Infoscience

Journal article

Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites

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 chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants contg. 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. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calcd. by the theory of Forster, permit to fix the ligand in space and define the structure of the receptor in a mol. 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 exptl. distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the anal. of spatial relationships in integral membrane proteins such as receptors or channels. [on SciFinder (R)]

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