaying agonist-independent constitutive activity that is mainly determined by its N-terminal domain. We previously reported that zebrafish MC4R exhibited a much higher basal cAMP level in comparison to mammalian MC4Rs. However, the functional evolution of constitutive activities in chordate MC4Rs remains to be elucidated. Here we cloned and compared the constitutive activities of MC4Rs from nine vertebrate species and showed that the additive action of the N-terminus with the extracellular region or transmembrane domain exhibited a combined pharmacological effect on the MC4R constitutive activity. In addition, we demonstrated that four residues of F149, Q156, V163, and K164 of the second intracellular loop played a vital role in determining MC4R constitutive activity. This study provided novel insights into functional evolution and identified a key motif essential for constitutive modulation of MC4R signaling.

Keywords: MC4R, constitutive activity, second intracellular loop

Melanocortin-4 receptor (MC4R), a G protein-coupled receptor (GPCR), is widely expressed in the central nervous system, especially in the paraventricular nucleus of the hypothalamus.1 The hypothalamic paraventricular nucleus serves as a key area for controlling appetite and food intake in mammals,2−5 and numerous studies have elucidated a vital role of MC4R in the embryonic growth and regulation of energy homeostasis in various vertebrates.6,7 The deficiency of MC4R is the most common monogenic cause of human obesity, underscoring its pivotal role in regulating fat homeostasis,8 and knockout of Mc4r caused obesity and hyperphagia in mice, suggesting a functional similarity between humans and mice.2 Therefore, the manipulation of signal transduction induced by MC4R has been widely accepted as a potential therapeutic approach to treat abnormal feeding behavior and metabolic disorders. MC4R is regularly stimulated by α-MSH and activates downstream adenylate cyclase (AC), leading to the accumulation of cyclic adenosine monophosphate (cAMP) by coupling to the heterotrimeric stimulatory G protein (Gαs), which subsequently triggers protein kinase A (PKA) cascades.9 In addition to the ligand-induced stimulation, MC4Rs have a unique feature of constitutive activity, allowing them to couple to G proteins in the absence of agonism.10,11 Several residues within transmembrane domain 3 (TM3) and TM6 have been identified to contribute to the constitutive regulation of human MC4R.12,13 Some MC4R mutations found only in obese individuals showed similar ligand binding affinity and expression but exhibited lower constitutive activity than wild-type ones.14 These studies suggest the high relevance of the constitutive activity of MC4R in maintaining energy homeostasis.

Constitutive activity, or basal activity, is the agonist-independent activity that is widely seen in many GPCRs and is associated with the intrinsic nature of the transmembrane domain of a receptor, which allows the receptors to spontaneously undergo a conformational transition without ligand agonism.15 Previous studies have shown that the constitutive regulation of MC4R is maintained by the N-terminal domain, and obesity-associated mutations in this region could attenuate the constitutive level.11,16 Moreover, the third intracellular loop of MC4R also plays an important role in the regulation of its constitutive activity.17,18

Beyond mammalian species, we have previously demonstrated that the zebrafish MC4R exhibited a higher constitutive activity than its counterparts in mice and human, at least in vitro cell lines.7 However, the evolutionary correlation of the constitutive activity of MC4R in other chordate species has not yet been explored. In this study, we cloned and compared the constitutive activities of MC4Rs from nine vertebrate species, including sea lamprey, elephant shark, zebrafish, coelacanth, frog, turtle, duck, mouse, and human. We comparatively analyzed the motif required for distinct constitutive levels of MC4R between species. Our results showed that the additive action of the N-terminus with the extracellular region or transmembrane domain exhibited a combined pharmacological effect on MC4R constitutive regulation. We also demonstrated that four residues of F149, Q156, V163, and K164 in the second intracellular loop played a vital role in determining MC4R constitutive activity by regulating the coupling of Gαs protein. This study elucidated the functional evolution of the basal pharmacological level and identified a novel key motif required for constitutive modulation of MC4R signaling.

Materials and Methods

Sequences Alignment

Protein sequences utilized in this study were acquired from NCBI database with the accession numbers as follows: sea lamprey (Petromyzon marinus, slMC4b, BK007095.1), elephant shark (Callorhinchus milii, esMC4R, XM_007895520.1), zebrafish (Danio rerio, zMC4R, NM_173278.1), coelacanth (Latimeria chalumnae, coMC4R, XM_006014267.1), frog (Xenopus tropicalis, fMC4R, XM_004915313.3), turtle (Pelodiscus sinensis, tMC4R, ENSPSIT00000009212.1), duck (Anas platyrhynchos, dMC4R, XM_005016243.2), mouse (Mus musculus, mMC4R, NM_016977.4), and human (Homo sapiens, hMC4R, NM_005912.2). The transmembrane domains of MC4R were analyzed by TMHMM v.2.0, Tmpred and Octopus and alignment was performed by DNAMAN with default parameters.

