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J Biol Chem, Vol. 273, Issue 17, 10428-10435, April 24, 1998
From the Departments of A novel fluorescent photoaffinity
cross-linking probe,
formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-
Identification of a Ligand Binding Site in the Human Neutrophil
Formyl Peptide Receptor Using a Site-specific Fluorescent Photoaffinity
Label and Mass Spectrometry
,,,,
Microbiology and
¶ Chemistry and Biochemistry, Montana State University,
Bozeman, Montana 59717-3520
-N-fluorescein
(fMBpaFYK-fl), was synthesized and used to identify binding site
residues in recombinant human phagocyte chemoattractant formyl peptide
receptor (FPR). After photoactivation, fluorescein-labeled membranes
from Chinese hamster ovary cells were solubilized in octylglucoside and
separated by tandem anion exchange and gel filtration chromatography. A
single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein
immunoaffinity matrix. Matrix-assisted laser desorption ionization mass
spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to
Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues
83-87). This peptide was further cleaved with trypsin, repurified by
antifluorescein immunoaffinity, and subjected to matrix-assisted laser
desorption ionization mass spectrometry. A tryptic fragment with
mass = 1582 was observed, which is the mass of fMBpaFYK-fl
cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin
cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger,
B. L., and Jesaitis, A. J. (1997) J. Immunol.
159, 4045-4054) and the photochemical cross-linking data.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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