Abstract
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.
Highlights
** Present address: Combined Program in Pediatric Gastroenterology and Nutrition, Enders Bldg. 1209, Children’s Hospital, 300 Longwood Ave., Boston, MA 02115
The phagocyte chemotactic receptors, including the formyl peptide receptor (FPR),1 the lipoxin A4 receptor, the C5a receptor, the platelet-activating factor receptor, and the interleukin-8 receptor are involved in inflammation and are all members of the G protein-coupled receptor (GPCR) superfamily
We report that a fluorescent photoaffinity analog of fMLF, formyl-Met-p-benzoyl-L-phenylalanine-PheTyr-Lys-⑀-N-fluorescein, efficiently photocrosslinks to FPR residues 83– 85
Summary
Peptide Synthesis—fMet-p-Benzoyl-L-phenylalanine-Phe-Tyr-Lys was synthesized from Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acids using a Milligen 9050 peptide synthesizer and purified by reverse phase HPLC. The antifluorescein-Sepharose was washed extensively (Ͼ72 h) with phosphate-buffered saline, 1.5 M NaCl, and 50% ethylene glycol It was washed quickly (Ͻ5 min) with 1% triethylamine, 1.5 M NaCl, 50% ethylene glycol, and equilibrated in 10 mM HEPES (pH 7.4) and 1% octyl glucoside just prior to use. HPLC Analysis of Photolabeled FPR—Membranes from CHO cells expressing FPR and wild type control CHO cell membranes were prepared as described above, resuspended in 0.7 ml of Buffer A containing 3% octyl glucoside, sonicated, and centrifuged at 150,000 ϫ g for 30 min. The samples were filtered through BA 85 0.45-m filters (Schleicher & Schuell), the filters were washed with 5 ml of 10 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM Mg2ϩ buffer, and the bound [35S]GTP␥S was measured by liquid scintillation counting
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