Receptor-antagonist interactions in the complexes of agouti and agouti-related protein with human melanocortin 1 and 4 receptors

The molecular interactions between human melanocortin receptor-1 and -4 (hMCIR and hMC4R) and their endogenous antagonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP), were assessed by studying the effects of site-directcd mutations on the binding affinity of I-125-ASIP[90-132(L89Y) and I-125-AGRP(86-132). Mutations of homologous residues from transmembrane helices (TMHs) 3 and 6 and extracellular loop (EL) 3 (D121A, T124A, F257A, and F277M in hMCIR and D126A, I129A F261A, and M281F in hMC4R) impaired binding of both antagonists to hMC4R and binding of the ASIP fragment to hMCIR. However, the mutations in TMH2 (E94A in hMCIR and E100A in hMC4R), TMH7 (F280A in hMCIR and F284A in hMC4R), and EL2 (Y183S, H184S, and D184H in hMCIR) only significantly affected binding of the ASIP fragment. The dependence of agonist binding on the dithiothreitol concentration followed a monophasic curve for wild-type hMC4R and its C40A, C271A, and C279A mutants and a biphasic curve for hMClR, suggesting the presence of at least one structurally and functionally essential disulfide bond in both wild-type receptors and the hMC4R mutants. Models of complexes of both receptors with the ASIP fragment and hMC4R with the AGRP fragment were calculated using constraints from the experimental structures of rhodopsin and AGRP fragments, a set of deduced hydrogen bonds, supplemented by two proposed disulfide bridges and receptor-ligand contacts, derived from our mutagenesis data. In the models of the ASIP fragment complexed with both receptors, the core ligand tripeptide, Arg-Phe-Phe, positioned between TMHs 3 and 6, is shifted toward TMHs 2 and 7 relative to its position in the AGRP-hMC4R model, while the N-terininal loop and two central disulfides of the antagonists interact with EL2 of the receptors.