Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions.

Neuropeptide Y (NPY) and peptide YY (PYY) share approximately 70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share approximately 30% sequence identity. Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i.e. Tyr2.64, Val6.58 and Tyr7.31. Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3-36) and NPY(13-36) as well as the non-peptide antagonist BIIE0246. Results from mutants where Tyr2.64 has been substituted by Ala suggest that Tyr2.64 is involved in the interaction with all investigated ligands whereas position Tyr7.31 seems to be more important for interaction with the truncated peptide PYY(3-36) than with intact NPY. Surprisingly, substitution of Tyr7.31 with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY. The third position, Val6.58, did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr2.64 and Tyr7.31 in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1. This suggests that the receptor-ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.

Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region.

The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a K(d) of 0.80 +/- 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a K(d) of 0.14 +/- 0.01 nm (mean +/- SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.

Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study.

Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions. The present study was triggered by contradictions among previously published reports and conclusions that seemed inconsistent with sequence comparisons across species and receptor subtypes. Our results show that Asp287, at the border between TM (transmembrane) region 6 and EL3 (extracellular loop 3) influences peptide binding, while two aspartic residues in EL2 do not, in agreement with some previous studies but in disagreement with others. A hydrophobic pocket of the Y1 receptor consisting of Tyr100 (TM2), Phe286 (TM6) and His298 (EL3) has been proposed to interact with the amidated C-terminus of NPY, a theory that is unsupported by sequence comparisons between Y1, Y2 and Y5. Nevertheless, our results confirm that these amino acid residues are critical for peptide binding, but probably interact with NPY differently than proposed previously. Studies with the Y1-selective antagonist SR120819A identified a new site of interaction at Asn116 in TM3. Position Phe173 in TM4 is also important for binding of this antagonist. In contrast with previous reports, we found that Phe173 is not crucial for the binding of BIBP3226, another selective Y1 receptor antagonist. Also, we found that position Thr212 (TM5) is important for binding of both antagonists. Our mutagenesis results and our three-dimensional model of the receptor based on the high-resolution structure of bovine rhodopsin suggest new interactions for agonist as well as antagonist binding to the Y1 receptor.