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The human complement fragment receptor, C5L2, is a recycling decoy receptor.

Scola AM, Johswich KO, Morgan BP, Klos A, Monk PN - Mol. Immunol. (2008)

Bottom Line: However, we detected neither an intracellular Ca(2+) response nor beta-arrestin redistribution in mutated C5L2, suggesting that the potential for G protein coupling is completely absent in this receptor and that, in humans, C5L2 may have functions that are unrelated to signaling.In confirmation of this, we detected constitutive ligand-independent internalization of C5L2 that resulted in the rapid accumulation of C5a and its stable metabolite, C5a des Arg, within the cell with only a small net change in cell surface receptor levels.Internalization was found to be through a clathrin-dependent mechanism that led to the retention and, in cells natively expressing C5L2, the degradation of the ligand within an intracellular compartment.

View Article: PubMed Central - PubMed

Affiliation: Academic Neurology Unit, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.

ABSTRACT
C5L2 is a 7 transmembrane domain receptor for complement fragment C5a that, unlike the classical C5a receptor, C5aR, does not couple to G proteins. However, in mice where C5L2 has been deleted, the response to C5a is altered, suggesting that C5L2 may have a signaling function. In order to investigate whether human C5L2 also has some capacity to transduce signals, we have attempted to produce a signaling competent form of human C5L2 by inserting C5aR sequences at three key G protein activation motifs. However, we detected neither an intracellular Ca(2+) response nor beta-arrestin redistribution in mutated C5L2, suggesting that the potential for G protein coupling is completely absent in this receptor and that, in humans, C5L2 may have functions that are unrelated to signaling. In confirmation of this, we detected constitutive ligand-independent internalization of C5L2 that resulted in the rapid accumulation of C5a and its stable metabolite, C5a des Arg, within the cell with only a small net change in cell surface receptor levels. Internalization was found to be through a clathrin-dependent mechanism that led to the retention and, in cells natively expressing C5L2, the degradation of the ligand within an intracellular compartment. In contrast, the classical C5a receptor, C5aR, internalized ligand much more slowly and a majority of this ligand was released back into the extracellular environment in an apparently undegraded form. These data suggest that a major function of human C5L2 is to remove active complement fragments from the extracellular environment.

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C5a does not stimulate increases in intracellular Ca2+ or translocation of β-arrestin in RBL cells transfected with C5L2. (A) Fluo4-AM loaded RBL cells transfected with C5aR, wild type C5L2, C5L2 with C5aR-like sequences at three key signaling motifs (C5L2+++) or untransfected controls (NX) were stimulated with C5a (left panel) or C5a des Arg (right panel). Changes in the intracellular Ca2+ concentration were measured by flow cytometry as changes in fluorescence integrated over 70 s and shown as a percentage of fluorescence in unstimulated cells. Results shown are means of three separate experiments performed in duplicate ± S.E.M. (B) Transfected RBL cells adhered to coverslips were treated with 100 nM C5a for 15 min, fixed, permeabilized, incubated with antibody specific for β-arrestin 1. Arrows show intracellular accumulation of β-arrestin 1 in C5aR-transfected cells. Bar equal to 10 μm.
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fig1: C5a does not stimulate increases in intracellular Ca2+ or translocation of β-arrestin in RBL cells transfected with C5L2. (A) Fluo4-AM loaded RBL cells transfected with C5aR, wild type C5L2, C5L2 with C5aR-like sequences at three key signaling motifs (C5L2+++) or untransfected controls (NX) were stimulated with C5a (left panel) or C5a des Arg (right panel). Changes in the intracellular Ca2+ concentration were measured by flow cytometry as changes in fluorescence integrated over 70 s and shown as a percentage of fluorescence in unstimulated cells. Results shown are means of three separate experiments performed in duplicate ± S.E.M. (B) Transfected RBL cells adhered to coverslips were treated with 100 nM C5a for 15 min, fixed, permeabilized, incubated with antibody specific for β-arrestin 1. Arrows show intracellular accumulation of β-arrestin 1 in C5aR-transfected cells. Bar equal to 10 μm.

