Type I beta-turn conformation is important for biological activity of the melanocyte-stimulating hormone analogues

In order to define which structure of alpha-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and s electivity, we analysed receptor-binding and cAMP-generating activity in Ch inese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized alpha-MSH analogues. C ompared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-MSH-ND re vealed a preference for the MC4R, whereas its IC50 and EC50 values were com parable to those of MTII reported previously. Truncation of Ahx4 and Asp5 o f alpha-MSH-ND remarkably decreased the receptor-binding and cAMP-generatin g activity. Meanwhile, maximum cAMP-generating activity was observed at a h igher concentration (10(-5) M) of alpha-MSH-ND(6-10), and MC4R preference w as changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to bo th receptors. Whereas the solution conformation of alpha-MSH-ND revealed a stable type I beta-turn structure, [Gln6]alpha-MSH-ND(6-10) revealed a tigh t gamma-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of alpha-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor bin ding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]alpha-MSH-ND exhibited a stable type I' beta-turn co mprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly r educed binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-t urn conformation around Lys6-DPhe7-Arg8. From the above results, we conclud e that a type I beta-turn conformation comprising the residues Asp5-His6-(D -Phe7)-Arg8 was important for receptor binding and activation, as well as t he selectivity of MSH analogues.