Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry
| Autor: | Heini M. Miettinen, John S. Mills, Michael J. Vlases, Susan Wimer-Mackin, Edward A. Dratz, David R. Barnidge, Jan Sunner, Algirdas J. Jesaitis |
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| Rok vydání: | 1998 |
| Předmět: |
Models
Molecular Receptors Peptide Neutrophils Affinity label Molecular Sequence Data Peptide CHO Cells Mass spectrometry Biochemistry Cricetinae medicine Animals Humans Amino Acid Sequence Binding site Receptors Immunologic Molecular Biology Peptide sequence Fluorescent Dyes chemistry.chemical_classification Formyl peptide receptor Chromatography Binding Sites biology Chemistry Chinese hamster ovary cell Cell Membrane Affinity Labels Cell Biology Trypsin Receptors Formyl Peptide N-Formylmethionine Leucyl-Phenylalanine Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Mutagenesis Site-Directed medicine.drug |
| Zdroj: | The Journal of biological chemistry. 273(17) |
| ISSN: | 0021-9258 |
| Popis: | A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (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. |
| Databáze: | OpenAIRE |
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