IDENTIFICATION OF METHIONINE AS THE SITE OF COVALENT ATTACHMENT OF A P-BENZOYL-PHENYLALANINE-CONTAINING ANALOG OF SUBSTANCE-P ON THE SUBSTANCE-P (NK-1) RECEPTOR
R. Kage et al., IDENTIFICATION OF METHIONINE AS THE SITE OF COVALENT ATTACHMENT OF A P-BENZOYL-PHENYLALANINE-CONTAINING ANALOG OF SUBSTANCE-P ON THE SUBSTANCE-P (NK-1) RECEPTOR, The Journal of biological chemistry, 271(42), 1996, pp. 25797-25800
Previously we have been able to restrict the site of covalent attachme
nt of a photolabile and radiolabeled derivative of substance P (SP), p
-benzoylphenylalanine(8)-SP (Bpa(8).SP), to residues 178-183 located o
n 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 specif
ic amino acid in this sequence that serves as the site of covalent att
achment for I-125-Bolton-Hunter reagent (EH)-Bpa(8)-SP, we have employ
ed here a novel solid-phase approach to cyanogen bromide cleavage of t
he 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 adsorb
ed onto C-18-derivatized silica gel and cleaved with cyanogen bromide,
A single radiolabeled fragment containing 63% of the photoincorporate
d radioactivity was generated and purified by high performance liquid
chromatography. Mass spectrometric analysis identified a single molecu
lar ion with a molecular mass of 1751.4 +/- 2, establishing that upon
irradiation the bound photoligand forms a covalent link with the methy
l group of a methionine residue at the peptide binding site, In view o
f our previous findings, this methionine is Met-181 on the primary seq
uence of the SP receptor.