The melanocortin receptors (MCRs)
are important for numerous biological
pathways, including feeding behavior and energy homeostasis. In addition
to endogenous peptide agonists, this receptor family has two naturally
occurring endogenous antagonists, agouti and agouti-related protein
(AGRP). At the melanocortin-4 receptor (MC4R), the AGRP ligand functions
as an endogenous inverse agonist in the absence of agonist and as
a competitive antagonist in the presence of agonist. At the melanocortin-3
receptor (MC3R), AGRP functions solely as a competitive antagonist
in the presence of agonist. The molecular interactions that differentiate
AGRP’s inverse agonist activity at the MC4R have remained elusive
until the findings reported herein. Upon the basis of homology molecular
modeling approaches, we previously postulated a unique interaction
between the D189 position of the hMC4R and Asn114 of AGRP. To further
test this hypothesis, six D189 mutant hMC4Rs (D189A, D189E, D189N,
D189Q, D189S, and D189K) were generated and pharmacologically characterized
resulting in the discovery of differences in inverse agonist activity
of AGRP and an 11 macrocyclic compound library. These data support
the hypothesized interaction between the hMC4R D189 position and Asn114
residue of AGRP and define critical ligand–receptor molecular
interactions responsible for the inverse agonist activity of AGRP
at the hMC4R.