The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling
| Autor: | Ingebrigt Sylte, Claire Bihoreau, Luce Hendricksen, Catherine Robin-Jagerschmidt, Svein G. Dahl, Alain Calvet, Claude Bénicourt |
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| Rok vydání: | 1998 |
| Předmět: |
Models
Molecular Glycosylation Protein Conformation Recombinant Fusion Proteins Molecular Sequence Data Mutant CHO Cells Biology Ligands Biochemistry Protein Structure Secondary chemistry.chemical_compound Endocrinology Cricetinae mental disorders Extracellular Animals Neuropeptide Y Amino Acid Sequence Cloning Molecular Binding site Site-directed mutagenesis Receptor Molecular Biology Binding Sites Base Sequence Cell Membrane Neuropeptide Y receptor Molecular biology humanities Rats Receptors Neuropeptide Y Transmembrane domain chemistry COS Cells Mutagenesis Site-Directed |
| Zdroj: | Molecular and Cellular Endocrinology. 139:187-198 |
| ISSN: | 0303-7207 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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