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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 polymorponuclear neutrophils is responsible for internalizing, retaining and degrading C5a and C5a des Arg. Neutrophils were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The total radioactivity in each sample is given for A–D and the bars show the percentage of the total radioactivity per sample found in cell pellet, precipitated or non-precipitated protein from supernatant and are the mean ± S.E.M. of two 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 < 0.005.
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fig11: C5L2 in polymorponuclear neutrophils is responsible for internalizing, retaining and degrading C5a and C5a des Arg. Neutrophils were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The total radioactivity in each sample is given for A–D and the bars show the percentage of the total radioactivity per sample found in cell pellet, precipitated or non-precipitated protein from supernatant and are the mean ± S.E.M. of two 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 < 0.005.

Mentions: Finally, we wanted to observe C5L2-mediated degradation of C5a/C5a des Arg in primary cells. Human PMN are known to express both C5aR and C5L2 (Okinaga et al., 2003). Using the competitive 125I-C5a binding assay, we found that the surface expression of C5L2 in PMN is weak compared to that of C5aR (Bmax = 4617 ± 1373 versus 97306 ± 42658 molecules per cell, mean ± standard error, 4 donors). The percentage of 125I-C5a bound to C5L2 (i.e. in the presence of the C5aR antagonist AcF[OPdChaWR]) relative to C5aR varied between donors (range: 2.4–29.7%; median 4.5%), suggesting a wide natural variation in C5L2 expression. As observed in HeLa cells, degradation of 125I-C5a des Arg in PMN appears to occur faster than 125I-C5a (Fig. 11A–D). Total neutrophil uptake of C5a (comparing absolute CPM values) was decreased by AcF[OPdChaWR] more markedly than in HL-60 cells, C5L2 being accountable for only 25% (Fig. 11A and B). In contrast, there was virtually no effect of C5aR antagonist AcF[OPdChaWR] on the total uptake of 125I-C5a des Arg by PMN (Fig. 11C and D), confirming that C5L2 is the main receptor for this processed form of C5a. Taken together, these data suggest that C5L2, in an appropriate cellular milieu, target ligands for 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 polymorponuclear neutrophils is responsible for internalizing, retaining and degrading C5a and C5a des Arg. Neutrophils were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The total radioactivity in each sample is given for A–D and the bars show the percentage of the total radioactivity per sample found in cell pellet, precipitated or non-precipitated protein from supernatant and are the mean ± S.E.M. of two 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 < 0.005.
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fig11: C5L2 in polymorponuclear neutrophils is responsible for internalizing, retaining and degrading C5a and C5a des Arg. Neutrophils were loaded with 125I-C5a or 125I-C5a des Arg at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and the supernatant subjected to TCA precipitation. The total radioactivity in each sample is given for A–D and the bars show the percentage of the total radioactivity per sample found in cell pellet, precipitated or non-precipitated protein from supernatant and are the mean ± S.E.M. of two 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 < 0.005.
Mentions: Finally, we wanted to observe C5L2-mediated degradation of C5a/C5a des Arg in primary cells. Human PMN are known to express both C5aR and C5L2 (Okinaga et al., 2003). Using the competitive 125I-C5a binding assay, we found that the surface expression of C5L2 in PMN is weak compared to that of C5aR (Bmax = 4617 ± 1373 versus 97306 ± 42658 molecules per cell, mean ± standard error, 4 donors). The percentage of 125I-C5a bound to C5L2 (i.e. in the presence of the C5aR antagonist AcF[OPdChaWR]) relative to C5aR varied between donors (range: 2.4–29.7%; median 4.5%), suggesting a wide natural variation in C5L2 expression. As observed in HeLa cells, degradation of 125I-C5a des Arg in PMN appears to occur faster than 125I-C5a (Fig. 11A–D). Total neutrophil uptake of C5a (comparing absolute CPM values) was decreased by AcF[OPdChaWR] more markedly than in HL-60 cells, C5L2 being accountable for only 25% (Fig. 11A and B). In contrast, there was virtually no effect of C5aR antagonist AcF[OPdChaWR] on the total uptake of 125I-C5a des Arg by PMN (Fig. 11C and D), confirming that C5L2 is the main receptor for this processed form of C5a. Taken together, these data suggest that C5L2, in an appropriate cellular milieu, target ligands for 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.

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