title: 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 Mr) 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 EC50 = 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.
authors.affiliation: Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Walid S
authors.initials: WS
authors.lastname: Maaty
authors.affiliation: Departments of Microbiology.
authors.forename: Connie I
authors.initials: CI
authors.lastname: Lord
authors.affiliation: Departments of Microbiology.
authors.forename: Jeannie M
authors.initials: JM
authors.lastname: Gripentrog
authors.affiliation: Departments of Microbiology.
authors.forename: Marcia
authors.initials: M
authors.lastname: Riesselman
authors.affiliation: Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Gal
authors.initials: G
authors.lastname: Keren-Aviram
authors.affiliation: Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.affiliation: Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Dratz
authors.affiliation: Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.affiliation: Departments of Microbiology. Electronic address: umbaj@montana.edu.
authors.forename: Algirdas J
authors.initials: AJ
authors.lastname: Jesaitis
mesh_terms: D003571:Cytochalasin B / Q000494:pharmacology; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism / Q000494:pharmacology*; D018375:Neutrophil Activation / Q000187:drug effects* / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D044042:Receptors, Formyl Peptide / Q000819:agonists / Q000378:metabolism*; D044067:Receptors, Lipoxin / Q000378:metabolism
chemical_list: C469986:FPR1 protein, human; C105101:FPR2 protein, human; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine
keywords: 7-Helix Receptor; Chemokines; Cytochalasin B; Epitope Mapping; FPR Phosphorylation; Inflammation; LC/MS/MS; Mass Spectrometry (MS); Neutrophil; f-Met-Leu-Phe