IDENTIFICATION OF A LIGAND-BINDING SITE IN THE HUMAN NEUTROPHIL FORMYL PEPTIDE RECEPTOR USING A SITE-SPECIFIC FLUORESCENT PHOTOAFFINITY LABEL AND MASS-SPECTROMETRY
Js. Mills et al., IDENTIFICATION OF A LIGAND-BINDING SITE IN THE HUMAN NEUTROPHIL FORMYL PEPTIDE RECEPTOR USING A SITE-SPECIFIC FLUORESCENT PHOTOAFFINITY LABEL AND MASS-SPECTROMETRY, The Journal of biological chemistry, 273(17), 1998, pp. 10428-10435
A novel fluorescent photoaffinity cross-linking probe, -L-phenylalanin
e-Phe-Tyr-Lys-epsilon-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 s
olubilized in octylglucoside and separated by tandem anion exchange an
d gel filtration chromatography, A single peak of fluorescence was obs
erved 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 desor
ption ionization mass spectrometry identified a major species with mas
s = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linke
d to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 8
3-87), This peptide was further cleaved with trypsin, repurified by an
tifluorescein immunoaffinity, and subjected to matrix-assisted laser d
esorption ionization mass spectrometry, A tryptic fragment with mass =
1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to V
al-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. Immun
ol. 159, 4045-4054) and the photochemical cross-linking data.