Plasmids

The wild-type MC4Rs of humans, mice, ducks, turtles, frogs, and zebrafish were cloned from the corresponding genomic library, and the coelacanth MC4R and sea lamprey MCb were synthesized by Generay Biotechnology (Shanghai, China) and subcloned into pc-DNA3.1(+) vector with 2× Flag tag at the N-terminus of each GPCR. Overlapping PCR was carried out to obtain the specific mutations of human or zebrafish MC4R. zICL2-hMC4R was generated by replacing ICL2 of hMC4R with the zebrafish ICL2. Two partitions were overlapped and extended with 10 rounds of Pfu polymerase(94 °C 15 s, 60 °C 30 s, 72 °C 15 s). Finally, the primers of the full-length fragments were applied and amplified with multiple rounds of PCR (94 °C 15 s, 65 °C 30 s,72 °C 60 s). The expression efficiencies of all chimeric hMC4R and zMC4R were quantified by ELISA upon transfection (Figure S1).

Cell Culture and Transfection

Human embryonic kidney 293T (HEK293T) cells were cultured with 5% CO2 at 37 °C. Cells were maintained in a high-glucose DMEM medium (Hyclone) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin. Transfection was performed with indicated plasmids using polyethylenimine (PEI) Transfection Reagent.

MC4R Constitutive Activity Measurement

HEK293T cells were seeded in 24-well plates and allowed to reach approximately 70% confluency before transfection. For the measurement of MC4R constitutive activity, cells were cotransfected with the following plasmids containing the specific MC4R variant (250 ng/well). pRL-TK, which encodes renilla luciferase with a TK promoter, serves as an internal control for transfection efficiency. pCre-Luc, which encodes firefly luciferase with a CRE promoter, whose expression is driven by cAMP-responsive elements, thus providing a readout for intracellular cAMP levels. Thirty hours post-transfection, the intracellular cAMP level, reflected by firefly luciferase activity, was assessed using the Dual-Glo Luciferase Assay System (Promega). Specifically, cells were lysed to release both fireflies and renilla luciferase. Subsequently, luciferin (for firefly luciferase) and coelenterazine (for renilla luciferase) were added in sequence to measure the respective chemiluminescence signals. The ratio of firefly to renilla luciferase activity provides a normalized measure of cAMP levels within the cells. Chemiluminescence was recorded using a Spectramax M5 plate reader. Basal activities were quantified based on the luciferase-to-renilla signal ratio. To account for variations in membrane trafficking efficiencies among MC4R variants, we calculated a relative basal activity by further normalizing to the cell surface protein abundance, as detected by surface ELISA assay (Figures S1 and S2).

Gαs and β-Arrestin 2 Recruitment Assay

A NanoBiT protein–protein interaction system (Promega) was employed to monitor the process of Gαs coupling and β-arrestin 2 recruitment of hMC4R, hMC4R-zICL2, zMC4R, and zMC4R-hICL2. NanoLuc functions as a synthetic luciferase that emits a stronger chemiluminescence signal than a traditional firefly luciferase. It consists of two segments: the larger LgBiT and the smaller SmBiT. When these segments are brought close together, an active NanoLuc is created, which produces chemiluminescence upon encountering its specific substrate. Leveraging this property, the NanoBiT system can effectively quantify Gαs coupling and the β-arrestin 2 recruitment process. MC4Rs were cloned into pBiT1.1-C[TK/LgBiT], GNAS (Gαs) into pBiT2.1-C[TK/SmBiT], and AARB2 (β-arrestin 2) into pBiT2.1-N[TK/SmBiT]. To monitor the basal recruitment level of specific downstream protein, Gαs for example, the MC4R-LgBiT and the Gαs-SmBiT constructs were transfected into HEK293T cells seeded at a concentration of 4 × 104 cells/well into a white 96-well plate precoated by poly-d-lysine. The total amount of the two plasmids was 200 ng/well. One day after the transfection, the medium was changed to HBSS with 25 mM HEPES 30 min before the test. The activity of NanoBiT was collected through a BioTek Synergy H1 plate reader (Agilent) at 37 °C. After collecting the background signal for 10 min, substrates were added into each well and the basal NanoBiT signal was quantified for 10 min (1 min intervals). The final constitutive recruitment signals were defined as the integration of chemiluminescence signals in the last 5 min (6 time points). The stimulated level of each group was represented by an area under curve (AUC) fold change calculating the drug-stimulated group divided by the negative control group after the addition of setmelanotide.

