The Bioactive Conformation of Neuropeptide Y Analogues at the Human Y₂‐Receptor

Abstract

Several attempts to investigate the bioactive conformation of neuropeptide Y have been made so far. As cyclic peptides are much more rigid than linear ones, we decided to synthesise cyclic analogues of the C-terminal dodekapeptide amide neuropeptide Y Ac-25-36. Cyclisation was performed by side chain lactamisation of ornithine or lysine and glutamic or aspartic acid. The affinity of the 19 peptides ranged from K, 0.6 nM to greater than 10000 nM. We found that the size, position, orientation, configuration, and the location of the cycle plays an important role for receptor recognition. Circular dichroic studies have been performed to characterise the secondary structure of each peptide. Receptor binding studies were carried out on human neuroblastoma cell lines SK-N-MC (Yl) and SMS-KAN (Y,), and on rabbit kidney membranes (Y2).The pharmacological and spectral data showed that the a-helix content was not the predominant factor for high Y,-receptor affinity. Instead, the location and the size of the hydrophobic lactam bridge, and the conserved C-terminal tetrapeptide (Arg-Glu-Arg-Tyr) seemed to be the main parameters.Using molecular dynamics, the structures of four cyclic peptides (i,i+4) have been investigated and compared with the previously published NMR structure of one of the cyclic peptide analogues. Significant differences have been found in the overall three-dimensional fold of the peptides. The distances between the N-and the C-terminus allow discrimination between peptides with high binding affinity and those with low binding affinity, because of the correlation that was found with the measured affinity. Thus, this study suggests that a turn-like structure and the orientation of the C-terminus towards the N-terminus play major roles for high affinity binding of cyclic dodecapeptides to the Y,-receptor.None of the cyclic segments exhibits significant affinity to the Y,-receptor. Thus, these results support the hypothesis of a discontinuous binding site of neuropeptide Y at the Y ,-receptor. Gerald et al., 1995;Gehlert et al., 1996a), Y n P , - (Bard et al., 1995;Lundell et al., 1995), Y,- (Gerald et al., 1996), and Y,- (Weinberg et al., 1996) receptors have been cloned recently and it was shown that they Dedication. Dedicated to Prof. Dieter Seebach o n the occasion of his 60th birthday.Note. The designation of peptide analogues used in this study is summarised in Table 1. ETH Zurich, Winterthurer Str. 190, CH-8057 Zurich, Switzerland all belong to the G-protein-coupled hormone family (BeckSickinger, 1996). Whereas NPY shows high affinity to Yl-, Yz-, Ys-, and Y,-receptors, pancreatic polypeptide (PP) has been found to be the endogenous ligand of the Y,-receptor, which is also called the PP,-receptor (Gehlert et al., 1996b).The Y,-receptor subtype is particularly found in the peripheral nervous system. Moreover, Y ,-receptors have been identified in coronary vessels (guinea pigs, rats) and veins (guinea pigs). All available evidence suggests that the peripheral bloodpressure-elevating effects of NPY are m...

Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family

“…It was concluded that high affinity is correlated with a tight hairpin, where the N-and C-terminal ends are very close to each other. In contrast, a more open conformation is characterized by minor affinity [88]. This suggests that the bioactive conformation of the ligand at the Y 2 -receptor should consist of a tightly closed structure in which the N-and C-terminal parts are close and oriented to each other in a well-defined way.…”

“…In order to stabilize the conformation of the Cterminal dodecapeptide 25 -36 of NPY, Rist and coworkers [88] introduced a lactam bridge of the type i− i+4 and varied its position along the sequence (from i= 25 to i =28) as well as its orientation (CO NH or NH CO) and size (Lys/Glu or Orn/Asp) ( Table 2). The two most potent ligands at the Y 2 -receptor, corresponding to the lactamizations Glu 27 -Lys 31 and Lys 28 -Glu 32 , showed an affinity of 0.8 and 0.6 nM, respectively.…”

Biophysical methods to study ligand-receptor interactions of neuropeptide Y

“…The ability of NPY to bind to so many different receptor subtypes can be explained by its high molecular flexibility. An attempt of investigating the local structural features that distinguish the bioactive conformations of NPY at the different receptors has been performed in our group some years ago 99, 100. The structural freedom of the C‐terminal dodecapeptide was restricted by cyclization.…”

Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK‐N‐MC

“…These SK-N-MC cells selectively expresses one kind of NPY receptors which was shown to be the Y1 receptor [7,8]. Furthermore, these cells are frequently used to investigate binding as well as signal transduction of neuropeptide Y [9].Internalization studies are based on the distinction between the cell surface associated ligand and the ligand within the cell. Intact living cells are incubated with the labeled ligand at a temperature between 30 and 37 8C.…”

“…These SK-N-MC cells selectively expresses one kind of NPY receptors which was shown to be the Y1 receptor [7,8]. Furthermore, these cells are frequently used to investigate binding as well as signal transduction of neuropeptide Y [9].…”