C. Robinjagerschmidt et al., THE LIGAND-BINDING SITE OF NPY AT THE RAT Y-1 RECEPTOR INVESTIGATED BY SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING, Molecular and cellular endocrinology, 139(1-2), 1998, pp. 187-198
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
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