Surface Epitope Detection by Cell-Based Surface Enzyme Linked Immunosorbent Assay (ELISA)

To examine the expression level of wild-type and mutant MC4R on the cell surface, surface epitope detection by ELISA was conducted in HEK293T cells. HEK293T cells were seeded in a 24-well plate coated with 0.2% poly-d-lysine. Plasmids were transiently transfected the following day. After 24 h transfection, cells were fixed with 4% paraformaldehyde for 20 min and then washed three times with D-PBS for 5 min each time. Next, cells were incubated for 40 min with blocking buffer (5% milk in D-PBS with/without 0.1% triton X-100), followed by incubation with Rabbit anti-Flag antibodies (1:2000) for 2 h at room temperature. After incubation, cells were washed three times with D-PBS and incubated for 2 h with HRP-conjugated Goat anti Rabbit IgG antibodies (1:2000) at room temperature. After washing again, these cells were incubated with TMB 250 μL/well for 15 min (protected from light). Then, the reaction liquid was transferred to a 96-well plate, and the reaction was terminated by sulfuric acid (2 mol/L). Then the 24-well plate was air-dried, 250 μL of Janus green was added for 5 min, and the 24-well plate was slowly washed with water. 250 μL of hydrochloric acid (0.3 mol/L) was added to each well for cell lysis and was transferred to another 96-well plate. The absorbance values at 450 and 595 nm, respectively, were recorded by Spectramax ID3 multimode microplate reader, and the OD450/OD595 presents the protein abundance.

Conformational Structural Analysis

To predict the 3D structures of chimeric hMC4R and zMC4R, we employed ColabFold2 (https://github.com/sokrypton/ColabFold) whose algorithm is based on AlphaFold2 and RoseTTAFold accelerated by a fast multiple sequence alignment (MSA) using MMseqs2.19 We uploaded MC4R amino sequences and performed 12 recycles with ColabFold2 on google colab to generate 5 different predicted PDB files assessed by pLDDT and pTMscore using the template previously generated by cryo-electron microscopy.20 The protein structures with the highest rank from each chimera were aligned and rendered in Open-Source PyMOL (https://github.com/schrodinger/pymol-open-source).

Statistical Analysis

All experiments were repeated at least three times in separate experiments. Data were shown as the mean ± SD (standard deviation) and analyzed using GraphPad Prism v7.0 software. For comparisons between two independent groups, the Student’s t test was employed. For comparisons among multiple groups, one-way ANOVA followed by Dunnett’s multiple comparisons test was utilized to compare each group to the control. p-value of <0.05 was considered statistically significant. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results

Evolutionary Comparison of Constitutive Activities of MC4R Signaling

First, we synthesized and examined the constitutive activities of MC4Rs from nine Chordate species, including sea lamprey (slMC4R), elephant shark (esMC4R), zebrafish (zMC4R), coelacanth (coMC4R), frog (fMC4R), turtle (tMC4R), duck (dMC4R), mouse (mMC4R), and human (hMC4R). In comparison to the mock condition (empty vector), MC4Rs of tetrapods (human, mouse, duck, turtle, and frog) and coelacanth, a transitional species from fish to amphibians, did not show significant constitutive activity. On the contrary, the constitutive activities of species lower than coelacanth such as sea lamprey, zebrafish, and elephant shark were much higher than other species (Figure 1A).

Figure 1.

Figure 1

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Distinct MC4R constitutive activities between species. (A) Constitutive comparison of MC4R from various species. Species abbreviations are as follows: h: human, m: mouse, d: duck, t: turtle, f: frog, co: coelacanth, z: zebrafish, es: elephant shark, sl: sea lamprey. Significance levels are denoted as n.s. (no significance) and *** (P < 0.001). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test, comparing each species to the mock group (pcDNA3.1 only). (B) Alignment of the N-terminal sequence of MC4R from 22 species, from fishes to mammals. Conserved residues were highlighted in blue. The “HLWNRS” motif was framed with a red square.

