PROBING THE STRUCTURE AND FUNCTION OF THE TACHYKININ NEUROKININ-2 RECEPTOR THROUGH BIOSYNTHETIC INCORPORATION OF FLUORESCENT AMINO-ACIDS ATSPECIFIC SITES

A general method for understanding the mechanisms of ligand recognitio n and activation of G protein coupled receptors has been developed, A study of ligand-receptor interactions in the prototypic seven-transmem brane neurokinin-a receptor (NK2) using this fluorescence-based approa ch is presented, A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the ai d of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expres sion in Xenopus oocytes, Fluorescence-labeled NK2 mutants containing a n unique robenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD- Dap) residue at either site 103, in the first extracellular loop, or 2 48, in the third cytoplasmic loop, were functionally active, The fluor escent NK2 mutants were investigated by microspectrofluorimetry in a n ative membrane environment, Intermolecular distances were determined b y measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 h eptapeptide antagonist, These distances, calculated by the theory of F orster, 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 me thod of nonsense codon suppression, as well as the first measurement o f 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 su ch as receptors or channels.