Molecular characterization of the melanin concentrating hormone/receptor complex: Identification of critical residues involved in binding and activation

A molecular model of the human melanin-concentrating hor-mone (MCH) peptide was constructed and docked into a heli-cal, 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. Substi-tution 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 Ca21 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