Accumulative evidence demonstrated that the constitutive activity of MC4R was maintained mainly by its N-terminus, especially the “HLWNRS” motif.11,16 Therefore, we performed sequence alignment and compared the N-termini of multiple chordate species. We found that the “HLWNRS” motif was conserved only in the tetrapod and coelacanth (Figure 1B), indicating that the high constitutive activity of fish species may not only result from the sole “HLWNRS” motif.

Constitutive Activity by the Substitution of hMC4R’s N-terminus with zMC4R’s

To explore the distinct difference in constitutive activity between higher and lower vertebrates, we selected zMC4R and hMC4R for further parallel comparative analysis because they showed the most robust difference in the basal cAMP levels (Figures 1A and 2A). We measured the relative basal activity in which the cAMP level is normalized by a surface ELISA amount of MC4R protein (Figure S1). We compared the protein sequences of zMC4R and hMC4R and subsequently substituted the N-terminus of hMC4R with that of zMC4R, abbreviated as zN-hMC4R (Figure 2B). Results showed a significant elevation of the constitutive activity of zN-hMC4R compared to that of the wild-type hMC4R (Figure 2C). In contrast, the constitutive activity of zMC4R dramatically decreased when its N-terminus was substituted by that of hMC4R (abbreviated as hN-zMC4R) (Figure 2D). We also found that the constitutive activity of hN-zMC4R was still obviously higher than that of hMC4R and zN-hMC4R despite its decreased trend compared with zMC4R. This result suggested that the N-terminus only played a partial role and may require additive action with other unknown domains to determine the overall constitutive activity of MC4R.

Figure 2.

Figure 2

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Requirement of the second intracellular loop for the modulation of hMC4R constitutive activity. (A) Transmembrane analysis and sequence alignment of hMC4R and zMC4R. Green lines represent extracellular regions (N-terminus and ECLs), red lines represent transmembrane regions (TM), and black lines represent intracellular regions (ICLs and C-terminus). (B) Diagrams of various MC4R substitutions. (C) Effects of the N-terminus on the constitutive activity of hMC4R. *** P < 0.001, student’s t test, compared to hMC4R. (D) Effects of the N-terminus on the constitutive activity of zMC4R. *** P < 0.001, student’s t test, compared to zMC4R. (E) Constitutive activity of all 15 substitutions. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test, compared to hMC4R. # P < 0.05, ## P < 0.01, ### P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test, compared to zN-hMC4R. Black square, wildtype MC4R; blue square, N-terminus substituted MC4R; purple square, N-terminus as well as special adjacent region substituted MC4R; orange square, ICL substituted MC4R. Basal activities were normalized by cell surface abundance in (C–E).

Superimposition of N-Terminus and Extracellular Loop or Transmembrane Domain of zMC4R on Modulating hMC4R Constitutive Activity

In order to screen other potential functional domains involved in the constitutive modulation of MC4R, the extracellular loops and transmembrane domains of zN-hMC4R were substituted by the corresponding regions of zMC4R, abbreviated as zN+ECL1-hMC4R, zN+ECL2-hMC4R, zN+ECL3-hMC4R, zN+TM1-hMC4R, zN+TM2-hMC4R, zN+TM3-hMC4R, zN+TM4-hMC4R, zN+TM5-hMC4R, zN+TM6-hMC4R and zN+TM7-hMC4R, respectively (Figure 2B). These constructs were designed to investigate the additive effect between the N-terminus of zebrafish MC4R and its adjacent domains. The constitutive activities were measured, and all the substitutions exhibited a higher level with 3–14 folds of hMC4R activity (Figure 2E). Meanwhile, the constitutive activities of zN+TM1-hMC4R, zN+TM3-hMC4R, zN+TM4-hMC4R, zN+TM5-hMC4R, and zN+TM7-hMC4R also significantly increased in comparison to that of zN-hMC4R, which demonstrated a combined effect of the N-terminus and these transmembrane domains. However, all constitutive activities were still much lower than that of native zMC4R, which indicated another potential mechanism lying under this evolved structure.

Elevation of hMC4R Constitutive Activity by the Intracellular Loops of zMC4R

The structural analysis showed several Gαs-binding sites in the ICL2 and ICL3 of hMC4R.20 The pharmacological effect of multiple intracellular loops of zMC4R on the constitutive activity of hMC4R was further assessed. Likewise, the four intracellular loops of hMC4R were substituted by the corresponding regions of zMC4R and abbreviated as zICL1-hMC4R, zICL2-hMC4R, zICL3-hMC4R, and zC-hMC4R, respectively (Figure 2B). As shown, all the four substitutions elevated the hMC4R constitutive activities, in which the zICL2-hMC4R showed the highest cAMP level (Figure 2E).

