Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites.
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.
Turcatti
G
G
Geneva Biomedical Research Institute, Glaxo Wellcome, CH-1228 Geneva, Switzerland.
Nemeth
K
K
Edgerton
M D
MD
Meseth
U
U
Talabot
F
F
Peitsch
M
M
Knowles
J
J
Vogel
H
H
Chollet
A
A
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D004735:Energy Transfer; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins; D016153:Genes, Suppressor; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D018041:Receptors, Neurokinin-2 / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D002712:Chlorides; D017931:DNA Primers; D005456:Fluorescent Dyes; D008024:Ligands; D012346:RNA, Transfer, Amino Acyl; D018041:Receptors, Neurokinin-2; D019204:GTP-Binding Proteins