Novel binding motif of ACTH analogues at the melanocortin receptors

The melanocortin receptor (MCR) subtype family is a member of theGPCRsuperfamily, and each of them has a different pharmacological profile with regard to the relative potency of the endogenous and synthetic melanocortin peptides. α-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R, and MC5R. In this study, we examined the role of Phe⁷ in ACTH on human (h) MC1R, MC3R, and MC4R binding and signaling. Our results indicate that substitution of Phe⁷ with D-Nal(2′)⁷ in ACTH1-24 yields a pharmacological profile different from that for substitution of Phe7 with D-Nal(2′)⁷ in MSH in hMC1R, hMC3R, and hMC4R. N-D-Nal(2′)⁷-ACTH1-24 is an agonist at hMC3R and hMC4R which did not change the peptide from an agonist to an antagonist at hMC3R and hMC4R. Further experiments indicate that N-D-Nal(2′) ⁷-ACTH1-17 is the minimal peptide required for hMC3Rand hMC4R activation. Single-amino acid substitution studies of D-Nal(2′) ⁷-ACTH1-17 indicate that amino acid residues 15-17 in N-D-Nal(2′)⁷-ACTH1-17 are crucial for hMC3R and hMC4R activation. Substitutions of these amino acid residues reduced or abolished agonist activity at hMC3R and hMC4R. Conformational studies revealed a new β-turn (Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹) in N-D-Nal(2′)⁷-ACTH1-17, compared to the β-turn-like structure at NDP-α-MSH (His⁶-D-Phe⁷-Arg ⁸-Trp⁹). Our results suggest that NDP-α-MSH and N-D-Nal(2′)⁷-ACTH1-17 do not share the same binding site; the highly basic C-terminal fragment (Lys¹⁵-Lys¹⁶-Arg ¹⁷) of N-D-Nal(2′)⁷-ACTH1-17 induced a new β-turn, and this shift contributed the selective agonist activity at hMC3R and hMC4R.