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. 2022 Aug 2;119(31):e2201249119.
doi: 10.1073/pnas.2201249119. Epub 2022 Jul 25.

Agonist concentration-dependent changes in FPR1 conformation lead to biased signaling for selective activation of phagocyte functions

Junlin Wang  1 , Richard D Ye  1
Affiliations

Agonist concentration-dependent changes in FPR1 conformation lead to biased signaling for selective activation of phagocyte functions

Junlin Wang et al. Proc Natl Acad Sci U S A. .

Abstract

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-β/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+ mobilization from intracellular stores and Ca2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and β-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

Keywords: GPCRs; biased signaling; calcium mobilization; phagocytes.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Functions induced by fMLF at different concentrations. (A and B) The fMLF dose curves for chemotaxis, degranulation, and superoxide production were derived from stimulated dHL-60 cells (A) and peripheral blood neutrophils (B). Chemotaxis was conducted for 2 h at 37 °C and data were subject to checkerboard analysis. For degranulation, the cells were stimulated for 15 min at 37 °C, and the released β-hexosaminidase (A) and β-glucuronidase (B) were quantified. Superoxide production was measured in real time based on the chemiluminescence of isoluminol. (C) Effects of ethylene glycol tetraacetic acid (EGTA; 1 mM) and PTX (500 ng/mL, 6 h) on fMLF-induced Ca2+ mobilization. (D) Dose curves of Ca2+ flux in response to different concentrations of fMLF in the presence or absence of 1 mM EGTA. (E and F) The effects of PTX and EGTA on degranulation (E) and superoxide production (F) were also measured in dHL-60 cells stimulated with 1 μM fMLF. Data shown are means ± SEM from three independent experiments, each with triplicate measurements. **P < 0.01. CPS, counts per second; FL, fluorescence intensity; Max, maximum.
Fig. 2.
Fig. 2.
fMLF-induced changes in cytoplasmic Ca2+ concentration. (A) dHL-60 cells were loaded with Fluo-4 AM and Fura Red AM for 1 h at 37 °C and treated with a point source of fMLF (1 nM and 1 μM, upper left corner of the viewing field). Arrows indicate direction of cell movement. Confocal microscopy images were taken from 30 cells and representative images are shown. (Scale bar, 5 μm.) (B–D) Fluorescence intensity (FL) of Fluo-4 (green lines) and Fura Red (red lines) was measured along the cell section (marked 0 to 7) and background fluorescence at each of the 92 data points was subtracted. (B–D) The processed data from control (B; no agonist), 1 nM fMLF (C), and 1 µM fMLF (D) were plotted. (E–G) Ratios of Fluo-4 to Fura Red from control (E; no agonist), 1 nM fMLF (F), and 1 µM fMLF (G) are plotted. Data are shown as means ± SEM based on 30 cells. (F) The average values collected from the first 3 μm were significantly lower than the average values in the last 1 μm, indicating lower cytoplasmic Ca2+ concentration in the leading edge of the migrating cells. *P < 0.05, **P < 0.01. AU, arbitrary units.
Fig. 3.
Fig. 3.
Assessment of fMLF-induced conformational changes using a NanoBRET-based FPR1 biosensor. (A) Schematic representation of a FlAsH-NanoBRET–based FPR1 biosensor. The Nanoluc (donor) was fused to the C terminus of FPR1. A tetracysteine tag (CCPGCC) for FlAsH (acceptor) binding was inserted into the third intracellular loop (IL3) to form the biosensor, FPR1-IL3-Luc, which detects alterations in distance between the donor and the acceptor. (B and C) The functional integrity of the FPR1 biosensor was examined by fMLF (1 μM)-induced internalization (B) and Ca2+ mobilization (C), and was found to be indistinguishable from the wild-type FFP1. (D and E) Measurement of fMLF-induced conformational changes using the FPR1 biosensor. HeLa cells expressing FPR1-IL3-Luc were loaded with 1 μM FlAsH-EDT2 and then coelenterazine H (nanoluc substrate) was added to 5 μM for 5 min at 37 °C. BRET ratio was recorded for 100 s before and 100 s after stimulation in the presence of fMLF at different concentrations. In (E), PTX was added to 500 ng/mL and the cells were incubated for 6 h before fMLF treatment. The data shown are means ± SEM from 3 independent experiments, each measured in triplicate. *P < 0.05, **P < 0.01.
Fig. 4.
Fig. 4.
Effect of different concentrations of fMLF on the recruitment of β-arr1 and β-arr2. (A and B) HeLa cells were cotransfected with FPR1-Clover (green fluorescence) and mRuby2-tagged β-arr1 (A) and β-arr2 (B). After 24 h, the cells were treated with different concentrations of fMLF for 10 min at 37 °C. Confocal microscopy images showing membrane translocation of the β-arrestins (red fluorescence) were taken and overlaid to show colocalization of these tagged proteins (yellow). The experiments were performed using a confocal microscope with a 40× oil objective, and representative pictures are shown. (Scale bar, 10 μm.) (C) schematic representation of a NanoBiT-based β-arrestin recruitment assay showing luminescence emission when the β-arrestin protein is recruited to FPR1 in the plasma membrane. (D) Quantification of FPR1-dependent recruitment of β-arr1 and β-arr2. HeLa cells cotransfected with FPR1-SmBiT and LgBiT–β-arrestins. Twenty-four hours after transfection, the substrate coelenterazine H (10 μM) was added and cells were incubated for 25 min at 37 °C before measurement of basal luminescence emission. The agonist fMLF was then added at the indicated concentrations and luminescence emission was measured again. Data (count per second) were plotted as a function of fMLF concentrations with the maximal luminescence emission (10 μM) set as 100% response. Data shown are mean ± SEM based on three independent experiments. Max, maximum; PBS, phosphate-buffered saline; term, terminus.
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