Next, the 15 substitutions were parallelly compared to better understand their contributions to the constitutive activity of hMC4R. Our results indicated that the substitution of ICL2 significantly elevated the hMC4R constitutive activity with almost 38-fold higher than hMC4R. However, although all the substitutions exhibited a significant contribution to the elevation of hMC4R constitutive activity, the overall constitutive levels were still largely lower than that of zMC4R (Figure 2E).

Determination of MC4R Constitutive Activity by the Second Intracellular Loop

Given the pronounced basal activity of zICL2-hMC4R, we hypothesized that ICL2 played a significant role in contributing to the differences in constitutive modulation observed between species. Only four different amino acid residues exist between the ICL2 region of hMC4R and zMC4R (Figure 2A). Subsequently, we replaced each residue of hMC4R with the corresponding residue from zMC4R to investigate whether substitutions at the same site could influence its function. Additionally, we used alanine substitution to assess the significance of a specific amino acid. These proteins are labeled as F149I hMC4R, Q156R hMC4R, V163Q hMC4R, K164R hMC4R, F149A hMC4R, Q156A hMC4R, V163A hMC4R, and K164A hMC4R (Figure 3A). The constitutive activity of these eight substitutions exhibited a significantly higher activity than hMC4R (6- to 19-fold of hMC4R activity; Figure 3B).

Figure 3.

Figure 3

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Requirement of residues within the ICL2 region for MC4R constitutive activity. (A) Diagrams of various hMC4R substitutions within the second intracellular loop. (B) Effects of the second intracellular loop on the constitutive activity of hMC4R. *** P < 0.001 one-way ANOVA compared to wildtype hMC4R (WT). (C) Diagrams of various zMC4R substitutions within the second intracellular loop. (D) Effects of different substitutions within ICL2 region on hMC4R constitutive activity. n.s. no significance, *** P < 0.001 one-way ANOVA, followed by Dunnett’s multiple comparisons test, compared to wildtype zMC4R (WT). Basal activities were normalized by cell surface abundance in B and D.

Similarly, the four different amino acid residues of zMC4R were also substituted with the corresponding residue of hMC4R or alanine, abbreviated as I147F zMC4R, R154Q zMC4R, Q161 V zMC4R, R162 K zMC4R, I147A zMC4R, R154A zMC4R, Q161A zMC4R, and R162A zMC4R (Figure 3C). Surprisingly, the normalized basal activities of I147F zMC4R, R154Q zMC4R, Q161 V zMC4R, and R162 K zMC4R mutations only decreased to 32.8, 28.2, 34.4, and 34.9%, respectively, which were at a similar trend. However, the alanine substitutions significantly reduced zMC4R basal activity by 96.3 to 99.1% (Figure 3D).

To obtain an evolutionary vision of the ICL2 of MC4R, we also performed sequence alignment of the ICL2 region from different species and showed that the distinct residues from different species were identical with the sites of different residues between hMC4R and zMC4R. The I147 zMC4R and R154 zMC4R were conserved in species below tetrapods, which suggested an important regulatory role that only the I147F zMC4R and R154Q zMC4R significantly decreased the basal activity of zMC4R (Figure 3D). Meanwhile, because we did not find any sole residue that was responsible for the entire constitutive activity of MC4R (Figure 3D), the zMC4R ICL2 region was further substituted with that of hMC4R (abbreviated as hICL2-zMC4R) and the constitutive activity of hICL2-zMC4R was measured. As expected, the substitution of the hMC4R ICL2 region sharply decreased 94% constitutive activity of zMC4R (Figure 4B). The NanoBiT protein–protein interaction detection assay was then introduced to clarify the undergoing Gαs coupling and β-arrestin 2 recruitment in the absence of any MC4R ligands (Figure 4C, G). As expected, the Gαs coupling level of hMC4R was slightly increased when its ICL2 was substituted, whereas the coupling signal of zICL2-hMC4R dramatically decreased compared with zMC4R (Figure 4E). The regulatory pattern of β-arrestin 2 recruitment of four constructs was similar to Gαs coupling (Figure 4I). Notably, when these mutants were challenged with corresponding agonists, the pharmacological levels of Gαs and β-arrestin 2 pathways were not quite different in the Gαs coupling of hICL2-zMC4R and the β-arrestin 2 recruitment of zICL2-hMC4R, or slightly changed in the Gαs coupling of zICL2-hMC4R and the β-arrestin 2 recruitment of hICL2-zMC4R (Figure 4D, F, H, J). Taken together, the decisive effect of the ICL2 region on modulating MC4R constitutive activity was dependent on the additive action of these four residues which influenced Gαs coupling and β-arrestin 2 recruitment.

