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 hormone (MCH) peptide was constructed and docked into a helical, bacteriorhodopsin-based model of the recently identified human MCH receptor. From this hormone-receptor com plex, potential sites of agonist-receptor interaction were identified, and site-directed mutagenesis was used to substitute residues predicted to resi de within the receptor binding pocket. Substitution of Asp(123) (3.32) in t he third transmembrane domain of the receptor resulted in a loss of detecta ble I-125-MCH binding and of MCH-stimulated Ca2+ flux; cell surface express ion of the mutant receptor was not affected. Arg(11) and Arg(14) of the MCH ligand were identified as potential sites of interaction with Asp(123) (3. 32). [Ala(14)]-MCH was equipotent to native MCH in its ability to bind to a nd activate the wild-type MCH receptor, whereas [Ala(11)]-MCH displayed a 3 000-fold reduction in binding affinity and a complete loss of measurable fu nctional activity. Furthermore, [Lys(11)]-MCH and [D-Arg(11)]-MCH displayed reduced affinity for the receptor. [Lys(11)]-MCH was observed to be a part ial agonist, eliciting approximately 67% of the native peptide's activity i n a Ca2+ flux assay, and [D-Arg(11)]-MCH was determined to be a functional antagonist with a K-b valve of 15.8 mu M. These data provide evidence that a basic moiety with specific stereochemical requirements at this site is ne eded for receptor activation. We conclude that both Asp(123) (3.32) in the MCH receptor and Arg(11) in the MCH peptide are required for the formation of the MCH peptide/receptor complex and propose that they form a direct int eraction that is critical for receptor function.