Functions of acidic transmembrane residues in human melanocortin-3 receptor binding and activation

The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis, inflammation, and cardiovascular function. Inactivating mutations in MC3R gene are associated with childhood obesity. How MC3R binds to its ligands has rarely been studied. In the present study, we systematically mutated all ten acidic residues in transmembrane (TM) domains and measured the cell surface expression levels as well as ligand binding and signaling properties of these mutants. Our results showed that of the 19 mutants stably expressed in HEK293 cells, all were expressed on the cell surface, although some mutants had decreased levels of cell surface expression. We showed that with the superpotent analog [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (MSH), E92, E131, D154, D158, D178, and D332 are important for ligand binding. D121 and D332 are important for binding and signaling. Further experiments using other ligands such as D-Trp(8)-gamma-MSH, alpha-MSH and gamma-MSH showed that different ligands induce or select different conformations. In summary, we showed that acidic residues in TMs 1 and 3 are important for ligand binding whereas the acidic residues in TMs 2 and 7 are important for both ligand binding and signaling.