C-Terminal Tail Phosphorylation of N-Formyl Peptide Receptor: Differential Recognition of Two Neutrophil Chemoattractant Receptors by Monoclonal Antibodies NFPR1 and NFPR2

Abstract

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni2+-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45–75 kDa species (peak Mr ∼60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of Mr ∼40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting ∼60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337–346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.

The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

“…This antibody strongly detected a broad band between 35 and 60 kDa in FPR1-transfected HEK 293 cells ( Fig. 2A, left lane), in good agreement with published data for FPR1 in neutrophils and FPR1-transfected CHO cells using the same antibody (25) and in human HL-60 neutrophils detected by radioligand affinity labeling (28). No corresponding band was found in wild type HEK 293 cells ( Fig.…”

“…SDS PAGE was performed for 60 -90 min at ϳ100 V on Mini-Protean TGX 4 -15% gels (BioRad). The samples were mixed with 2ϫ sample buffer prepared as previously described (25) or with 4ϫ NuPAGE LDS sample buffer, both containing 2-mercaptoethanol (35 l/ml). Immunoblotting was performed with Immobilon-P PVDF membranes (Millipore, Billerica, MA), blocked overnight with 5% milk in TBST.…”

“…We addressed the structural state of FPR1 protein from FHL 124 cell membranes, as defined by Western blot detection using a previously described second FPR1-specific antibody, the monoclonal reagent NFPR2 (25). This antibody strongly detected a broad band between 35 and 60 kDa in FPR1-transfected HEK 293 cells ( Fig.…”

Formyl Peptide Receptor-1 Activation Enhances Intestinal Epithelial Cell Restitution through Phosphatidylinositol 3-Kinase-Dependent Activation of Rac1 and Cdc42

“…This Ab recognizes the C-terminal tail sequence 337-NSTLPSAEUA-346, which is sensitive to phosphorylation and thus recognizes a nonphosphorylated receptor (41). Rabbit polyclonal anti-Rac1 and Cdc42 were obtained from Santa Cruz Biotechnology.…”

“…Mouse anti-FPR mAb, NFPR2, was generated as previously described (41). This Ab recognizes the C-terminal tail sequence 337-NSTLPSAEUA-346, which is sensitive to phosphorylation and thus recognizes a nonphosphorylated receptor (41).…”