Probing the Structure and Function of the Tachykinin Neurokinin-2 Receptor through Biosynthetic Incorporation of Fluorescent Amino Acids at Specific Sites

Abstract

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.

Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

“…Given the broad range of structures and activities for GPCRs, as well as their undeniable pharmaceutical importance, it has been appreciated for some time that unnatural amino acids could provide a valuable probe of this essential class of membrane receptors. An early study incorporated the fluorescent unnatural amino acid NBD-Dap into the NK2 receptor and showed that exposure to tachykinin did produce measurable electrophysiological currents in Xenopus oocytes (due to opening of Ca 2ϩ -activated Cl Ϫ channels that are endogenous to the oocyte) (29). A very recent study used an orthogonal tRNAsynthetase pair to incorporate a benzophenone-containing unnatural amino acid into the Ste2p GPCR (30) and the CCR5 receptor (31).…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…This codon can be used in combination with the appropriate tRNA to introduce non-natural amino acids at the position of interest and does not require the deletion of other amino acids. Until now, only one publication has used this technique for fluorescence resonance energy transfer studies at a GPCR, the NK2 receptor (Turcatti et al, 1996). The limitations of the technique were due to the difficulties in producing large amounts of chemically aminoacylated suppressor tRNA that needs to be injected into large numbers of cells to produce significant amounts of protein.…”

“…First, one can study the interaction between ligand and receptor as the FRET TECHNIQUES FOR GPCR ACTIVATION AND SIGNALING initial step in the signaling cascade (Turcatti et al, 1996;Castro et al, 2005). Second, FRET between two different labeled receptor-bound fluorescent ligands has been used to detect oligomeric GPCR complexes, not only in transfected cells but also in native tissue (Albizu et al, 2010).…”

Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

“…Highly sensitive measurements of unique channel activities made the absolute expression yield non-critical, with single cell expression being sufficient. However, these and other studies (26) have been mostly limited to oocytes. The low yield and the technical difficulties to produce and deliver chemically aminoacylated suppressor tRNA into a large number of mammalian cells made this method impractical for general studies of GPCRs.…”