Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor 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. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that read-through at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodol. Fluorescence-labeled NK2 mutants contg. an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionally active. 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 in a native membrane environment. These distances confirmed the seven transmembrane topol. for G protein-coupled receptors and detd. a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helixes required for receptor function. [on SciFinder (R)]