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

Abstract

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

Keywords: MC4R; allosteric ligand; biased signaling; constitutive activity; paralogs; rainbow trout.

Figure 1

Comparison of amino acid sequences between omMc4rs and hMC4R. Dark boxes indicated potential N-linked glycosylation sites. Shaded boxes show putative TMD 1–7. Open boxes show the conserved motifs (PMY, DRY, and DPxxY). Asterisk (*) indicates identical residues (fully conserved residue); colons (:) and period (.) indicate highly conserved and semi-conserved amino acids, respectively.

Figure 2

Amino acid sequence identities of omMc4rs and other MC4Rs.

Figure 3

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

Figure 4

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

Figure 5

Cell-surface and total expression of trout Mc4rs. (A) cell-surface expression; (B) total expression; (C) surface ratio (surface expression/total expression). HEK293T cells were transiently transfected with omMc4ra1, omMc4ra2, omMc4rb1, omMc4rb2, or hMC4R, as described in Materials and Methods. Data are mean ± SEM from three or four independent experiments. * Significantly different from hMC4R (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Ligand-binding properties of omMc4rs. HEK293T cells were transiently transfected with hMC4R or omMc4r plasmids, and the binding properties were measured 48 h later by displacing the binding of ¹²⁵I-NDP-MSH using different concentrations of unlabeled α-MSH (A), ACTH (1–24) (B), AgRP (83–132) (C), Ipsen 5i (D), ML00253764 (E), or MCL0020 (F). Data are expressed as % of hMC4R binding ± range from duplicate measurements within one experiment. The curves are representative of at least three independent experiments.

Figure 7

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

Figure 8

Effects of four ligands on the basal activities of omMc4rs. HEK293T cells were transiently transfected with four omMc4rs plasmids, and intracellular cAMP levels were measured 48 h later by RIA after stimulation without (A) or with 10 nM AgRP, 1 μM MCL0020, 1 μM ML00253764, or 1 μM Ipsen 5i at the four omMc4r isoforms (B), omMc4ra1; (C), omMc4ra2; (D), omMc4rb1; and (E), omMc4rb2 as described in Section 2. Data are mean ± SEM from three independent experiments. * Significantly different from basal activity of hMC4R or corresponding omMc4r (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 9

ERK1/2 signaling properties of trout Mc4rs. (A) Immunoblots of pERK1/2 in cells expressing omMc4ra1, omMc4ra2, omMc4rb1, or omMc4rb2. HEK293T cells were transiently transfected with omMc4ra1, omMc4ra2, omMc4rb1, omMc4rb2, or empty vector plasmids and were starved overnight 24 h after transfection. Cells were treated with vehicle only or with 10 nM AgRP, 1 μM Ipsen 5i, 1 μM ML00253764, or 1 μM MCL0020 for 5 min. Western blots were performed as described in Materials and Methods. (B) Densitometry results of basal pERK1/2 levels of omMc4ra1, omMc4ra2, omMc4rb1, and omMc4rb2. (C) Densitometry results of pERK1/2 level of omMc4ra1. (D) Densitometry results of pERK1/2 level of omMc4ra2. (E) Densitometry results of pERK1/2 level of omMc4rb1. (F) Densitometry results of pERK1/2 level of omMc4rb2. Data are mean ± SEM from at least three independent experiments. * Significantly different from basal or vector activity (* p < 0.05, and ** p < 0.01).