THE LIGAND-BINDING SITE OF NPY AT THE RAT Y-1 RECEPTOR INVESTIGATED BY SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING

The ligand binding site of neuropeptide Y (NPY) at the rat Y-1 (rY(1), ) receptor was investigated by construction of mutant receptors and [H -3]NPY binding studies. Expression levels of mutant receptors that did not bind [H-3]NPY were examined by an immunological method. The singl e mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abol ished [H-3]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [H-3]NPY binding. Simil arly, the double mutation Leu34Arg/Asp199Ala totally abrogated the bin ding of [H-3]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [H-3]NPY 2.7- and 5.2-fold, respectively, The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affecte d [H-3]NPY binding. A receptor with a deletion of the segment Asn2-Glu 20 or with simultaneous mutations of the three putative N-terminal gly cosylation sites; displayed no detectable [H-3]NPY binding, due to abo lished 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 bind ing of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a pr oper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial f or correct expression of the rY(1) receptor at the cell surface. (C) 1 998 Elsevier Science Ireland Ltd. All rights reserved.