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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 differentiated HL-60 and HeLa cells is responsible for internalizing C5a. Bt2cAMP-differentiated HL-60 and HeLa cells were loaded with 125I-C5a at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], 100 μM of phenylarsine oxide or 50 μg/ml nystatin, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and washed in acidic buffer to strip surface bound ligand. The results show uptake of radioactivity compared to controls incubated at 4 °C and are the mean ± S.E.M. of three to six separate experiments performed in duplicate. The amount of radioactivity actually internalized by HL-60 cells is shown for each condition. Significantly different from control at the same time-point by two-way ANOVA with Bonferroni post-test; *p < 0.05, ***p < 0.005.
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fig8: C5L2 in differentiated HL-60 and HeLa cells is responsible for internalizing C5a. Bt2cAMP-differentiated HL-60 and HeLa cells were loaded with 125I-C5a at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], 100 μM of phenylarsine oxide or 50 μg/ml nystatin, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and washed in acidic buffer to strip surface bound ligand. The results show uptake of radioactivity compared to controls incubated at 4 °C and are the mean ± S.E.M. of three to six separate experiments performed in duplicate. The amount of radioactivity actually internalized by HL-60 cells is shown for each condition. Significantly different from control at the same time-point by two-way ANOVA with Bonferroni post-test; *p < 0.05, ***p < 0.005.

Mentions: It is possible that transfected cells lack aspects of the cellular machinery for the degradation of ligand internalized by C5L2. We tested this possibility in two cell lines: bt2-cAMP differentiated HL-60 cells, which endogenously express C5aR and C5L2, and also in HeLa cells, which only endogenously express C5L2 (Johswich et al., 2006). Using 125I-C5a (and removing remaining free ligand by a washing step before cells were warmed up to 37 °C), internalization was rapid and sensitive to the clathrin inhibitor, phenylarsine oxide, in both cell types (Fig. 8A–C). Nystatin moderately inhibited internalization in HL-60 cells (by ∼50%) but no effect of nystatin (<10%) was observed when C5aR was blocked by AcF[OPdChaWR], i.e. when only internalization of 125I-C5a by C5L2 was measured. As the total amount of acid wash resistant radioactivity in nystatin-treated cells was almost identical in cells with or without AcF[OPdChaWR] (1989 ± 216 CPM versus 1926 ± 81 CPM per 106 cells), only C5aR internalization seems to be dependent on caveolin in differentiated HL-60 cells. Thus, C5L2-mediated, clathrin-dependent internalization of ligand also occurs in cells that naturally express this receptor.


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 differentiated HL-60 and HeLa cells is responsible for internalizing C5a. Bt2cAMP-differentiated HL-60 and HeLa cells were loaded with 125I-C5a at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], 100 μM of phenylarsine oxide or 50 μg/ml nystatin, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and washed in acidic buffer to strip surface bound ligand. The results show uptake of radioactivity compared to controls incubated at 4 °C and are the mean ± S.E.M. of three to six separate experiments performed in duplicate. The amount of radioactivity actually internalized by HL-60 cells is shown for each condition. Significantly different from control at the same time-point by two-way ANOVA with Bonferroni post-test; *p < 0.05, ***p < 0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2697321&req=5

fig8: C5L2 in differentiated HL-60 and HeLa cells is responsible for internalizing C5a. Bt2cAMP-differentiated HL-60 and HeLa cells were loaded with 125I-C5a at 4 °C for 1 h and (where indicated) 10 μM of the C5aR antagonist AcF[OPdChaWR], 100 μM of phenylarsine oxide or 50 μg/ml nystatin, then extensively washed and shifted to 37 °C for the specified times. Cells were harvested and washed in acidic buffer to strip surface bound ligand. The results show uptake of radioactivity compared to controls incubated at 4 °C and are the mean ± S.E.M. of three to six separate experiments performed in duplicate. The amount of radioactivity actually internalized by HL-60 cells is shown for each condition. Significantly different from control at the same time-point by two-way ANOVA with Bonferroni post-test; *p < 0.05, ***p < 0.005.
Mentions: It is possible that transfected cells lack aspects of the cellular machinery for the degradation of ligand internalized by C5L2. We tested this possibility in two cell lines: bt2-cAMP differentiated HL-60 cells, which endogenously express C5aR and C5L2, and also in HeLa cells, which only endogenously express C5L2 (Johswich et al., 2006). Using 125I-C5a (and removing remaining free ligand by a washing step before cells were warmed up to 37 °C), internalization was rapid and sensitive to the clathrin inhibitor, phenylarsine oxide, in both cell types (Fig. 8A–C). Nystatin moderately inhibited internalization in HL-60 cells (by ∼50%) but no effect of nystatin (<10%) was observed when C5aR was blocked by AcF[OPdChaWR], i.e. when only internalization of 125I-C5a by C5L2 was measured. As the total amount of acid wash resistant radioactivity in nystatin-treated cells was almost identical in cells with or without AcF[OPdChaWR] (1989 ± 216 CPM versus 1926 ± 81 CPM per 106 cells), only C5aR internalization seems to be dependent on caveolin in differentiated HL-60 cells. Thus, C5L2-mediated, clathrin-dependent internalization of ligand also occurs in cells that naturally express this receptor.

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