The C5a receptor (C5aR) C5L2 is a modulator of C5aR-mediated signal transduction

Abstract

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and beta-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the beta-arrestin pathway. Association of C5L2 with beta-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the beta-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.

FIGURE 1.

Expression analysis of human C5L2 and C5aR. A, the relative mRNA abundance for C5L2 (right), relative to the C5aR (left) in the human cells and tissues shown, is indicated on a scale of 0–12, where 0 (pale green) is at the low extreme and 12 (red) is most abundant. B, flow cytometric staining of murine pre-B L1.2 cells transfected with human C5aR (left) or human C5L2 (right), using anti-human C5L2 mAb, 1D9, anti-human C5aR, 7F3, or isotype control mAbs. C, inhibition of ¹²⁵I-C5a binding to human C5L2-L1.2 transfectants by anti-C5L2, 1D9 (○), or anti-C5aR, 7F3 (■). Hybridoma medium (HAT) was used as a negative control (+). Data are expressed as the mean ± S.E. of triplicate determinations and representative of three independent experiments.

FIGURE 2.

C5L2 is an intracellular receptor in human PMNs. A, the anti-C5L2 blocking mAb 1D9 does not block binding of ¹²⁵I-C5a to human PMNs. Purified human PMNs were incubated with 0.05 nm ¹²⁵I-C5a and increasing concentrations of unlabeled C5a in the presence of 1 μg/ml anti-C5L2, 1D9, anti-C5aR, 3C5, or isotype-matched control. After 60 min at 22 °C, unbound ligand was separated, and the cell-associated ligand was determined by gamma counting. Data are the mean ± S.E. of duplicate determinations, representative of at least four independent experiments. Inset, shown is an example of human PMN binding of ¹²⁵I-C5a under the same conditions but using the C5aR antagonist, NDT2001 (NDT, 500 nm) to block interactions with the C5aR. B, flow cytometric analysis of C5L2 (solid line) and the C5aR (dashed line) on membrane intact (left) and saponin-permeabilized (right) human PMNs. The histograms for isotype-matched control mouse IgG are shaded in gray. Data are representative of 12 independent experiments.

FIGURE 3.

Ligand-dependent co-localization of the C5a receptors in human PMNs. A, confocal microscopic localization of C5L2 in human PMNs was determined using human neutrophils adherent to fibronectin-coated coverslips. Cells were loaded with anti-C5L2 and anti-C5aR at 4 °C for 30 min then warmed to 37 °C for 10 min to allow endocytosis. Cells were then fixed, permeabilized, and immunostained with Alexa488 anti-mouse IgG (green) and Alexa555 anti-rabbit IgG (red). Nuclei were visualized with DAPI. Bar, 10 μm. B, purified human PMNs adherent to fibronectin-coated coverslips were incubated with 0 or 100 nm C5a at 37 °C for the times indicated. Cells were then fixed and permeabilized with saponin and immunostained with mouse anti-human C5L2, 1D9, and rabbit anti-human C5aR, followed by Alexa555 anti-mouse IgG (red) and Alexa488 anti-rabbit IgG (green). Nuclei were visualized with DAPI. Bar, 10 μm.

FIGURE 4.

Antibody blockade of C5L2 does not alter ligand uptake or internalization. A, C5a binding and internalization under conditions of receptor recycling were assessed using human PMNs incubated with 2 nm ¹²⁵I-C5a in the presence of 5 μg/ml anti-C5L2, 4C8, anti-C5aR, 3C5, or isotype control. Total and internalized (resistant to stripping at low pH) ligand (cpm) were determined as a function of time at 37 °C. Data represent the mean ± S.E. of duplicate determinations from five independent experiments. B, to evaluate ligand internalization under conditions of single pass endocytosis, PMNs were preincubated with antibodies as in A and 2 nm ¹²⁵I-C5a for 60 min at 0 °C. Cells were then washed and transferred to 37 °C, and internalized ligand was determined as a function of time by washing at low pH as above. Data represent the mean ± S.E. of duplicate determinations from three independent experiments.

FIGURE 5.

C5L2 suppresses C5a-mediated chemotaxis and ERK1/2 activation in PMNs. A, calcein-loaded human PMNs were preincubated with anti-C5L2, 1D9, or isotype control IgG, and chemotactic responses to C5a were assessed in modified Boyden chambers. Data are the mean ± S.E. of triplicate determinations, representative of five independent experiments. B, C5a-mediated mobilization of intracellular calcium in cells preincubated with 5 μg/ml anti-C5L2, 1D9, or isotype control IgG. Data are representative of four to five replicates in two independent experiments. C, representative Western blot analysis of the phospho-ERK1/2 signal generated in human PMNs in the presence of the anti-human C5L2 antibodies, 1D9 or 4C8, or isotype control at 5 μg/ml, as a function of time after addition of 1 nm C5a. Blots were stripped and reprobed for total ERK1/2 to ensure equivalent loading of samples. D, densitometric quantitation of the relative phospho-ERK1/2 signal generated by 1–3 min of incubation with 1 nm C5a in cells treated with anti-human C5L2 antibodies, 1D9 or 4C8, normalized to isotype control IgG. Data represent the mean ± S.E. of 9 independent experiments; the significance of differences between anti-C5L2 treatment and isotype control are indicated.

FIGURE 6.

Co-localization of C5L2 with β-arrestin in human PMNs is increased following C5a stimulation. A, purified human PMNs adherent to fibronectin-coated coverslips were saponin treated fixed and immunostained with mouse anti-C5L2 or anti-C5aR and rabbit anti-β-arrestin-1, followed by Alexa488 anti-mouse IgG (green) and Alexa555 anti-rabbit IgG (red) in the absence of C5a or 10 min after addition of 100 nm C5a. Nuclei were visualized with DAPI. Bar, 10 μm. B, PMNs incubated with 0 (−) or 100 nm (+) C5a for 10 min at 37 °C were cross-linked with dithiobis(succinimidyl proprionate), detergent-solubilized, and immunoprecipitated (IP) with goat anti-β-arrestin (lanes 3 and 4) or an irrelevant goat IgG (lanes 1 and 2). Immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) for C5L2 or β-arrestin-1. Molecular mass markers (kDa) are indicated on right. Data are representative of three independent experiments.

FIGURE 7.

C5L2 activation is a consequence of activation of the C5aR. Human PMNs were incubated with C5a in the presence of 5 μg/ml isotype control IgG or anti-C5aR antibody 3C5, as indicated, and then permeabilized and stained with rabbit anti-C5L2 and goat anti-β-arrestin-1 antibodies, followed by Alexa555-labeled anti-rabbit IgG and Alexa488-labeled anti-goat IgG. Nuclei were visualized with DAPI. Bars, 10 μm.

FIGURE 8.

Schematic representation of the mechanism by which C5L2 negatively modulates C5a-C5aR-mediated ERK1/2 activation in human neutrophils. C5L2 functions as an intracellular receptor, becoming activated only after ligand binding to the C5aR. Receptor activation induces phosphorylation by G protein receptor kinases (GRK), facilitating their association with β-arrestin. The C5aR-β-arrestin complex activates ERK1/2, whereas the C5L2-β-arrestin complex inhibits ERK1/2, and the net signal is a result of the balance of the two.