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

• Published in: The Journal of biological chemistry 1996-10, Vol.271 (42), p.25797-25800
• Main Authors: Kage, R, Leeman, S E, Krause, J E, Costello, C E, Boyd, N D
• Format: Article
• Language: English
• Subjects: Affinity Labels - metabolism; Animals; Binding Sites; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Cyanogen Bromide - metabolism; Female; Methionine - metabolism; Models, Chemical; Ovary - metabolism; Receptors, Neurokinin-1 - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substance P - analogs & derivatives; Substance P - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.42.25797

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., Kage, 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 125 I-Bolton-Hunter reagent (BH)-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 125 I-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.