MOLECULAR EVOLUTION OF GPCRS: Melanocortin/melanocortin receptors

  1. Natalie Lesinski
  1. Department of Biological Sciences, University of Denver, Denver, Colorado 80210, USA
    1Department of Biology, University of Akron, Akron, Ohio 44325, USA
  1. Correspondence should be addressed to R M Dores; Email: rdores{at}
  1. Figure 1

    Schematic of human melanocortin receptors (MCRs). These cartoons of human MCRs are roughly drawn to scale to show the positioning of the seven transmembrane-spanning domains (red numbered boxes), and the relative size of the N-terminal domains (blue boxes), and the C-terminal domains (green boxes) for each receptor. The functions ascribed for each receptor are adapted from Gantz & Fong (2003).

  2. Figure 2

    Schematic of the organization of gnathostome POMC. These cartoons (not drawn to scale) show the POMC organizational plan for tetrapods, teleosts, and cartilaginous fishes. All POMC sequences have a single copy of the β-endorphin sequence located at the C-terminal of the precursor. Tetrapod POMCs have a γ-MSH (γ) sequence in the N-terminal of the precursor, an ACTH/α-MSH (α) sequence in the middle of the precursor, and a β-MSH (β) sequence near the C-terminal of the precursor. Teleost POMCs lack a γ-MSH, but have retained the ACTH/α-MSH and the β-MSH sequences. Cartilaginous fish POMCs have retained the γ-MSH, ACTH/α-MSH, and the β-MSH sequences and have an additional δ-MSH (δ) sequence N-terminal to the β-MSH sequence. The sequences of dogfish (Amemiya et al. 1999) melanocortins are shown. The HFRW motif is highlighted in red and the RKRRP motif in green.

  3. Figure 3

    In-silico simulations of ACTH(1–24) tertiary structure from rainbow trout (Oncorhynchus mykiss, ACC #Q04617), dogfish (Squalus acanthias, ACC #P01197), African clawed frog (Xenopus laevis ACC #NP_001080838), and human (ACC #NP_001030333). Sequences were submitted to ab initio modeling via the Quark algorithm (Xu & Zhang 2012), which returned many similar structures per species. Models 1 and 2 represent two possible structures generated from ab initio modeling, followed by extensive energy minimization simulation. From this population of models, we can draw inference about general ACTH(1–24) structure among vertebrates. The first two structures per species were energy minimized using the YASARA Software (de Groot et al. 1997) over 100 000 NS. The HFRW motif is shown in magenta and the R/KKRRP motif in gray. HFRW is more constrained than the rest of the structures and maintains a uniform distance from R/KKRRP in all but one structure (rainbow trout model 1).

  4. Figure 4

    Ligand selectivity of Xenopus tropicalis melanocortin receptors (MCRs): MC1R, MC3R, MC4R, and MC5R. The dose–response curves for X. tropicalis MCRs stimulated with either X. tropicalis ACTH(1–24), α-MSH, β-MSH, or γ-MSH are presented for (A) MC1R; (B) MC3R; (C) MC4R; and (D) MC5R). The X. tropicalis MCR plasmids were separately transiently transfected into CHO cells with the cAMP reporter plasmid CRE-luciferase (Chepurny & Holz 2007) using a Cell Line Nucleofector Solution T Kit (Amaxa, Inc., as described by Liang et al. (2011). The cells were plated on a 96-well plate at a density of 1×105 cells/well and stimulated 48 h after transfection with the ligands listed above (New England Peptide, Boston, MA, USA) in serum-free CHO media for 4 h at 37 °C at concentrations ranging from 10−6 to 10−12 M. After a 4 h incubation period, 100 μl of Bright-Glo luciferase assay reagent (Promega, Inc.) were applied to each well and incubated at room temperature for 5 min. Luminescence was then measured using a Bio-Tek Synergy HT plate reader (Winooski, VT, USA). Previous studies have provided evidence that the CHO cells used in this study do not endogenously express mrap genes (Reinick et al. 2012b). All dose–response curves were done in triplicate. Average values and s.e.m. were graphed using the KaleidaGraph Software (, and the EC50 value for each ligand was determined. The curves were not constrained through the 0/0 origin. The EC50 values were compared using the Student's t-test, P<0.05, n=3.

  5. Figure 5

    Ligand selectivity of Xenopus tropicalis MC2R. (A) The dose–response curves for X. tropicalis MC2R stimulated with either X. tropicalis ACTH(1–24) or α-MSH. These transfections were performed as described in the legend to Fig. 4 with the exception that a mouse Mrap cDNA construct was included for both transfections. In the absence of mouse MRAP, X. tropicalis MC2R cannot be functionally expressed (Liang et al. 2011). Mouse MRAP was used in this experiment due to the fact that at present a X. tropicalis mrap ortholog has not been detected in the genome of X. tropicalis. (B) A summary of the EC50 values for the ligand selectivity experiments in Figs 4 and 5A is presented, n=3.

  6. Figure 6

    Overview of the hypothalamus/pituitary/adrenal–interrenal (HPA–I) axis and the interaction between MRAP and MC2R. (A) This figure presents a schematic of the HPA–I axis. CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropin; (+), stimulate; red dashed line, negative feedback. (B) MC2R monomers (oval figure) are inserted into the membrane of the rough endoplasmic reticulum (RER). MRAP1 monomers (rectangle) are also inserted into the RER membrane. MRAP1 monomer: the circle indicates the N-terminal of the monomer and the diamond the C-terminal of the monomer. The green box represents the transmembrane domain of the MRAP1 monomer. The red box is the site of the motif for reverse topology. For mouse MRAP1, the amino acid sequence of this motif is LKANKNS (Hinkle & Sebag 2009). The yellow box represents the activation motif in the MRAP1 monomer. For mouse MRAP1, the amino acid sequence of this motif is LDYI (Hinkle & Sebag 2009). At the plasma membrane, the MC2R/MRAP1 complex is composed of a MC2R dimer and two MRAP1 dimers (Cooray et al. 2011).

  7. Figure 7

    NPY/POMC circuit in the hypothalamus. This diagram depicts the orexigenic NPY/AGRP neuron and the anorexigenic POMC neurons, each forming a synapse with a neuron in the anorexigenic circuit that expresses the melanocortin 4 receptor (MC4R). The red circles represent AGRPs and NPY being released at the synapse from an NPY/AGRP neuron, and the green circles represent α-MSH, β-MSH, γ-MSH, and β-endorphin being released from a POMC neuron.

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