Key amino acid residues in the melanocortin-4 receptor for nonpeptide THIQ specific binding and signaling

Abstract

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and glucose homeostasis. Synthetic nonpeptide compound N- (3R)-1 4-tetrahydroisoquinolinium-3-ylcarbonyl-(1R)-1-(4-chlorobenzyl)-2-4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl-2-oxoethylamine (THIQ) is a potent agonist at MC4R but not at hMC2R. In this study, we utilized two approaches (chimeric receptor and site-directed mutagenesis) to narrow down the key amino acid residues of MC4R responsible for THIQ binding and signaling. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TMs) of the human MC4R (hMC4R) with the homologous regions of hMC2R were constructed. Our results indicate that the cassette substitutions of these TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter THIQ binding affinity and potency except the substitution of the hMC4R TM3, suggesting that the conserved amino acid residues in these TMs of the hMC4R are main potential candidates for THIQ binding and signaling while non conserved residues in TM3 of MC4R may also be involved. Nineteen MC4R mutants were then created, including 13 conserved amino acid residues and 6 non-conserved amino acid residues. Our results indicate that seven conserved residue [E100 (TM2), D122 (TM3), D126 (TM3), F254 (TM6), W258 (TM6), F261 (TM6), H264 (TM6)] are important for THIQ binding and three non-conserved residues [N123 (TM3), I129 (TM3) and S131 (TM3)] are involved in THIQ selectivity. In conclusion, our results suggest that THIQ utilize both conserved and non-conserved amino acid residues for binding and signaling at hMC4R and non conserved residues may be responsible for MC4R selectivity.

Figure 1

Sequence comparison between nonpeptide THIQ and peptide NDP-MSH.

Figure 2

The procedure of the triazole-based non peptide THIQ synthesis

Figure 3

Schematic representation of the chimeric human melanocortin receptors utilized in these studies. Panel A schematically depicts the seven transmembrane structures of the “wild-type” (WT) MC4R (drawn with heavy lines) and MC2R (drawn with thin lines). Panel B depicts the structure of the chimeric MC4R with the substituted TMs of the MC2R.

Figure 4

Binding affinity and potency of THIQ at the wild-type hMC4R and hMC2R. Panel A depicts the binding affinity of THIQ as determined by inhibition of ¹²⁵I NDP-MSH binding. Panel B demonstrates the ability of THIQ to stimulate the production of intracellular cAMP. Data points represent the mean ± SEM of at least 3 independent experiments.

Figure 5

Binding affinity and potency of THIQ at the chimeric receptors. Panel A depicts the binding affinity of THIQ as determined by inhibition of ¹²⁵I NDP-MSH binding. Nonspecific binding was determined by measuring the amount of ¹²⁵I-label bound on the cells in the presence of excess 10⁻⁶ M unlabeled ligand. Specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Panel B demonstrates the ability of THIQ to stimulate the production of intracellular cAMP. Data points represent the mean ±SEM of at least three independent experiments.

Figure 6

Receptor sequence comparison between MC4R and MC2R. The mutated conserved TM residues in these experiments are denoted by bold. The mutations of the residues affected THIQ binding were highlighted by bold italic.

Figure 7

Binding affinity and potency of THIQ at the mutations of the conserved amino acid residues of the hMC4Rs. Panel A shows the binding affinity of unlabeled THIQ to displace ¹²⁵I NDP-MSH. Nonspecific binding was determined by measuring the amount of ¹²⁵I-label bound on the cells in the presence of excess 10⁻⁶ M unlabeled ligand. Specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Panel B shows that THIQ to stimulate cAMP production. Data points represent the mean ±SEM of at least three independent experiments with duplicated wells.

Figure 8

Binding affinity and potency of THIQ at the mutations of the conserved amino acid residues of the hMC4Rs. Panel A shows the binding affinity of unlabeled THIQ to displace ¹²⁵I NDP-MSH. Nonspecific binding was determined by measuring the amount of ¹²⁵I-label bound on the cells in the presence of excess 10⁻⁶ M unlabeled ligand. Specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Panel B shows that THIQ to stimulate cAMP production. Data points represent the mean ±SEM of at least three independent experiments with duplicated wells.

Figure 9

Binding affinity and potency of THIQ at the mutations of non-conserved amino acid residues of the hMC4Rs. Panel A shows the amino acid residue differences between hMC4R and hMC2R. Panel B shows the binding affinity of unlabeled THIQ to displace ¹²⁵I NDP-MSH. Nonspecific binding was determined by measuring the amount of ¹²⁵I-label bound on the cells in the presence of excess 10⁻⁶ M unlabeled ligand. Specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Panel C shows that THIQ to stimulate cAMP production. Data points represent the mean ±SEM of at least three independent experiments with duplicated wells.

Figure 10

Two-dimensional representation of a proposed three dimensional model illustrating the synthetic melanocortin THIQ docked inside the hMC4R. Three receptor binding pockets are hypothesized. The first is a predominantly ionic pocket formed by Asp¹²² and Asp ¹²⁶. The second a hydrophobic pocket formed by aromatic residues in TM6., F²⁶¹ and H²⁶⁴ are included. The third one is THIQ specific, including N123 and I129.