title: Acidic residues in extracellular loops of the human Y1 neuropeptide Y receptor are essential for ligand binding.
abstract: To investigate whether negatively charged residues of the human Y1 neuropeptide Y (NPY) receptor are required for ligand binding, a series of mutants were constructed in which aspartic acid and glutamic acid residues present in putative extracellular domains of the Y1 receptor were systematically replaced by alanines. The mutant cDNAs were transiently expressed in HeLa cells using a vaccinia virus-derived expression system, and their ability to bind NPY was evaluated. The level of expression of mutants unable to bind NPY was also tested immunologically. In addition, the ability of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. Substitution of aspartic acids and glutamic acids of the N-terminal first extracellular domain had no effect on binding. On the other hand, substitution of acidic residues present in the second, third, and fourth extracellular loops resulted in proteins unable to bind 125I-NPY. These results demonstrate that the extracellular loops of the human Y1 NPY receptor are essential portions of its ligand binding domain.
authors.affiliation: Division of Hypertension, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Walker
authors.forename: M
authors.initials: M
authors.lastname: Munoz
authors.forename: R
authors.initials: R
authors.lastname: Martinez
authors.forename: M C
authors.initials: MC
authors.lastname: Peitsch
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000378:metabolism*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
chemical_list: D008024:Ligands; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y