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N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR.

Hartt JK, Barish G, Murphy PM, Gao JL - J. Exp. Med. (1999)

Bottom Line: The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides.The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2.The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

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FPR2 is a chemotactic receptor. HEK 293 cells stably transfected with mouse FPR, FPR2, or Fpr-rs1 were incubated in a microchemotaxis chamber for 5 h, and the number of migrating cells was counted. Data shown are from a single experiment representative of more than five separate experiments in each panel with a consistent pattern. (A) FPR2 transfectants with equal concentration of fMLF in the upper and lower chambers (fMLF/fMLF, open circles), or with fMLF in only the lower chamber (Medium/fMLF, filled circles). *Statistically significant difference between the points shown and baseline migration in the absence of fMLF, P < 0.0005 by Student's t test. (B) FPR transfectants (circles) and Fpr-rs1 transfectants (squares).
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Figure 3: FPR2 is a chemotactic receptor. HEK 293 cells stably transfected with mouse FPR, FPR2, or Fpr-rs1 were incubated in a microchemotaxis chamber for 5 h, and the number of migrating cells was counted. Data shown are from a single experiment representative of more than five separate experiments in each panel with a consistent pattern. (A) FPR2 transfectants with equal concentration of fMLF in the upper and lower chambers (fMLF/fMLF, open circles), or with fMLF in only the lower chamber (Medium/fMLF, filled circles). *Statistically significant difference between the points shown and baseline migration in the absence of fMLF, P < 0.0005 by Student's t test. (B) FPR transfectants (circles) and Fpr-rs1 transfectants (squares).

Mentions: Given the reactivity of FPR2 to fMLF observed in the calcium flux assay, we next tested its ability to mediate chemotaxis. FPR2-expressing cells migrated in a concentration-dependent manner in response to fMLF with a threshold of ∼1 μM. The EC50 was ∼5 μM. In all experiments, the upward phase of the concentration–response curve was consistently superimposable with that of the calcium flux assay for the same FPR2-expressing cell line. Mouse FPR-expressing HEK 293 cell migration followed a clear-cut bell-shaped concentration–response curve whose EC50 was shifted ∼10–100-fold to the left relative to the FPR2 curve (Fig. 3). The activity was specific for both receptors, since HEK 293 cells transfected with the related gene Fpr-rs1 and selected with G-418 did not exhibit concentration-dependent migration in response to fMLF when tested with concentrations ranging from 0.1 nM to 100 μM (Fig. 3 B). To distinguish chemotaxis from chemokinesis, equal concentrations of fMLF were added to the upper and lower chambers of the chemotaxis apparatus. In this configuration, no dose-dependent cell migration was observed (Fig. 3 A). Therefore, FPR2 can not only cause intracellular signaling, but can also use those signals to elicit a chemotactic action by the cell.


N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR.

Hartt JK, Barish G, Murphy PM, Gao JL - J. Exp. Med. (1999)

FPR2 is a chemotactic receptor. HEK 293 cells stably transfected with mouse FPR, FPR2, or Fpr-rs1 were incubated in a microchemotaxis chamber for 5 h, and the number of migrating cells was counted. Data shown are from a single experiment representative of more than five separate experiments in each panel with a consistent pattern. (A) FPR2 transfectants with equal concentration of fMLF in the upper and lower chambers (fMLF/fMLF, open circles), or with fMLF in only the lower chamber (Medium/fMLF, filled circles). *Statistically significant difference between the points shown and baseline migration in the absence of fMLF, P < 0.0005 by Student's t test. (B) FPR transfectants (circles) and Fpr-rs1 transfectants (squares).
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Figure 3: FPR2 is a chemotactic receptor. HEK 293 cells stably transfected with mouse FPR, FPR2, or Fpr-rs1 were incubated in a microchemotaxis chamber for 5 h, and the number of migrating cells was counted. Data shown are from a single experiment representative of more than five separate experiments in each panel with a consistent pattern. (A) FPR2 transfectants with equal concentration of fMLF in the upper and lower chambers (fMLF/fMLF, open circles), or with fMLF in only the lower chamber (Medium/fMLF, filled circles). *Statistically significant difference between the points shown and baseline migration in the absence of fMLF, P < 0.0005 by Student's t test. (B) FPR transfectants (circles) and Fpr-rs1 transfectants (squares).
Mentions: Given the reactivity of FPR2 to fMLF observed in the calcium flux assay, we next tested its ability to mediate chemotaxis. FPR2-expressing cells migrated in a concentration-dependent manner in response to fMLF with a threshold of ∼1 μM. The EC50 was ∼5 μM. In all experiments, the upward phase of the concentration–response curve was consistently superimposable with that of the calcium flux assay for the same FPR2-expressing cell line. Mouse FPR-expressing HEK 293 cell migration followed a clear-cut bell-shaped concentration–response curve whose EC50 was shifted ∼10–100-fold to the left relative to the FPR2 curve (Fig. 3). The activity was specific for both receptors, since HEK 293 cells transfected with the related gene Fpr-rs1 and selected with G-418 did not exhibit concentration-dependent migration in response to fMLF when tested with concentrations ranging from 0.1 nM to 100 μM (Fig. 3 B). To distinguish chemotaxis from chemokinesis, equal concentrations of fMLF were added to the upper and lower chambers of the chemotaxis apparatus. In this configuration, no dose-dependent cell migration was observed (Fig. 3 A). Therefore, FPR2 can not only cause intracellular signaling, but can also use those signals to elicit a chemotactic action by the cell.

Bottom Line: The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides.The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2.The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

Show MeSH
Related in: MedlinePlus