Mentions: Although several studies have failed to detect signaling by C5L2, others have demonstrated some capacity for signal transduction (Cain and Monk, 2002; Chen et al., 2007; Johswich et al., 2006; Kalant et al., 2005; Lee et al., 2008; Okinaga et al., 2003; Rittirsch et al., 2008). It is possible that C5L2 has a small capacity to transduce ligand-binding signals that is difficult to detect due to attenuation by the lack of highly conserved motifs (DRY in the 3rd transmembrane domain, NPXXY in the 7th transmembrane domain) and/or the short 3rd intracellular loop. To investigate this possibility, we made a mutant of C5L2 (C5L2+++) in which the motifs from the efficiently coupled C5a receptor, C5aR were substituted for the C5L2 sequence: D131LC-D131RF; N287PMLF–N287PMLY and the entire third intracellular loop sequence of C5aR. Signaling capacity was measured using the highly sensitive fluorescent intracellular free Ca2+ ([Ca2+]i) assay. When C5aR expressing RBL cells were stimulated with C5a (from 0.1 nM) there was a dramatic increase in fluorescence (Fig. 1A, left panel). In contrast, RBL cells transfected with wild type C5L2 or the mutant C5L2+++ showed no increase in fluorescence upon stimulation with concentrations of C5a of up to 100 nM, suggesting that no G protein-coupled signaling was occurring (Fig. 1A). Similarly, C5a des Arg could stimulate an increase in [Ca2+]i in C5aR-transfected RBL cells at concentrations of 10 nM and above but no changes were detectable in C5L2 or C5L2+++ transfected cells at any concentration used (Fig. 1A, right panel). Constructs of C5L2 with individual motifs mutated to their equivalents in C5aR had the same responses to both C5a and C5a des Arg (data not shown). A previous report showed some coupling of a C5L2 mutant (D131RC) to Gα16 in transiently co-transfected HEK cells (Okinaga et al., 2003). Signaling here may have been detected due to very high levels of receptor expression and also because the promiscuous Gα16 is less discriminating than the endogenous Gαi in RBL cells. Nevertheless, these results confirm that signaling by human C5L2 is highly repressed and there are multiple structural features that prevent Gαi activation by C5L2. β-Arrestin is a cytosolic protein that mediates the desensitization and internalization of GPCR and has also been shown to mediate G protein-independent signaling by 7 transmembrane domain receptors (reviewed by Defea (2008)). It has been found to translocate from the cytosol to the plasma membrane and associate with GPCR only once the receptors have been bound by their ligands and phosphorylated by GPCR kinases. In keeping with our previous findings (Kalant et al., 2005), there was obvious ligand-induced translocation of β-arrestin in RBL cells expressing human C5aR (Fig. 1B). β-Arrestin was seen throughout the cytosol prior to ligand stimulation but appeared to accumulate in granular structures after 15 min stimulation with 100 nM C5a. In contrast to previous studies that showed distinctive translocation of exogenous GFP-tagged β-arrestin in co-transfected HEK cells after stimulation with C5a (Kalant et al., 2005), we found there was no obvious translocation of β-arrestin 1 after C5a stimulation of human C5L2 (Fig. 1B). Human C5L2 is unable to induce the translocation of endogenous β-arrestin 1 in the RBL cell line.


The human complement fragment receptor, C5L2, is a recycling decoy receptor.

Scola AM, Johswich KO, Morgan BP, Klos A, Monk PN - Mol. Immunol. (2008)

