The use of photolabelled 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 incorporated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-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-Bz)Phe and acylated at the N-terminus by a biotinyl sulfone reporter via a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety 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 spectrometry analysis allowed identification of the NK-1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsin/Staphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop Thr173-Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified residue.
Girault
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S
Laboratoire de Chimie Organique Biologique associé au CNRS, Université Pierre et Marie Curie, Paris, France.
Sagan
S
S
Bolbach
G
G
Lavielle
S
S
Chassaing
G
G
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D010450:Endopeptidases; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D018040:Receptors, Neurokinin-1 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015290:Second Messenger Systems; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013373:Substance P / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D000345:Affinity Labels; D010446:Peptide Fragments; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D013373:Substance P; D010450:Endopeptidases