Figure 4.

Figure 4

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Determination of the ICL2 region for MC4R constitutive activity. (A) Alignment of the sequence of MC4R ICL2 from different species. Conserved residues were highlighted in blue. (B) Constitutive activity of hICL2 substituted zMC4R. *** P < 0.001 one-way ANOVA, followed by Dunnett’s multiple comparisons test, compared to hICL2-hMC4R. (C) Diagram of NanoBiT luciferase reconstruction between MC4R-LgBiT (Large NanoBiT) and GNAS-SmBiT (Small NanoBiT). (D) Normalized Gαs coupling time-resolved curve of different MC4R subtypes with or without the addition of agonist (setmelanotide) where the background signals (first 10 min) were subtracted, and the basal signals (11–20 min) were normalized to one. (E) Gαs coupling levels of ICL2 substituted MC4Rs. (F) Stimulated Gαs coupling levels of ICL2 substituted MC4Rs. (G) Diagram of NanoBiT luciferase reconstruction between MC4R-LgBiT and SmBiT-ARRB2. (H) Normalized β-arrestin 2 recruitment time-resolved curve of different MC4R subtypes with or without the addition of agonist (setmelanotide) where the background signals (first 10 min) were subtracted, and the basal signals (11–20 min) were normalized to one. (I) β-arrestin 2 recruitment of ICL2 substituted MC4Rs. (J) Stimulated β-arrestin 2 recruitment of ICL2 substituted MC4Rs. n.s. no significance, * P < 0.05, *** P < 0.001 one-way ANOVA followed by Dunnett’s multiple comparisons test. Basal activities were normalized by cell surface abundance in B.

Conformational Structural Analysis of the Second Intracellular Loop of MC4R Proteins

By utilizing the AlphaFold2-based structure predictive platform, ColabFold2, we generated and compared the chimeric MC4R protein structures with wild-type ones. We first compared hMC4R and zMC4R to get full superimposed images and found that their main divergences were located mainly in the N-terminus. A unique α helix loss was found in wildtype hMC4R, which may make this fragment less hydrophobic and impact the ligand binding or the formation of a divalent ion pocket in the center of MC4R (Figure 5A). We also aligned the N-terminus and the results matched the divergence shown in the wildtype comparation. The other segments of MC4R did not change obviously (Figure 5B, D). However, the ICL2 of either hMC4R or zMC4R could not bring dramatic structural changes to the wild-type MC4R (Figure 5C–E). In addition, most predicted IDDTs (pLDDT) of each structure generated by ColabFold were higher than 70 and considered a reliable prediction (Figure S3).

Figure 5.

Figure 5

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Predicted structural alignments of MC4R and its chimeras. (A) Front and top views of the alignment of wildtype hMC4R and zMC4R. Green, hMC4R; red, zMC4R. (B) Front and top view of alignment of wildtype hMC4R and zN-hMC4R. Green, hMC4R; red, human part of zN-hMC4R; blue, zebrafish N-terminus of zN-hMC4R. (C) Front and side view of the alignment of wildtype hMC4R and zICL2-hMC4R. Green, hMC4R; red; main human part of zICL2-hMC4R; blue, zebrafish ICL2 fragment of zICL2-hMC4R. (D) Front and top view of alignment of wildtype zMC4R and hN-zMC4R. green, zMC4R; red, zebrafish part of hN-zMC4R; blue, human N-terminus of hN-zMC4R. (E) Front and side view of the alignment of wildtype zMC4R and hICL2-zMC4R. green, zMC4R; red, main zebrafish part of hICL2-zMC4R; blue, human ICL2 fragment of hICL2-zMC4R.

