THE USE OF PHOTOLABELED PEPTIDES TO LOCALIZE THE SUBSTANCE-P-BINDING SITE IN THE HUMAN NEUROKININ-1 TACHYKININ RECEPTOR

The amino acid p-benzoyl-L-phenylalanine, (p-Bz)Phe, has been incorpor ated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Me t-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-B z)Phe and acylated at the N-terminus by a biotinyl sulfone reporter vi n a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone mo iety 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 spectrornetry analysis allowed identif ication of the NK-1 fragments obtained after photolysis and proteolyti c digestion. Trypsin digestion and combined trypsin/Staphylococcus arr reus V8 protease enzymatic cleavage established that the site of coval ent attachment of the photolabelled SP resides in the second extracell ular loop, Thr173-Arg177. Cyanogen bromide cleavage shows that the pro be is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified r esidue.