Interactions of the melanocortin-4 receptor with the peptide agonist NDP-MSH

Abstract

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His(6)-Phe(7)-Arg(8)-Trp(9), which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle(4),D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R(*)), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level. The mutation of residues in the human MC4R--such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)--to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between D-Phe(7) on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp(9). For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the beta(2)-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the beta(2)-adrenergic receptor.

Fig. 1

(a) Three-dimensional image of the R^⁎ hMC4R model. GαCT(340–350)K341L) is indicated in purple. (b) Three-dimensional image zoomed to the area of the E/DRY motif, with interacting residues indicated. The outer helices were removed for clarity. (c) Three-dimensional image zoomed to the area of the NPxxY motif (in yellow); Asp146 and Arg147 are indicated in blue, with interacting residues identified (see the text for further details).The images were generated using PyMOL.

Fig. 1

(a) Three-dimensional image of the R^⁎ hMC4R model. GαCT(340–350)K341L) is indicated in purple. (b) Three-dimensional image zoomed to the area of the E/DRY motif, with interacting residues indicated. The outer helices were removed for clarity. (c) Three-dimensional image zoomed to the area of the NPxxY motif (in yellow); Asp146 and Arg147 are indicated in blue, with interacting residues identified (see the text for further details).The images were generated using PyMOL.

Fig. 2

(a) Three-dimensional structure indicating relevant residues mentioned in this study: Leu106 (ECL1); Asp122, Ile125, and Asp126 (TM3); Cys196 (TM5); Cys257 and His264 (TM6); and Met292 (TM7). The position of the NDP-MSH peptide is presented in purple. A zoomed-in image of the region is presented in (b). The images were produced using PyMOL. (c) A MOE two-dimensional depiction of the interactions involving the core of the peptide (chain B) and the MC4R R^⁎ model (chain A). The two-dimensional interaction caption is presented in (d).

Fig. 2

(a) Three-dimensional structure indicating relevant residues mentioned in this study: Leu106 (ECL1); Asp122, Ile125, and Asp126 (TM3); Cys196 (TM5); Cys257 and His264 (TM6); and Met292 (TM7). The position of the NDP-MSH peptide is presented in purple. A zoomed-in image of the region is presented in (b). The images were produced using PyMOL. (c) A MOE two-dimensional depiction of the interactions involving the core of the peptide (chain B) and the MC4R R^⁎ model (chain A). The two-dimensional interaction caption is presented in (d).

Fig. 3

Interactions between peptide analogues and key residues in the binding pocket. Membrane samples prepared from HEK293 cells expressing the hMC4R with Cys mutations in key positions and incubated separately with 100 nM of each biotinylated cysteine-substituted NDP α-MSH peptide in the absence (T, total binding) or in the presence  (NS, nonspecific binding) of 50 μM SHU9119.

Fig. 4

Detection of WT MC4R cross-linked with the cysteine-substituted peptide analogues. Membrane samples prepared from HEK293 cells expressing the hMC4R were incubated separately with 100 nM of each biotinylated cysteine-substituted NDP α-MSH peptide in the absence (T, total binding) or in the presence  (NS, nonspecific binding) of 50 μM SHU9119. Peptide 1 has cysteine at position 1 of the ligand; peptide 2 has cysteine at position 2, and so forth. Peptide 7L has l-cysteine at position 7, whereas peptide 7D has d-Cys at the position. Cys-to-Cys cross-linking was induced by further incubation in the presence of CuP. Samples were then analyzed by SDS-PAGE, followed by Western blot analysis and detection of biotin using streptavidin polyperoxidase and chemiluminescence.

Fig. 5

Detection of residue on the MC4R that interacts with the N-terminus of the agonist. Membrane samples prepared from HEK293 cells expressing the MC4R were incubated separately with 100 nM of each biotinylated cysteine-substituted NDP-MSH peptide 2 (T, total binding). The WT receptor (lane 1) was also incubated in the presence of 50 μM SHU9119 (NS, nonspecific binding) and 1 mM GTPγS. Peptide 2 has cysteine at position 2 of the NDP-MSH ligand. Samples were then analyzed by SDS-PAGE, followed by Western blot analysis and detection of biotin using streptavidin polyperoxidase and chemiluminescence. The absence of cross-linking is an indication of the position of interaction of peptide 2 and is located at C196 (lane 6).

Fig. 6

Effect of the D189A mutation of the MC4R on MTII and NDP-MSH agonist stimulation. Activity studies of the MC4R were performed in HEK293 cells stably expressing CRE-Luciferase and with transient transfection of the MC4R. Activity is expressed as a percentage of the luminescence response compared to 10 mM forskolin (a direct adenylate cyclase stimulator). The development of the CRE-Luciferase assay was described by Stables et al. Twenty-four hours posttransfection, HEK293 CRE-Luciferase cells were plated and cultured for a further 12–18 h. The medium was replaced, and the appropriate concentration of the agonist NDP-MSH was added. After incubation, Luclite® reagent (Perkin-Elmer) containing the substrate for the luciferase was added to each well. Luciferase activity was determined by scintillation counting. The data are representative of at least three independent experiments performed in triplicate and analyzed in triplicate by one-site competition plots using GraphPad PRISM 3.02 software. NDP-MSH pEC₅₀ values were shifted from 8.27 ± 0.27 to 5.15 ± 0.12 (over 1000-fold) when D189 was mutated to Ala, respectively; however, only a small shift from 7.78 ± 0.13 to 7.33 ± 0.18 (less than 3-fold) was seen for the truncated agonist MTII.

Fig. 7

Detection of residue on the MC4R that interacts with the C-terminus of the agonist. Membrane samples prepared from HEK293 cells expressing the MC4R were incubated separately with 100 nM of each biotinylated cysteine-substituted NDP-MSH peptide 13 in the absence (T, total binding) or in the presence  (N, nonspecific binding) of 50 μM SHU9119 and 1 mM GTPγS. Peptide 13 has cysteine at position 13 of the NDP-MSH ligand. Cysteine-to-cysteine cross-linking was induced by further incubation in the presence of CuP. Samples were then analyzed by SDS-PAGE, followed by Western blot analysis and detection of biotin using streptavidin polyperoxidase and chemiluminescence. The absence of cross-linking is an indication of the position of interaction of peptide 13. Molecular mass is expressed in kilodaltons. The MC4R/peptide complex is located at 37 kDa.