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 rec eptor by suppression of UAG nonsense codons with the aid of a chemical ly misacylated synthetic tRNA specifically designed for the incorporat ion of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra-or intrac ellular loops and proximal transmembrane domains established that read through at some UAG sites may represent a limitation to the range of a pplicability of the nonsense suppression methodology. Fluorescence-lab eled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl- diaminopropionic acid residue at known sites were shown to be function nally active, Intermolecular distances were determined by measuring th e fluorescence resonance energy transfer (FRET) between the fluorescen t unnatural amino acid and a fluorescently labeled NK2 heptapeptide an tagonist in a native membrane environment. These distances confirmed t he seven transmembrane topology for G protein-coupled receptors and de termined 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 p acking of the helices required for receptor function.