A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligandreceptor 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 Fö rster, 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 proteincoupled 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.

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to M agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.Neurotransmitters stimulate target cells upon interacting with two major categories of regulatory proteins, ligand-gated ion channels (1-3), and G protein-coupled receptors (4). For ligand-gated ion channels, response activation and termination are mediated by a single molecule that carries the neurotransmitter site and the effector site (1). In contrast, responses mediated by G protein-coupled receptors result from a complex cascade of transient and sequential interactions between the receptor and a series of distinct messengers and effector proteins, which belong to distinct subcellular compartments (5-9).Tachykinins or neurokinins form a family of related neuropeptides found throughout central and peripheral nervous tissues. Their biological activities related to pain transmission (10, 11), smooth muscle contraction, vasodilation, or neurogenic inflammation are mediated by at least three distinct G protein-coupled receptors, NK1, NK2, and NK3, that show preferential binding for the endogenous agonists substance P, neurokinin A (NKA), 1 and neurokinin B (NKB), respectively (12). All three receptor isotypes belong to the family of seven transmembrane regulatory proteins and are coupled to an intracellular calcium release response primarily mediated by the pertussis toxin-insensitive heterotrimeric GTP-binding protein Gq/G11.In the present work, we have used fluorescence resonance energy transfer-based detection of neurokinin A binding to its G protein-coupled receptor, the NK2 tachykinin receptor, to detect real-time interactions on living cells and to study the spatial distribution of response activation and desensitization at the single cell level. Tachykinin NK2 receptors w...

INTRODUCTIONThe antitumour effect of endotoxins has been known for over a century because of remarkable observations regarding spontaneous regression of

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Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the N ␣ position were synthesized and characterized. Peptides modified at the ␣-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18,000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyllabeled peptides indicating that the micro-environment of the binding site is more hydrophobic for the nonpeptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl-or fluoresceinyllabeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.Many peptide hormones and neuropeptides act via known receptors belonging to the superfamily of G protein-coupled receptors characterized by a seven membrane-spanning topology. There is considerable interest in understanding ligandreceptor recognition and the mechanisms of action of both non-peptide ligands and natural peptides for peptide receptors. The tachykinin substance P (SP) 1 is a peptide transmitter that plays an important role in pain perception and neurogenic inflammation (1, 2). The cellular actions of SP are mediated by the tachykinin (neurokinin) NK1 receptor, a G protein-coupled receptor. Therefore, the NK1 receptor has been the target for the development of multiple non-peptide antagonists. The prototype NK1 non-peptide antagonist is the quinuclidine compound CP96,345, which acts as a high affinity and highly selective non-peptide inhibitor of SP in both binding and functional a...