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 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 alpha-MSH analogues. Compared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-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 alpha-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 alpha-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both 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 tight 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 binding, 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 comprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-turn conformation around Lys6-DPhe7-Arg8. From the above results, we conclude 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 the selectivity of MSH analogues.
Li
S Z
SZ
Department of Internal Medicine, College of Medicine, Yonsei University, Seoul, Korea.
Lee
J H
JH
Lee
W
W
Yoon
C J
CJ
Baik
J H
JH
Lim
S K
SK
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000494:pharmacology*
D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin; D011994:Recombinant Proteins; D000521:alpha-MSH; D000242:Cyclic AMP