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

Chapman KL, Kinsella GK, Cox A, Donnelly D, Findlay JB - J. Mol. Biol. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

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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.
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fig4: 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.

Mentions: Experiments were performed to cross-link the NDP-MSH Cys analogues to the WT receptor. Figure 4 illustrates the cross-linking of peptide analogues to the native receptor when Cys is present near the amino-terminus of the peptide (particularly at position 2) or near the carboxyl-terminus of the peptide (positions 12 and 13). These data suggested that one or more Cys residues in the WT receptor (there are 15 Cys residues in MC4R) make a direct contact with residues close to the amino-terminus and carboxyl-terminus of the agonist NDP-MSH. The Kd values for cysteine substitutions at positions 1, 2, 10, and 11 were not significantly different from the native peptide (Student's t test, p > 0.05; data not shown). In contrast, binding affinity was not readily detectable with cysteine substitutions at positions His-Phe-Arg-Trp (unpublished data).


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

Chapman KL, Kinsella GK, Cox A, Donnelly D, Findlay JB - J. Mol. Biol. (2010)

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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3101337&req=5

fig4: 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.
Mentions: Experiments were performed to cross-link the NDP-MSH Cys analogues to the WT receptor. Figure 4 illustrates the cross-linking of peptide analogues to the native receptor when Cys is present near the amino-terminus of the peptide (particularly at position 2) or near the carboxyl-terminus of the peptide (positions 12 and 13). These data suggested that one or more Cys residues in the WT receptor (there are 15 Cys residues in MC4R) make a direct contact with residues close to the amino-terminus and carboxyl-terminus of the agonist NDP-MSH. The Kd values for cysteine substitutions at positions 1, 2, 10, and 11 were not significantly different from the native peptide (Student's t test, p > 0.05; data not shown). In contrast, binding affinity was not readily detectable with cysteine substitutions at positions His-Phe-Arg-Trp (unpublished data).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

Show MeSH
Related in: MedlinePlus