MOLECULAR MODELING OF THE NPY BINDING-SITE ON THE Y-1 RECEPTOR

A three-dimensional model of the neuropeptide Y (NPY) - rat Y-1 (rY(1) ) receptor complex and of the NPY13-36 - rY(1) receptor complex was co nstructed by molecular modeling based on the electron density projecti on map of rhodopsin and on site-directed mutagenesis studies of neurop eptide receptors. In order to further guide the modeling, the nucleoti de sequences encoding Trp287, Cys295 and His297 in the third extracell ular loop of the rY(1) receptor, were altered by site-directed mutagen esis experiments. Single-point mutated receptors were expressed in COS -7 cells, and tested for their ability to bind radio labelled NPY (H-3 -NPY). Mutations of Trp287 and His297 completely abolished binding of H-3-NPY. The Cys295Ser mutation only slightly decreased the binding of H-3-NPY, suggesting that the involvement of Cys295 in a disulphide bo nd is not essential for maintaining the correct three-dimensional stru cture of the binding site for NPY. Molecular dynamics simulations of N PY-rY(1) receptor interactions suggested that Asp199, Asp103 and Asp28 6 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 o f NPY. The simulations also suggested that His297 acts as a hydrogen a cceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a bindi ng pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests t hat amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in bindin g to NPY and NPY13-36.