The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling.
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.
Robin-Jagerschmidt
C
C
Jouveinal, Fresnes, France.
Sylte
I
I
Bihoreau
C
C
Hendricksen
L
L
Calvet
A
A
Dahl
S G
SG
Bénicourt
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane; D003001:Cloning, Molecular; D006224:Cricetinae; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D017476:Receptors, Neuropeptide Y / Q000032:analysis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins
D008024:Ligands; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C097212:neuropeptide Y-Y1 receptor