Molecular Characterization of the Melanin-Concentrating Hormone/Receptor Complex: Identification of Critical Residues Involved in Binding and Activation

Abstract

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.