Complete L‐Alanine Scan of Neuropeptide Y Reveals Ligands Binding to Y₁ and Y₂ Receptors with Distinguished Conformations

Abstract

The synthesis of more than fifty 36-residue oligopeptide analogs of neuropeptide Y (NPY) and their affinity to human Y, and Y, receptors is described. Each amino acid of the natural sequence was replaced by L-alanine, the four alanine residues at position 12, 14, 18 and 23 were replaced by glycine. Additional residues .were exchanged to closely related ones in order to characterize the prerequisites for binding. A combination of automated single and multiple peptide synthesis using fluoren-9-ylmethoxycarbonyl/tert-butoxy strategy was applied. The purified peptides were characterized by electrospray mass spectrometry, analytical HPLC and amino acid analysis. Binding was investigated by displacement of '251-labelled neuropeptide Y from human neuroblastoma cell lines Whereas Pro2 and the integrity of the neuropeptide Y loop is important for the binding to the Y, receptor, exchanges within the C-terminal helix affect the affinity to the Y, receptor. The Cterminal pentapeptide amide is important for both receptors and probably represents the binding site. However, Arg33 and Arg35 may not be exchanged by L-alanine in the Y, system, whereas Arg35 and Tyr36 are the most susceptible residues in the Y, system. In order to distinguish between conformational effects and direct hormone/receptor interaction via the side chains of neuropeptide Y, circular dichroic studies of the alanine-containing peptides were performed and structure affinity relationships are discussed.Comparing the affinities of the neuropeptide Y analogs to Y, and Y, receptors significant differences were found for the two binding sites, which suggests a different active conformation of neuropeptide Y at the two subtypes of receptors. Using molecular dynamics calculations, two distinct conformations were identified which are in good agreement with the data obtained by structure/ affinity investigations. SK-N-MC and SMS-KAN.Neuropeptide Y (NPY) is a 36-amino-acid amide which exhibits a high similarity to the pancreatic polypeptide and peptide YY. It was isolated from pig brain and sequenced (Tatemoto) in 1982.Information about the secondary structure of pancreatic polypetptide hormones has been obtained through the work of Blundell and co-workers who performed X-ray analyses on avian (turkey) pancreatic polypeptide Wood, 1982., Glover et al., 1983). Residues 1-8 of the crystalline avian pancreatic polypeptide form a type I1 proline helix followed by a loop (residues 9-14) and an a-helical segment (residues 15-32). The four C-terminal amino acids adopt a relatively flexible loop-like conformation. Hydrophobic interactions of the proline helix and the amphiphilic ahelix lead to the hairpin structure. Comparative circular dichroic measurements of different pancreatic polypeptides and Correspondence to A. G.

Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor

“…We and others have shown recently that smaller centrally or N-terminally truncated analogs such as [ NPY are full Y2 receptor agonists [9] or show at least good binding properties [12][13][14][15]. In addition, the L-Ala scan of NPY 1-36 suggests that only the C-terminal amino acids are involved in receptor binding and we speculated that the N-terminal residues only serve for the stabilization of the molecule [16]. We have now succeeded in developing a small molecule, which only consists of the C-terminal 12 amino acids, and which has been stabilized by an intramolecular lactam bridge.…”

“…1). These residues had been identified by means of the c-alanine scan not to contribute to the binding of the peptide [16]. Furthermore, activity studies have been performed: as previously described [10], bath applications of NPY cause concentration-dependent, reversible inhibitions of Ca 2+ channel current amplitudes in SH-SY5Y human neuroblastoma cells …”

“…Membrane suspensions were prepared as described previously [16]. propionyl-NPY (3H-NPY) and different concentrations of the peptide in a total volume of 250 ~tl for 2 h at room temperature as described recently [16].…”

Novel analogues of neuropeptide Y with a preference for the Y1‐receptor

“…10 000 nm) at the Y 1 -receptor [44]. Exchange of each residue of NPY individually by l-Ala revealed that the most sensitive positions are: Pro2, Pro5, Arg19, Tyr20 and the C-terminal positions 27±36, especially the two Arg residues at positions 33 and 35 [45]. The importance of the Pro residues 2 and 5 and of the Tyr residues 20 and 27 is probably due to their role in stabilizing the hairpin-like structure of the hormone by means of a hydrophobic core [46].…”

“…This suggests that important positions are sensitive to both side chain orientation and to the side chain itself. Although the Ala scan of NPY showed no essential importance of the central positions of NPY for the binding to the Y 1 -receptor [45], central truncation of NPY by connecting the N-and C-terminus by a spacer (e.g. 6-amino hexanoic acid) led, in almost all cases, to a significant reduction or complete loss of affinity [48].…”