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Volume 271, Number 33, Issue of August 16, 1996 pp. 19991-19998
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Probing the Structure and Function of the Tachykinin Neurokinin-2 Receptor through Biosynthetic Incorporation of Fluorescent Amino Acids at Specific Sites

(Received for publication, February 22, 1996, and in revised form, May 16, 1996)

Gerardo Turcatti , Karin Nemeth , Michael D. Edgerton , Ulrich Meseth , François Talabot , Manuel Peitsch , Jonathan Knowles , Horst Vogel and André Chollet

From the Geneva Biomedical Research Institute, Glaxo Wellcome, CH-1228 Geneva, Switzerland and the  Institut de Chimie-physique, Ecole Polytechnique Fédérale, CH-1015 Lausanne, Switzerland

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels.

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