Discussion

Similar to numerous known GPCRs, MC4R shows agonist-independent G protein-coupling constitutive activity. In this study, we focused on MC4R from various species and aimed to find the key regions affecting distinct MC4R constitutive activity from evolutionary aspects.10,11,15 At present, most studies monitored obvious hMC4R constitutive activity and recruited as a positive control to compare with various mutations from obese patients.11,16,17 In our study, we first measured the constitutive activities of MC4R in human and zebrafish. Compared with hMC4R, zMC4R exhibited an obvious much higher basal activity. In our previous work, we demonstrated that α-MSH stimulated zMC4R signaling exhibited a higher constitutive activity compared to its counterparts of human and mice MC4R.7 In addition to zebrafish, multiple studies have reported the divergent constitutive activity in fish MC4R, such as spotted scat, grass carp and sweep eel.21−23 Several studies demonstrated that the extracellular N-terminal domain of hMC4R functioned as a tethered partial agonist to maintain its constitutive activity, especially the “HLWNRS” motif.11,16 According to the alignment of the MC4R N-terminus from different species, we found that the “HLWNRS” motif was only conserved in coelacanth and tetrapods, while it was not conserved in some Osteichthyes such as zebrafish and primitive species such as elephant shark or sea lamprey. In line with the sequence alignment, the constitutive activity of MC4R subtypes from distinct species that owned the “HLWNRS” motif was significantly lower than that of MC4R subtypes from fishes (zebrafish, elephant shark, and sea lamprey). Our observation was consistent with the work of Li et al., in which, the grass carp MC4R showed a higher constitutive activity than human MC4R. Meanwhile, the “HLWNRS” motif was absent in the MC4R N-terminal of grass carp.24 Therefore, it is reasonable to speculate that the N-terminus may be responsible for the “HLWNRS” induced constitutive differences between species. Coelacanth is a special animal considered a “lived fossil” at the midstate between terricolous and aqueous species, and the “HLWNRS” motif begins to form in this species. This motif evolves along the phylogenetic history, suggesting the essential role of MC4R in aiding animals to leave the water and adapt to terrestrial habitats and challenges.

Due to the huge difference of constitutive activity between hMC4R and zMC4R, the two MC4Rs were chosen for the subsequent investigation. Upon substitution of the hMC4R N-terminus with the zMC4R N-terminus, the constitutive activity of hMC4R was significantly elevated. On the contrary, the constitutive activity of hN-zMC4R dramatically decreased compared with zMC4R. Probably because of the extremely high basal activity of zMC4R, the constitutive activity of hN-zMC4R was still significantly higher than that of hMC4R. Similarly, the constitutive activity of zN-hMC4R was only 4-fold of hMC4R basal activity. Clinical studies have found some mutations of the N-terminus of hMC4R from obese patients, such as R7C/H, T11A/S, and R18C/H/L.8,11,25 The R7C mutation showed no effect on its constitutive activity, while R7H, T11S, and R18C/H/L mutations significantly decreased its constitutive activity.11,25 The R7 and R18 residues in hMC4R correspond to the H7 and H18 residues of zMC4R. However, the constitutive activity of zMC4R was significantly higher than that of hMC4R. Therefore, we speculated that the N-terminus might require synergy with other unknown regions to play a decisive role in modulating MC4R constitutive activity.

We next explored the synergetic effect of the N-terminus with the extracellular loops or transmembrane domains on hMC4R constitutive activity. Our results showed that the cosubstitution of the N-terminus and the extracellular loop or transmembrane domain significantly elevated the zN-hMC4R constitutive activity with the exception of zN+ECL2-hMC4R, zN+ECL3-hMC4R, zN+TM2-hMC4R, and zN+TM6-hMC4R. Previous studies reported that the TM1, TM3, and TM7 regions of MC4R could play important roles in the conformational changes upon receptor activation, and mutations in these regions caused retention of MC4R within the cell and could not be activated.26 More studies also found that T246A, L247A, I251A, G252A, W258A, and P260A mutations in the TM6 region significantly reduced hMC4R constitutive activity, while A244G, L250A and A259G mutations increased that.27 Similarly, mutations in I125A, D126A, S127A, V128A, I143A, and A144G in the TM3 region significantly reduced the constitutive activity of MC4R, and the I137A and L141A mutations significantly increased that.28 These results elucidated the effect of transmembrane regions on modulating MC4R constitutive activity. For the extracellular loops, most studies mainly focused on the surface expression and agonist-induced activity of MC4R mutations that naturally occurred and led to obesity. In our study, beyond the natural single amino acid mutations, we explored more on the interactions and functions inside MC4R. The cosubstitution of the N-terminus and the first or third extracellular loop both elevated the basal activity of hMC4R. However, the constitutive activity of all these ten substitutions still could not be compared with zMC4R, suggesting that the extremely high basal activity of zMC4R was not dependent on the synergetic effects of N-terminus with the extracellular loop or transmembrane domain.

