Abstract
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.
Highlights
103, in the first extracellular loop, or 248, in the third activation of G protein-coupled receptors and in the design of cytoplasmic loop, were functionally active
In this study we present a novel fluorescence spectroscopic approach to obtain structural information on integral membrane proteins as demonstrated for the Neurokinin (tachykinin)-2 receptor (NK2) neurokinin G protein-coupled receptor
The aim was to use fluorescence resonance energy transfer (FRET) techniques between a fluorescent donor group engineered in the NK2 receptor at known sites, and an acceptor fluorophore in the ligand, for the determination of a structural model for interaction of a peptide ligand with NK2
Summary
Materials—SP6 polymerase and T7 polymerase were from Promega (Madison, WI). FokI and BstNI endonucleases were from New England Biolabs (Beverly, MA). The amino acid-dinucleotide conjugate pdCpA-(N-Nvoc)Dap(NBD) was obtained in 35% yield after high performance liquid chromatography purification (column Machery-Nagel ET250/8/4 Nucleosil 5 C18, 1 ml/ min, gradient of acetonitrile in 50 mM sodium acetate, pH 4.5, detection at 260 or 460 nm) and lyophilization. This pdCpA-(N-Nvoc)Dap(NBD) conjugate (25 nmol) was dissolved in a mixture of 4 l of dimethyl sulfoxide, 10 l of ligase buffer (220 mM Hepes, pH 7.5, 80 g/ml bovine serum albumin, 1 mM ATP, 60 mM MgCl2 in diethylpyrocarbonatetreated water), and 21 l of diethylpyrocarbonate-treated water. The relative positions and orientation of the helices were derived from the study of Baldwin [26] and were based on the projection map of rhodopsin determined by electron diffraction [27]
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