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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 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.
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fig7: 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.

Mentions: In the model, the C-terminus of the peptide ligand is close to the extracellular end of TM6 and TM7, and ECL3 (Fig. 7). Western blot analysis of peptide 13 demonstrates cross-linking of the peptide to all of the mutant receptors, except for C257A-MC4R, implying that the carboxyl-terminus interacts with TM6 (Fig. 3). The Cys(6.47) residue is located on TM6 of the MC4R (Cys257) just below the hydrophobic pocket (Trp257, Phe261, Tyr268, and Ile269). It is conceivable that due to TM6 flexibility, Cys257 in the MC4R may be more accessible in the active state of the receptor.


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 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.
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Related In: Results  -  Collection

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

fig7: 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.
Mentions: In the model, the C-terminus of the peptide ligand is close to the extracellular end of TM6 and TM7, and ECL3 (Fig. 7). Western blot analysis of peptide 13 demonstrates cross-linking of the peptide to all of the mutant receptors, except for C257A-MC4R, implying that the carboxyl-terminus interacts with TM6 (Fig. 3). The Cys(6.47) residue is located on TM6 of the MC4R (Cys257) just below the hydrophobic pocket (Trp257, Phe261, Tyr268, and Ile269). It is conceivable that due to TM6 flexibility, Cys257 in the MC4R may be more accessible in the active state of the receptor.

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