RT Journal Article
SR Electronic
T1 Molecular Characterization of the Melanin-Concentrating Hormone/Receptor Complex: Identification of Critical Residues Involved in Binding and Activation
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 217
OP 225
DO 10.1124/mol.58.1.217
VO 58
IS 1
A1 Douglas Macdonald
A1 Nicholas Murgolo
A1 Rumin Zhang
A1 James P. Durkin
A1 Xiaorui Yao
A1 Catherine D. Strader
A1 Michael P. Graziano
YR 2000
UL http://molpharm.aspetjournals.org/content/58/1/217.abstract
AB A molecular model of the human melanin-concentrating hormone (MCH) peptide was constructed and docked into a helical, bacteriorhodopsin-based model of the recently identified human MCH receptor. From this hormone-receptor complex, potential sites of agonist-receptor interaction were identified, and site-directed mutagenesis was used to substitute residues predicted to reside within the receptor binding pocket. Substitution of Asp123(3.32) in the third transmembrane domain of the receptor resulted in a loss of detectable 125I-MCH binding and of MCH-stimulated Ca2+ flux; cell surface expression of the mutant receptor was not affected. Arg11 and Arg14 of the MCH ligand were identified as potential sites of interaction with Asp123(3.32). [Ala14]-MCH was equipotent to native MCH in its ability to bind to and activate the wild-type MCH receptor, whereas [Ala11]-MCH displayed a 3000-fold reduction in binding affinity and a complete loss of measurable functional activity. Furthermore, [Lys11]-MCH and [d-Arg11]-MCH displayed reduced affinity for the receptor. [Lys11]-MCH was observed to be a partial agonist, eliciting approximately 67% of the native peptide's activity in a Ca2+ flux assay, and [d-Arg11]-MCH was determined to be a functional antagonist with a K b valve of 15.8 μM. These data provide evidence that a basic moiety with specific stereochemical requirements at this site is needed for receptor activation. We conclude that both Asp123(3.32) in the MCH receptor and Arg11 in the MCH peptide are required for the formation of the MCH peptide/receptor complex and propose that they form a direct interaction that is critical for receptor function.