Functional Characterization of Melanocortin-3 Receptor Variants Identify a Loss-of-Function Mutation Involving an Amino Acid Critical for G Protein-Coupled Receptor Activation

Although melanocortin-4 receptor mutations are the cause of the most common monogenic form of obesity, the involvement of the melanocortin-3 receptor (MC3R) in the pathogenesis of obesity is unknown. Earlier studies failed to identify any mutations in obese patients except for the identification of two variants (K6T and I81V) that likely represent polymorphisms. However, a potential mutation (I183N) was recently reported from patients having high-fat contents. We report here the functional characterization of these variants. We show that K6T and I81V have ligand binding and signaling properties similar to wild-type (wt) MC3R, indicating that they are indeed polymorphisms. However, the other variant, I183N, completely lacks signaling in response to agonist stimulation, although it binds ligand with normal affinity and with only slightly decreased capacity. Coexpression of the wt and I183N MC3Rs showed that I183N does not exert dominant-negative activity on wt MC3R. These results provide supporting evidence for the hypothesis proposed in the original case report that MC3R mutation might be a genetic factor that confers susceptibility to obesity, likely due to haploinsufficiency. Further mutations at I183 revealed a discrete requirement for I183 in agonist-induced MC3R activation. The corresponding residue is also important for agonist-induced human melanocortin-4 receptor and lutropin receptor activation. In summary, we identify a residue that is critical for activation of G protein-coupled receptors.