The Use of Photolabelled Peptides to Localize the Substance‐P‐Binding Site in the Human Neurokinin‐1 Tachykinin Receptor

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Abstract
The amino acid p -benzoyl-l-phenylalanine, (p -Bz)Phe, has been incorporated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, to localize the agonist-binding domains of the human neurokinin-1 (NK-1) receptor overexpressed in a transfected mammalian cell line. The NK-1-specific agonist [Pro9]SP was modified at position 8 by (p -Bz)Phe and acylated at the N-terminus by a biotinyl sulfone reporter via a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety allowed easy and efficient removal of biotinylated fragments from the complex incubation mixture with streptavidin-coated beads. Direct elution from the beads with the matrix used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS), which was facilitated by saturation of streptavidin sites with biotin, and subsequent MALDI-TOF mass spectrometry analysis allowed identification of the NK-1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsin/Staphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop, Thr173–Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified residue.