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 chemically 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 readthrough at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodology. Fluorescence-labeled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionnally active. 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 in a native membrane environment. These distances confirmed the seven transmembrane topology for G protein-coupled receptors and determined 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 helices required for receptor function.
Turcatti
G
G
Geneva Biomedical Research Institute, Glaxo Wellcome, Switzerland.
Nemeth
K
K
Edgerton
M D
MD
Knowles
J
J
Vogel
H
H
Chollet
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D018041:Receptors, Neurokinin-2 / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines; D014981:Xenopus
D005456:Fluorescent Dyes; D008024:Ligands; D018041:Receptors, Neurokinin-2; D012235:Rhodamines; C005358:tetramethylrhodamine