In this study, we further examined the effects of intracellular loops on the MC4R constitutive activity. The four substitutions of hMC4R intracellular loops with the corresponding zMC4R intracellular loops all increased hMC4R constitutive activity. The study of Kim et al. suggested the importance of the third intracellular loop for regulating and maintaining the MC4R constitutive activity associated with G protein coupling,17 and the third intracellular loop was also observed to contribute to the hMC4R constitutive activity in our study. Besides, the second intracellular loop was confirmed to play a vital role in the conformational changes during receptor activation.26 In our study, the second intracellular loop substitution showed the highest basal activity compared to the other three intracellular loop substitutions, even among all the 15 substitutions. These findings suggested that the second intracellular loop must play a decisive role in constitutive MC4R constitutive activity. The second intracellular loop of zMC4R was next substituted with that of hMC4R. In line with our expectation, the constitutive activity of hICL2-zMC4R sharply decreased compared with that of zMC4R, thus demonstrating the decisive role of the second intracellular loop in modulating the constitutive activity. Among the second intracellular loop, only four amino acid residues are different between hMC4R and zMC4R. In these four different residues, the R156 receptor variant of toothed whales showed greater constitutive activity, while the Q156 receptor variant of the larger baleen whales was functionally less responsive to α-MSH.29 Coincidentally, Q156 was found in hMC4R and R154 in zMC4R. In accordance with the work of Zhao et al., the introduction of an R residue at position 156 of hMC4R significantly elevated its constitutive activity. On the contrary, the introduction of a Q residue at position 154 of the zMC4R decreased its constitutive activity.29 For hMC4R, the substitution of the rest of the residues elevated its constitutive activity. For zMC4R, the I147F mutation in zMC4R decreased its constitutive activity similar to the effects of R154Q, Q161V, and R162K mutations. Meanwhile, the alanine substitutions significantly reduced zMC4R constitutive activity similar to hICL2-zMC4R. Therefore, we concluded that the MC4R constitutive activity was dependent on all these four residues within ICL2.

In another similar study, researchers investigated the alternative function of ICL2 in human MC4R and found that five mutants (R147A, T150A, I151A, L155A, and Y157A) significantly decreased the constitutive activity of human MC4R.30 This study was based on alanine scanning mutagenesis, whereas our study focused on evolutionary organism-scale comparison. On the other hand, different loci were reported to influence the basal activity of MC4R, additionally showing the important function of the ICL2 region. The crystal structures of hMC4R at different phases were recently illustrated in which the ICL2 region was found to be responsible for the GPCR function such as G protein binding.31 Q156 of hMC4R was found to form a hydrogen bond with the backbone of D201 in the β2−β3 turn of Gαs, which was present in the NDP-α-MSH complex but not setmelanotide complex, and may contribute to the increase of constitutive activity.32 However, the other three mutations were not found in these and other related structural investigations.20,33 In combination with our evidence, the ICL region of MC4R significantly altered the Gαs coupling as well as the β-arrestin 2 recruitment. This discrepancy may suggest an unknown mechanism of alternative MC4R constitutive activity which needs to be revealed in future studies.

In summary, our study compared and found the difference of MC4R constitutive activity between two vertebrates, as well as the requirement of F149, Q156, V163, and K164 in the ICL2 region for modulating MC4R constitutive activity. This study provided novel structural and pharmacological insights into the molecular determinant responsible for modulating GPCR constitutive activity.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00169.

  • Surface expression of chimeric MC4R proteins; surface expression of MC4R ICL2 mutants; and plDDT of Alpha-fold2 prediction (PDF)

Author Contributions

∇ B.X. and J.Y. contributed equally to this work.

This work was supported by the National Key Research and Development Program of China (Grant No. 2019YFA0111400), the National Natural Science Foundation of China (Grant No. 32271165), and the Interdisciplinary Project in Ocean Research of Tongji University (Grant No. 2022-2-ZD-02).

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Pharmacology & Translational Sciencevirtual special issue “GPCR Signaling”.

Supplementary Material

pt3c00169_si_001.pdf (648.2KB, pdf)

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Supplementary Materials

pt3c00169_si_001.pdf (648.2KB, pdf)

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