The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling

• Published in: Molecular and cellular endocrinology Vol. 139; no. 1-2; pp. 187 - 198
• Main Authors: Robin-Jagerschmidt, C, Sylte, I, Bihoreau, C, Hendricksen, L, Calvet, A, Dahl, S G, Bénicourt, C
• Format: Journal Article
• Language: English
• Published: Ireland 30.04.1998
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cell Membrane; CHO Cells; Cloning, Molecular; COS Cells; Cricetinae; Ligands; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Neuropeptide Y - chemistry; Neuropeptide Y - genetics; Protein Conformation; Protein Structure, Secondary; Rats; Receptors, Neuropeptide Y - analysis; Receptors, Neuropeptide Y - chemistry; Recombinant Fusion Proteins
• ISSN: 0303-7207
• DOI: 10.1016/S0303-7207(98)00060-4

The ligand binding site of neuropeptide Y (NPY) at the rat Y1 (rY1,) receptor was investigated by construction of mutant receptors and [3H]NPY binding studies. Expression levels of mutant receptors that did not bind [3H]NPY were examined by an immunological method. The single mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abolished [3H]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [3H]NPY binding. Similarly, the double mutation Leu34Arg/Asp199Ala totally abrogated the binding of [3H]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [3H]NPY 2.7- and 5.2-fold, respectively. The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affected [3H]NPY binding. A receptor with a deletion of the segment Asn2-Glu20 or with simultaneous mutations of the three putative N-terminal glycosylation sites, displayed no detectable [3H]NPY binding, due to abolished expression of the receptor at the cell surface. Taken together, these results suggest that amino acids in the N-terminal part as well as in the first and second extracellular loops are important for binding of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a proper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial for correct expression of the rY1 receptor at the cell surface.