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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 does not undergo ligand-dependent internalization in transfected RBL cells. Transfected RBL cells were incubated with 100 nM C5a at 37 °C for 15 min and then fixed and permeabilized. Receptor was visualized with antibody specific for the N termini of C5aR or the N-terminal HA tag of C5L2. Bar equal to 10 μm.
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fig3: C5L2 does not undergo ligand-dependent internalization in transfected RBL cells. Transfected RBL cells were incubated with 100 nM C5a at 37 °C for 15 min and then fixed and permeabilized. Receptor was visualized with antibody specific for the N termini of C5aR or the N-terminal HA tag of C5L2. Bar equal to 10 μm.

Mentions: For many GPCR, ligand binding increases the receptor internalization rate, manifested as a reduction in cell surface receptor levels (Marchese et al., 2008). However, previous reports by our group and others (Cain and Monk, 2002; Okinaga et al., 2003) have shown that, unlike C5aR, C5L2 does not undergo ligand-induced internalization upon stimulation with ligand. The lack of internalization or re-distribution of C5L2 was confirmed by fluorescence microscopy of transfected RBL cells, where the ligand-induced accumulation of C5aR in discrete granular structures is obvious (Fig. 3). In contrast, C5L2 was primarily located in intracellular granular structures prior to the addition of ligand. Again, NPXXY-C5L2 behaved identically to the wild type receptor (data not shown).


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 does not undergo ligand-dependent internalization in transfected RBL cells. Transfected RBL cells were incubated with 100 nM C5a at 37 °C for 15 min and then fixed and permeabilized. Receptor was visualized with antibody specific for the N termini of C5aR or the N-terminal HA tag of C5L2. Bar equal to 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697321&req=5

fig3: C5L2 does not undergo ligand-dependent internalization in transfected RBL cells. Transfected RBL cells were incubated with 100 nM C5a at 37 °C for 15 min and then fixed and permeabilized. Receptor was visualized with antibody specific for the N termini of C5aR or the N-terminal HA tag of C5L2. Bar equal to 10 μm.
Mentions: For many GPCR, ligand binding increases the receptor internalization rate, manifested as a reduction in cell surface receptor levels (Marchese et al., 2008). However, previous reports by our group and others (Cain and Monk, 2002; Okinaga et al., 2003) have shown that, unlike C5aR, C5L2 does not undergo ligand-induced internalization upon stimulation with ligand. The lack of internalization or re-distribution of C5L2 was confirmed by fluorescence microscopy of transfected RBL cells, where the ligand-induced accumulation of C5aR in discrete granular structures is obvious (Fig. 3). In contrast, C5L2 was primarily located in intracellular granular structures prior to the addition of ligand. Again, NPXXY-C5L2 behaved identically to the wild type receptor (data not shown).

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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