Identification of methionine as the site of covalent attachment of a p-benzoyl-phenylalanine-containing analogue of substance P on the substance P (NK-1) receptor

Previously we have been able to restrict the site of covalent attachment of a photolabile and radiolabeled derivative of substance P (SP), p-benzoylphenylalanine(8)-SP (Bpa(8).SP), to residues 178-183 located on the second extracellular loop (E2) of the SP (NK-1) receptor (Boyd, N. D., Rage, R., Dumas, J. J., Krause, J. E., and Leeman, S. E. (1996) Proc, Natl, Acad, Sci, U. S. A. 93, 433-437), To ascertain the specific amino acid in this sequence that serves as the site of covalent attachment for I-125-Bolton-Hunter reagent (EH)-Bpa(8)-SP, we have employed here a novel solid-phase approach to cyanogen bromide cleavage of the photolabeled receptor followed by mass spectrometric analysis of a purified labeled fragment. SP receptors on transfected Chinese hamster ovary cells were photolabeled with isotopically diluted I-125-BH-Bpa(8)-SP. A membrane preparation of the photolabeled receptors was adsorbed onto C-18-derivatized silica gel and cleaved with cyanogen bromide, A single radiolabeled fragment containing 63% of the photoincorporated radioactivity was generated and purified by high performance liquid chromatography. Mass spectrometric analysis identified a single molecular ion with a molecular mass of 1751.4 +/- 2, establishing that upon irradiation the bound photoligand forms a covalent link with the methyl group of a methionine residue at the peptide binding site, In view of our previous findings, this methionine is Met-181 on the primary sequence of the SP receptor.