GPC receptors and not ligands decide the binding mode in neuropeptide Y multireceptor/multiligand system

Many G protein-coupled receptors belong to families of different receptor subtypes, which are recognized by a variety of distinct ligands. To study such a multireceptor/multiligand system, we investigated the Y-receptor family. This family consists of four G protein-coupled Y receptors in humans (hY(1)R, hy(2)R, hy(4)R, and hy(5)R) and is activated by the so-called NPY hormone family, which itself consists of three native peptide ligands named neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY). The hy(5)R shows high affinity for all ligands, although for PP binding, the affinity is slightly decreased. As a rational explanation, we suggest that Tyr(27) is lost as a contact point between PP and the hY(5)R in contrast to NPY or PYY. Furthermore, several important residues for ligand binding were identified by the first extensive mutagenesis study of the hy(5)R. Using a complementary mutagenesis approach, we were able to discover a novel interaction point between hY(5)R and NPY. The interaction between NPY(Arg(25)) and hy(5)R(ASp(2.68)) as well as between NPY(Arg(33)) and hy5R(ASp(6.59)) is maintained in the binding of PYY and PP to hY5R but different to the PP-hY(4)R and NPY-hY(1)R contact points. Therefore, we provide evidence that the receptor subtype and not the pre-orientated conformation of the ligand at the membrane decides the binding mode. Furthermore, the first hY(5)R model was set up on the basis of the crystal structure of bovine rhodopsin. We can show that most of the residues identified to be critical for ligand binding are located within the now postulated binding pocket.