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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.

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.

We have investigated the interaction of fluorescent peptide ligands with the G protein-coupled receptor NK2 using novel spectrofluorometric approaches. Several heptapeptide antagonists of structure PhCO-Xaa-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 were labelled on position 1 (Xaa) with the environment-sensitive nitrobenzoxadiazole (NBD) probe, differing only in the length of the spacer between the NBD group and the peptide. Upon binding of the labelled antagonist to NK2 receptors stably expressed in Chinese hamster ovary (CHO) cells, an increase in NBD fluorescence was observed when the spacer length was less than 10 A. Collisional quenching experiments using iodide and Co2+ ions were performed to define the accessibility of the NBD group on bound ligands to the solvent. By comparing ligands with spacer arms of varying lengths, we found that the binding pocket is buried at a depth of 5-10 A. In contrast, N-terminally NBD-labelled agonists, decapeptide neurokinin A (NKA) or heptapeptide Nle10-NKA[4-10], bound to the NK2 receptor were accessible to the solvent. Binding of fluorescent ligands to the NK2 receptor was accompanied by an enhancement in the fluorescence anisotropy. The changes in fluorescence properties were used to determine the kinetic parameters of antagonist binding and dissociation. These results indicate that the binding site on the NK2 receptor for the amino-terminal end of the heptapeptide antagonists is buried in the hydrophobic pocket of the receptor protein and clearly distinct from the binding site for the amino-terminal end of agonists, which is accessible to the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Several fluorescent probes for the NK2 receptor were designed, synthesized, and pharmacologically characterized. These fluorescent ligands are analogues of the selective NK2 heptapeptide antagonist N-alpha-benzoyl-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1, GR94800). They were obtained by substitution of 2,n-diaminoalkyl amino acid (n = 3-6) for Ala1 and the subsequent coupling of the fluorophore NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) or fluoresceinthiocarbamyl to the N-omega amino group. The fluorescent derivatives retained high binding affinities for the NK2 receptor in transfected CHO cells. In contrast, fluorescent derivatives made by replacing the N-alpha-benzoyl group of 1 by NBD or fluorescein were considerably less active. The effect on ligand potency of varying the length of the spacer arm between the peptide moiety and the fluorescent group was also studied. The most potent fluorescent antagonists were N-alpha-benzoyl-Dab(gamma-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (5B), pKi = 8.87 for NK2; N-alpha-benzoyl-Orn (delta-NBD)-Ala-D-Trp-Phe- D-Pro-Pro-Nle-NH2 (4B), pKi = 8.84; and N-alpha-benzoyl-Lys(epsilon-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (3B), pKi = 8.83. These three compounds were highly selective for NK2 over NK3 and NK1 receptors. We show that these fluorescent ligands are useful tools for the detection of NK2 receptor expression by flow cytometry. Additionally, these fluorescent probes should prove valuable for fluorescence microscopy and study of ligand-receptor interaction by spectrofluorimetry.