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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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Ligand uptake in HEK cells transfected with C5L2 is sensitive to inhibitors of clathrin-mediated internalization HEK cells were transiently transfected with either human C5L2 or C5aR, treated with NA = no addition; Ama = amantadine; CPZ = chlorpromazine; PAO = phenylarsine oxide; Nys = nystatin and incubated with 0.1 nM 125I-C5a for 2 h at either 4 or 37 °C, as indicated. The cells were subjected to extensive washing with either buffer at pH7.4 (buffer wash) or pH3 (acid wash). The data shown are from two separate experiments, mean ± S.D.
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fig7: Ligand uptake in HEK cells transfected with C5L2 is sensitive to inhibitors of clathrin-mediated internalization HEK cells were transiently transfected with either human C5L2 or C5aR, treated with NA = no addition; Ama = amantadine; CPZ = chlorpromazine; PAO = phenylarsine oxide; Nys = nystatin and incubated with 0.1 nM 125I-C5a for 2 h at either 4 or 37 °C, as indicated. The cells were subjected to extensive washing with either buffer at pH7.4 (buffer wash) or pH3 (acid wash). The data shown are from two separate experiments, mean ± S.D.

Mentions: A separate model system, transiently transfected HEK cells, also showed a temperature-dependent uptake of C5a by C5L2 but not by C5aR (Fig. 7A and B). After 2 h incubation at 37 °C, ∼90% of 125I-C5a could not be removed by an acid wash indicating that the majority of the C5a was internalized. The sensitivity of C5a uptake to a series of inhibitors of endocytosis was additionally tested in these cells (Fig. 7A and B): clathrin inhibitors amantadine (Schlegel et al., 1982), chlorpromazine and phenylarsene oxide (Frost and Lane, 1985) lowered the amounts of 125I-C5a that was resistant to acid wash whereas caveolin inhibitor nystatin had little effect. Intriguingly, there were decreases in the total binding of 125I-C5a at 37 °C to C5L2 in the presence of amantadine and chlorpromazine, suggesting that these inhibitors can decrease basal C5L2 expression.


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

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

Ligand uptake in HEK cells transfected with C5L2 is sensitive to inhibitors of clathrin-mediated internalization HEK cells were transiently transfected with either human C5L2 or C5aR, treated with NA = no addition; Ama = amantadine; CPZ = chlorpromazine; PAO = phenylarsine oxide; Nys = nystatin and incubated with 0.1 nM 125I-C5a for 2 h at either 4 or 37 °C, as indicated. The cells were subjected to extensive washing with either buffer at pH7.4 (buffer wash) or pH3 (acid wash). The data shown are from two separate experiments, mean ± S.D.
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Related In: Results  -  Collection

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fig7: Ligand uptake in HEK cells transfected with C5L2 is sensitive to inhibitors of clathrin-mediated internalization HEK cells were transiently transfected with either human C5L2 or C5aR, treated with NA = no addition; Ama = amantadine; CPZ = chlorpromazine; PAO = phenylarsine oxide; Nys = nystatin and incubated with 0.1 nM 125I-C5a for 2 h at either 4 or 37 °C, as indicated. The cells were subjected to extensive washing with either buffer at pH7.4 (buffer wash) or pH3 (acid wash). The data shown are from two separate experiments, mean ± S.D.
Mentions: A separate model system, transiently transfected HEK cells, also showed a temperature-dependent uptake of C5a by C5L2 but not by C5aR (Fig. 7A and B). After 2 h incubation at 37 °C, ∼90% of 125I-C5a could not be removed by an acid wash indicating that the majority of the C5a was internalized. The sensitivity of C5a uptake to a series of inhibitors of endocytosis was additionally tested in these cells (Fig. 7A and B): clathrin inhibitors amantadine (Schlegel et al., 1982), chlorpromazine and phenylarsene oxide (Frost and Lane, 1985) lowered the amounts of 125I-C5a that was resistant to acid wash whereas caveolin inhibitor nystatin had little effect. Intriguingly, there were decreases in the total binding of 125I-C5a at 37 °C to C5L2 in the presence of amantadine and chlorpromazine, suggesting that these inhibitors can decrease basal C5L2 expression.

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