In order to define which structure of α-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and selectivity, we analysed receptor-binding and cAMP-generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized α-MSH analogues. Compared with [Ahx4]α-MSH, the linear MTII designated as α-MSH-ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of α-MSH-ND remarkably decreased the receptor-binding and cAMP-generating activity. Meanwhile, maximum cAMP-generating activity was observed at a higher concentration (10-5 M) of α-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]α-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of α-MSH-ND revealed a stable type I β-turn structure, [Gln6]α-MSH-ND(6-10) revealed a tight γ-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of α-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]α-MSH-ND exhibited a stable type I' β-turn comprising Asp5, Gln6, D- Phe7 and Arg8. [Lys6]α-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]α-MSH-ND also demonstrated a γ-turn conformation around Lys6-DPhe7- Arg8. From the above results, we conclude that a type I β-turn conformation comprising the residues Asp5-His6-(D-Phe7)-Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.
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