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

• Published in: Biochemical journal 2006-01, Vol.393 (Pt 1), p.161-169
• Main Authors: Sjödin, Paula, Holmberg, Sara K S, Akerberg, Helena, Berglund, Magnus M, Mohell, Nina, Larhammar, Dan
• Format: Article
• Language: English
• Subjects: Arginine - analogs & derivatives; BIBP3226; Binding Sites; Cell Line; Conserved Sequence; G-protein-coupled receptor (GPCR); Humans; Ligands; MEDICIN; MEDICINE; Models, Molecular; mutagenesis; Mutagenesis, Site-Directed; Naphthalenes; neuropeptide Y (NPY); Protein Binding; Protein Conformation; Pyrrolidines; receptor Y1; Receptors, Neuropeptide Y - agonists; Receptors, Neuropeptide Y - antagonists & inhibitors; Receptors, Neuropeptide Y - genetics; Receptors, Neuropeptide Y - metabolism; three-dimensional model
• ISSN: 0264-6021; 1470-8728; 1470-8728
• DOI: 10.1042/BJ20050708

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.