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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 transfected CHO cells is more efficient than C5aR at internalizing and retaining C5a and C5a des Arg but ligand degradation does not occur in these cells. (Panels A and B) Ligand internalization: transfected cells expressing equal levels of receptor were loaded with 125I-C5a (A) or 100 nM 125I-C5a des Arg (B) at 4 °C for 1 h, then extensively washed and warmed to 37 °C for the indicated times. Cells were then washed in either ice-cold PBS or acidic medium to strip cell-surface ligand. Results are shown as percentage of radioactivity in acid-washed cells compared to cells washed in PBS and are the mean ± S.E.M. of 8–10 separate experiments performed in duplicate. (C–F) Ligand fate: cells expressing equal levels of receptor were loaded with 0.1 nM 125I-C5a (C and D) or 100 nM 125I-C5a des Arg (E and F) 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 total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of four separate experiments performed in duplicate. Significantly different from zero-time by two-way ANOVA with Bonferroni post-test *p < 0.05; ***p < 0.005, Student's t-test.
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fig6: C5L2 in transfected CHO cells is more efficient than C5aR at internalizing and retaining C5a and C5a des Arg but ligand degradation does not occur in these cells. (Panels A and B) Ligand internalization: transfected cells expressing equal levels of receptor were loaded with 125I-C5a (A) or 100 nM 125I-C5a des Arg (B) at 4 °C for 1 h, then extensively washed and warmed to 37 °C for the indicated times. Cells were then washed in either ice-cold PBS or acidic medium to strip cell-surface ligand. Results are shown as percentage of radioactivity in acid-washed cells compared to cells washed in PBS and are the mean ± S.E.M. of 8–10 separate experiments performed in duplicate. (C–F) Ligand fate: cells expressing equal levels of receptor were loaded with 0.1 nM 125I-C5a (C and D) or 100 nM 125I-C5a des Arg (E and F) 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 total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of four separate experiments performed in duplicate. Significantly different from zero-time by two-way ANOVA with Bonferroni post-test *p < 0.05; ***p < 0.005, Student's t-test.

Mentions: One feature of the chemokine decoy receptors is their ability to rapidly endocytose ligand. Further, ligands internalized by decoy receptor D6 are targeted for rapid degradation unlike ligands of the non-decoy receptor, CCR5 (Weber et al., 2004). We were interested to see if C5L2 also had this ability. Using 125I-C5a and C5a des Arg, we carried out ligand-uptake experiments, using an acid wash step to remove cell surface ligand. In transfected CHO cells which expressed high (and similar) levels of surface C5aR and C5L2, rapid internalization of both ligands was observed; this was significantly higher on cells expressing C5L2 as compared to cells expressing C5aR (Fig. 6A and B). There was almost no uptake of C5a des Arg by C5aR, probably due to the lower affinity of C5aR for this protein. Transfected RBL cells also showed C5L2-dependent uptake of C5a/C5a des Arg, although to a lesser extent, most likely due to lower receptor expression levels (data not shown). We also investigated whether the ligand endocytosed by C5L2 became degraded, estimated by the appearance of TCA-soluble radioactivity. As expected, there were significantly higher levels of 125I-C5a retained in the cell pellet of C5L2-transfected CHO cells (∼20% at 20, 60 and 120 min (p < 0.001)) compared to C5aR. However there was no significant increase in the degradation of 125I-C5a or 125I-C5a des Arg over time between C5aR and C5L2 (Fig. 6C–F). Ectopic expression of C5L2 appears, in this case, to confer the ability to store ligand internally but not to cause its degradation.


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 transfected CHO cells is more efficient than C5aR at internalizing and retaining C5a and C5a des Arg but ligand degradation does not occur in these cells. (Panels A and B) Ligand internalization: transfected cells expressing equal levels of receptor were loaded with 125I-C5a (A) or 100 nM 125I-C5a des Arg (B) at 4 °C for 1 h, then extensively washed and warmed to 37 °C for the indicated times. Cells were then washed in either ice-cold PBS or acidic medium to strip cell-surface ligand. Results are shown as percentage of radioactivity in acid-washed cells compared to cells washed in PBS and are the mean ± S.E.M. of 8–10 separate experiments performed in duplicate. (C–F) Ligand fate: cells expressing equal levels of receptor were loaded with 0.1 nM 125I-C5a (C and D) or 100 nM 125I-C5a des Arg (E and F) 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 total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of four separate experiments performed in duplicate. Significantly different from zero-time by two-way ANOVA with Bonferroni post-test *p < 0.05; ***p < 0.005, Student's t-test.
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fig6: C5L2 in transfected CHO cells is more efficient than C5aR at internalizing and retaining C5a and C5a des Arg but ligand degradation does not occur in these cells. (Panels A and B) Ligand internalization: transfected cells expressing equal levels of receptor were loaded with 125I-C5a (A) or 100 nM 125I-C5a des Arg (B) at 4 °C for 1 h, then extensively washed and warmed to 37 °C for the indicated times. Cells were then washed in either ice-cold PBS or acidic medium to strip cell-surface ligand. Results are shown as percentage of radioactivity in acid-washed cells compared to cells washed in PBS and are the mean ± S.E.M. of 8–10 separate experiments performed in duplicate. (C–F) Ligand fate: cells expressing equal levels of receptor were loaded with 0.1 nM 125I-C5a (C and D) or 100 nM 125I-C5a des Arg (E and F) 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 total radioactivity per sample found in cell pellet, precipitated and non-precipitated protein from supernatant and are the mean ± S.E.M. of four separate experiments performed in duplicate. Significantly different from zero-time by two-way ANOVA with Bonferroni post-test *p < 0.05; ***p < 0.005, Student's t-test.
Mentions: One feature of the chemokine decoy receptors is their ability to rapidly endocytose ligand. Further, ligands internalized by decoy receptor D6 are targeted for rapid degradation unlike ligands of the non-decoy receptor, CCR5 (Weber et al., 2004). We were interested to see if C5L2 also had this ability. Using 125I-C5a and C5a des Arg, we carried out ligand-uptake experiments, using an acid wash step to remove cell surface ligand. In transfected CHO cells which expressed high (and similar) levels of surface C5aR and C5L2, rapid internalization of both ligands was observed; this was significantly higher on cells expressing C5L2 as compared to cells expressing C5aR (Fig. 6A and B). There was almost no uptake of C5a des Arg by C5aR, probably due to the lower affinity of C5aR for this protein. Transfected RBL cells also showed C5L2-dependent uptake of C5a/C5a des Arg, although to a lesser extent, most likely due to lower receptor expression levels (data not shown). We also investigated whether the ligand endocytosed by C5L2 became degraded, estimated by the appearance of TCA-soluble radioactivity. As expected, there were significantly higher levels of 125I-C5a retained in the cell pellet of C5L2-transfected CHO cells (∼20% at 20, 60 and 120 min (p < 0.001)) compared to C5aR. However there was no significant increase in the degradation of 125I-C5a or 125I-C5a des Arg over time between C5aR and C5L2 (Fig. 6C–F). Ectopic expression of C5L2 appears, in this case, to confer the ability to store ligand internally but not to cause its degradation.

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