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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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C5L2 in HeLa cells is responsible for internalizing, retaining and degrading C5a and C5a des Arg. HeLa cells were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The results are shown as a percentage of the total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of two to three separate experiments performed in duplicate. Significantly different from controls at the zero time-point by two-way ANOVA with Bonferroni post-test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.005.
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fig10: C5L2 in HeLa cells is responsible for internalizing, retaining and degrading C5a and C5a des Arg. HeLa cells were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The results are shown as a percentage of the total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of two to three separate experiments performed in duplicate. Significantly different from controls at the zero time-point by two-way ANOVA with Bonferroni post-test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.005.

Mentions: A clear degradation of both C5a and C5a des Arg was also observed in both differentiated HL-60 and HeLa cells (Figs. 9 and 10), measured as the increase of TCA-soluble radioactivity with time. In HL-60 cells, 50–60% of ligand was apparently degraded after 240 min and, again, this effect was not inhibited by C5aR antagonist, strongly suggesting that ligand degradation occurs after uptake by C5L2 (Fig. 9A–D). To prove that cellular uptake was indeed a prerequisite for degradation of the radioligand, we treated differentiated HL-60 cells with the clathrin inhibitor, phenylarsine oxide, which abolished ligand degradation (Fig. 9E and F) and led to the release of intact ligand back into the supernatant. When the cells underwent ATP depletion by treatment with sodium azide and 2-deoxy-d-glucose, they also lost their capacity to degrade ligand (Fig. 9G and H) and again released undegraded ligand. 125I-C5a binding was normal (data not shown) in the presence of all of these inhibitors. Viability seemed to be affected to only a small extent by these treatments, as measured by trypan blue exclusion: 83.4 ± 0.67% (88.2 ± 1.82%) of azide/2-deoxy-d-glucose and 60.6 ± 5.35% (89.5 ± 1.68%) of phenylarsine oxide treated cells excluded dye (mean ± S.D., viability of untreated control cells in brackets). Importantly, C5a incubated with undifferentiated HL-60 cells which express neither C5aR nor C5L2 showed no degradation at all, even after 120 min (data not shown). HeLa cells internalized 125I-C5a rapidly but little degradation was apparent, even after 4 h (Fig. 10A). By contrast, 125I-C5a des Arg was both internalized and degraded rapidly, with most radiolabel in a TCA-soluble form within 20 min (Fig. 10B).


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

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

C5L2 in HeLa cells is responsible for internalizing, retaining and degrading C5a and C5a des Arg. HeLa cells were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The results are shown as a percentage of the total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of two to three separate experiments performed in duplicate. Significantly different from controls at the zero time-point by two-way ANOVA with Bonferroni post-test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.005.
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fig10: C5L2 in HeLa cells is responsible for internalizing, retaining and degrading C5a and C5a des Arg. HeLa cells were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The results are shown as a percentage of the total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of two to three separate experiments performed in duplicate. Significantly different from controls at the zero time-point by two-way ANOVA with Bonferroni post-test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.005.
Mentions: A clear degradation of both C5a and C5a des Arg was also observed in both differentiated HL-60 and HeLa cells (Figs. 9 and 10), measured as the increase of TCA-soluble radioactivity with time. In HL-60 cells, 50–60% of ligand was apparently degraded after 240 min and, again, this effect was not inhibited by C5aR antagonist, strongly suggesting that ligand degradation occurs after uptake by C5L2 (Fig. 9A–D). To prove that cellular uptake was indeed a prerequisite for degradation of the radioligand, we treated differentiated HL-60 cells with the clathrin inhibitor, phenylarsine oxide, which abolished ligand degradation (Fig. 9E and F) and led to the release of intact ligand back into the supernatant. When the cells underwent ATP depletion by treatment with sodium azide and 2-deoxy-d-glucose, they also lost their capacity to degrade ligand (Fig. 9G and H) and again released undegraded ligand. 125I-C5a binding was normal (data not shown) in the presence of all of these inhibitors. Viability seemed to be affected to only a small extent by these treatments, as measured by trypan blue exclusion: 83.4 ± 0.67% (88.2 ± 1.82%) of azide/2-deoxy-d-glucose and 60.6 ± 5.35% (89.5 ± 1.68%) of phenylarsine oxide treated cells excluded dye (mean ± S.D., viability of untreated control cells in brackets). Importantly, C5a incubated with undifferentiated HL-60 cells which express neither C5aR nor C5L2 showed no degradation at all, even after 120 min (data not shown). HeLa cells internalized 125I-C5a rapidly but little degradation was apparent, even after 4 h (Fig. 10A). By contrast, 125I-C5a des Arg was both internalized and degraded rapidly, with most radiolabel in a TCA-soluble form within 20 min (Fig. 10B).

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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