C5a does not stimulate increases in intracellular Ca2+ or translocation of β-arrestin in RBL cells transfected with C5L2. (A) Fluo4-AM loaded RBL cells transfected with C5aR, wild type C5L2, C5L2 with C5aR-like sequences at three key signaling motifs (C5L2+++) or untransfected controls (NX) were stimulated with C5a (left panel) or C5a des Arg (right panel). Changes in the intracellular Ca2+ concentration were measured by flow cytometry as changes in fluorescence integrated over 70 s and shown as a percentage of fluorescence in unstimulated cells. Results shown are means of three separate experiments performed in duplicate ± S.E.M. (B) Transfected RBL cells adhered to coverslips were treated with 100 nM C5a for 15 min, fixed, permeabilized, incubated with antibody specific for β-arrestin 1. Arrows show intracellular accumulation of β-arrestin 1 in C5aR-transfected cells. Bar equal to 10 μm.
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fig1: C5a does not stimulate increases in intracellular Ca2+ or translocation of β-arrestin in RBL cells transfected with C5L2. (A) Fluo4-AM loaded RBL cells transfected with C5aR, wild type C5L2, C5L2 with C5aR-like sequences at three key signaling motifs (C5L2+++) or untransfected controls (NX) were stimulated with C5a (left panel) or C5a des Arg (right panel). Changes in the intracellular Ca2+ concentration were measured by flow cytometry as changes in fluorescence integrated over 70 s and shown as a percentage of fluorescence in unstimulated cells. Results shown are means of three separate experiments performed in duplicate ± S.E.M. (B) Transfected RBL cells adhered to coverslips were treated with 100 nM C5a for 15 min, fixed, permeabilized, incubated with antibody specific for β-arrestin 1. Arrows show intracellular accumulation of β-arrestin 1 in C5aR-transfected cells. Bar equal to 10 μm.
Mentions: Although several studies have failed to detect signaling by C5L2, others have demonstrated some capacity for signal transduction (Cain and Monk, 2002; Chen et al., 2007; Johswich et al., 2006; Kalant et al., 2005; Lee et al., 2008; Okinaga et al., 2003; Rittirsch et al., 2008). It is possible that C5L2 has a small capacity to transduce ligand-binding signals that is difficult to detect due to attenuation by the lack of highly conserved motifs (DRY in the 3rd transmembrane domain, NPXXY in the 7th transmembrane domain) and/or the short 3rd intracellular loop. To investigate this possibility, we made a mutant of C5L2 (C5L2+++) in which the motifs from the efficiently coupled C5a receptor, C5aR were substituted for the C5L2 sequence: D131LC-D131RF; N287PMLF–N287PMLY and the entire third intracellular loop sequence of C5aR. Signaling capacity was measured using the highly sensitive fluorescent intracellular free Ca2+ ([Ca2+]i) assay. When C5aR expressing RBL cells were stimulated with C5a (from 0.1 nM) there was a dramatic increase in fluorescence (Fig. 1A, left panel). In contrast, RBL cells transfected with wild type C5L2 or the mutant C5L2+++ showed no increase in fluorescence upon stimulation with concentrations of C5a of up to 100 nM, suggesting that no G protein-coupled signaling was occurring (Fig. 1A). Similarly, C5a des Arg could stimulate an increase in [Ca2+]i in C5aR-transfected RBL cells at concentrations of 10 nM and above but no changes were detectable in C5L2 or C5L2+++ transfected cells at any concentration used (Fig. 1A, right panel). Constructs of C5L2 with individual motifs mutated to their equivalents in C5aR had the same responses to both C5a and C5a des Arg (data not shown). A previous report showed some coupling of a C5L2 mutant (D131RC) to Gα16 in transiently co-transfected HEK cells (Okinaga et al., 2003). Signaling here may have been detected due to very high levels of receptor expression and also because the promiscuous Gα16 is less discriminating than the endogenous Gαi in RBL cells. Nevertheless, these results confirm that signaling by human C5L2 is highly repressed and there are multiple structural features that prevent Gαi activation by C5L2. β-Arrestin is a cytosolic protein that mediates the desensitization and internalization of GPCR and has also been shown to mediate G protein-independent signaling by 7 transmembrane domain receptors (reviewed by Defea (2008)). It has been found to translocate from the cytosol to the plasma membrane and associate with GPCR only once the receptors have been bound by their ligands and phosphorylated by GPCR kinases. In keeping with our previous findings (Kalant et al., 2005), there was obvious ligand-induced translocation of β-arrestin in RBL cells expressing human C5aR (Fig. 1B). β-Arrestin was seen throughout the cytosol prior to ligand stimulation but appeared to accumulate in granular structures after 15 min stimulation with 100 nM C5a. In contrast to previous studies that showed distinctive translocation of exogenous GFP-tagged β-arrestin in co-transfected HEK cells after stimulation with C5a (Kalant et al., 2005), we found there was no obvious translocation of β-arrestin 1 after C5a stimulation of human C5L2 (Fig. 1B). Human C5L2 is unable to induce the translocation of endogenous β-arrestin 1 in the RBL cell line.

Bottom Line: However, we detected neither an intracellular Ca(2+) response nor beta-arrestin redistribution in mutated C5L2, suggesting that the potential for G protein coupling is completely absent in this receptor and that, in humans, C5L2 may have functions that are unrelated to signaling.In confirmation of this, we detected constitutive ligand-independent internalization of C5L2 that resulted in the rapid accumulation of C5a and its stable metabolite, C5a des Arg, within the cell with only a small net change in cell surface receptor levels.Internalization was found to be through a clathrin-dependent mechanism that led to the retention and, in cells natively expressing C5L2, the degradation of the ligand within an intracellular compartment.

View Article: PubMed Central - PubMed

Affiliation: Academic Neurology Unit, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.

ABSTRACT
C5L2 is a 7 transmembrane domain receptor for complement fragment C5a that, unlike the classical C5a receptor, C5aR, does not couple to G proteins. However, in mice where C5L2 has been deleted, the response to C5a is altered, suggesting that C5L2 may have a signaling function. In order to investigate whether human C5L2 also has some capacity to transduce signals, we have attempted to produce a signaling competent form of human C5L2 by inserting C5aR sequences at three key G protein activation motifs. However, we detected neither an intracellular Ca(2+) response nor beta-arrestin redistribution in mutated C5L2, suggesting that the potential for G protein coupling is completely absent in this receptor and that, in humans, C5L2 may have functions that are unrelated to signaling. In confirmation of this, we detected constitutive ligand-independent internalization of C5L2 that resulted in the rapid accumulation of C5a and its stable metabolite, C5a des Arg, within the cell with only a small net change in cell surface receptor levels. Internalization was found to be through a clathrin-dependent mechanism that led to the retention and, in cells natively expressing C5L2, the degradation of the ligand within an intracellular compartment. In contrast, the classical C5a receptor, C5aR, internalized ligand much more slowly and a majority of this ligand was released back into the extracellular environment in an apparently undegraded form. These data suggest that a major function of human C5L2 is to remove active complement fragments from the extracellular environment.

Show MeSH