ACIDIC RESIDUES IN EXTRACELLULAR LOOPS OF THE HUMAN-Y1 NEUROPEPTIDE-YRECEPTOR ARE ESSENTIAL FOR LIGAND-BINDING

To investigate whether negatively charged residues of the human Y1 neu ropeptide Y (NPY) receptor are required for ligand binding, a series o f mutants were constructed in which aspartic acid and glutamic acid re sidues present in putative extracellular domains of the Y1 receptor we re systematically replaced by alanines. The mutant cDNAs were transien tly expressed in HeLa cells using a vaccinia virus-derived expression system, and their ability to bind NPY was evaluated. The level of expr ession 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 asp artic acids and glutamic acids of the N-terminal first extracellular d omain had no effect on binding. On the other hand, substitution of aci dic residues present in the second, third, and fourth extracellular lo ops resulted in proteins unable to bind I-125-NPY. These results demon strate that the extracellular loops of the human Y1 NPY receptor are e ssential portions of its ligand binding domain.