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-benzoylphenylalanine8-SP (Bpa8-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 125I-Bolton-Hunter reagent (BH)-Bpa8-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 125I-BH-Bpa8-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.
Kage
R
R
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Leeman
S E
SE
Krause
J E
JE
Costello
C E
CE
Boyd
N D
ND
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D003488:Cyanogen Bromide / Q000378:metabolism; D005260:Female; D008715:Methionine / Q000378:metabolism*; D008956:Models, Chemical; D010053:Ovary / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013373:Substance P / Q000031:analogs & derivatives* / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D018040:Receptors, Neurokinin-1; C101870:substance P, Bpa(8)-; D013373:Substance P; D008715:Methionine; D003488:Cyanogen Bromide