Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils

Abstract

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N -formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N -formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 M r ) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu 312 –Arg 322 and Arg 323 –Lys 350 ) and extracellular FPR1 peptide (Ile 191 –Arg 201 ) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala 323 –Lys 350 only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr 325 , Ser 328 , Thr 329 , Thr 331 , Ser 332 , Thr 334 , and Thr 339 . No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC 50 = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies. Background: N -Formylated bacterial/mitochondrial peptides in infected/injured tissues are GPCR chemoattractant agonists for neutrophil FPRs. Results: C-terminal tail FPR phosphopeptides were identified by LC/MS/MS in tryptic digests of FPRs immunopurified from human blood neutrophils. Conclusion: FPR1 but not FPR2 is monophosphorylated at any one of seven C-terminal tail Ser/Thr residues after fMLF stimulation. Significance: Decoding of human neutrophil FPR phosphorylation may be important for controlling inflammation.