Elsevier

Molecular Immunology

Volume 46, Issue 15, September 2009, Pages 3086-3098
Molecular Immunology

C5a- and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I-C5L2 mutation

https://doi.org/10.1016/j.molimm.2009.06.007Get rights and content

Abstract

C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca2+ or chemotaxis changes.

Aims

(i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality.

Results

stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. β-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent β-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and 125I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of β-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis.

Conclusion

C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.

Introduction

C5L2, first identified as an orphan receptor by Ohno et al. (2000) and Lee et al. (2001), belongs to the seven transmembrane G-protein-coupled receptor (GPCR) superfamily and consists of 337 amino acids (37 kDa) (Ohno et al., 2000). C5L2 shares 38%, 33%, 29%, and 29% amino acid identity with C5a, C3a, fMLP, and ChemR23 receptors, respectively (Ohno et al., 2000). C5L2 is expressed in many tissues, including all adipose tissues, such as pectoral, perirenal, gonadal and inguinal fat depots, and brown adipose tissue, in addition to brain, kidney, liver, spleen, intestine and myeloid cells (Kalant et al., 2005). The function of C5L2 remains controversial with questions regarding: (i) ligand binding, (ii) activation and functional response and (iii) impact in C5L2 knockout animal models.

Both C5a and C5adesArg have been shown to bind C5L2, but with differing affinities (Cain and Monk, 2002). A functional receptor for C5a has been previously identified as CD88 (C5aR), which is a 39 kDa protein (350 amino acids) member of the anaphylatoxin receptor family which belongs to the rhodopsin-like GPCR superfamily (Gerard and Gerard, 1991). C5aR is expressed in granulocytes, macrophages, mast cells and dendritic cells (Chenoweth and Goodman, 1983). In addition, it has been shown that most organs express C5aR such as smooth muscle, liver, lung, kidney, spleen, small intestine, and heart (Fayyazi et al., 2000). C5a binding to C5aR induces inflammatory effects such as chemotaxis and oxidative burst (Burg et al., 1996), and β-hexosaminidase release (Cain and Monk, 2002). C5adesArg binds with lower affinity, resulting in decreased or absence of response (Perez, 1984). It has been well documented that stimulation of C5aR with C5a induces rapid phosphorylation (<2 min) and subsequent internalization (Giannini and Boulay, 1995). By 5 min post-stimulation, 60% of C5aR is internalized and after 10 min the internalized receptors within endosomes localize to the perinuclear region (Naik et al., 1997). Using analogous methodology and time frame, several studies have consistently shown that C5a binding to C5L2 did not result in stimulation of β-hexosaminidase release or increased intracellular calcium concentration (Cain and Monk, 2002, Ohno et al., 2000). These two criteria have typically been used to identify decoy chemokine receptors (Borroni et al., 2008). Accordingly, it has been suggested that C5L2 does not interact with G-proteins and acts as a decoy receptor for C5a. However there are many downstream effectors of G-proteins which are Ca2+independent (Brambilla et al., 2002, Kehrl, 1998), and this alone cannot be used to exclude a functional role for C5L2.

In fact, a body of evidence does suggest C5L2 functionality. C5a activation of C5L2 does result in phosphorylation of C5L2 (Okinaga et al., 2003), enhances the IgE effect on β-hexosaminidase release (Cain and Monk, 2002) and induces C5L2 internalization (Kalant et al., 2005), albeit on a slower time scale than with C5aR. Furthermore, macrophages and neutrophils from C5L2 knockout mice had decreased or absent C5a- and/or C3a-mediated Akt phosphorylation and ERK1/2 phosphorylation (in spite of expression of C5aR and C3aR) (Chen et al., 2007).

Acylation stimulating protein (ASP, also known as C3adesArg) is an anabolic hormone which regulates fat storage in tissues by stimulating triglyceride (TG) synthesis and glucose transport (Kalant et al., 2005). ASP (C3adesArg) and C3a have also been shown to bind C5L2 (Cain and Monk, 2002, Kalant et al., 2003, Kalant et al., 2005). By contrast, C3a, but not C3adesArg (ASP), binds to the C3a receptor (C3aR) (Kohl, 2001), and neither C3a nor ASP binds to C5aR (Kalant et al., 2005). In C5L2-HEK stably transfected cells sorted through fluorescent-ASP binding, ASP stimulates TG synthesis and glucose transport, while there is no effect in HEK 293 cells (Kalant et al., 2005). It has also been demonstrated that ASP-mediated C5L2 activation induced C5L2 phosphorylation, and increased β-arrestin-2-GFP translocation to the cell-surface membrane followed by enhanced formation of β-arrestin-2-GFP associated intracellular endosomes (Kalant et al., 2005). In the absence of ligand or C5L2, there was no change in β-arrestin-2-GFP distribution (Kalant et al., 2005). In addition, loss-of-function using antisense or si-RNA in C5L2 endogenously expressed in 3T3-L1 preadipocytes and human skin fibroblasts (HSF) cells resulted in a decrease in TG synthesis, coordinate with a reduction of C5L2 expression (Kalant et al., 2005). Furthermore, in 3T3-L1 cells, ASP induces ERK1/2 phosphorylation and Akt phosphorylation (Maslowska et al., 2006), consistent with the results in C5L2 KO mice described above (Chen et al., 2007), as well as phospholipase A2 and protein kinase C activation (Baldo et al., 1995, Maslowska et al., 2006). Blocking these signalling pathways blocks the ASP induced stimulation of TG synthesis (Maslowska et al., 2006), and in C5L2 KO mice, ASP no longer stimulates TG synthesis in adipocytes (Paglialunga et al., 2007). Thus the only functional receptor identified to date for ASP is C5L2.

As a lipogenic adipokine, ASP plays a potent role in post-prandial clearance in vivo and is linked to hyperlipidemic disorders, characterized by increased plasma triglycerides or apolipoprotein B containing lipoproteins (Cianflone et al., 2003). Moreover, hyperlipidemia is also associated with coronary artery disease, metabolic syndrome, insulin resistance and other associated metabolic diseases (Faraj et al., 2004). Genomic sequencing of the C5L2 coding region in subjects with coronary heart disease identified a naturally occurring C5L2 mutation with a substitution of serine 323 by isoleucine in the C-terminal intracellular region (S323I). This proband was characterized by increased circulating plasma ASP, triglycerides and apolipoprotein B. Increased circulating triglycerides are often associated with delayed plasma clearance due to decreased fatty acid uptake and esterification in storage tissues such as adipose tissue (Cianflone et al., 2003). Although this mutation was not detected in scanning an additional 2176 subjects, 8 out of 17 family members were identified as heterozygote for S323I-C5L2. The heterozygote family members had significantly higher plasma ASP, apolipoprotein B and triglyceride levels. Bioactivity and competition binding assays in cells obtained from C5L2 (+/−) subjects showed that ASP stimulation of TG synthesis and glucose transport was reduced by 50%, as was ASP binding, compared to cells obtained from siblings and un-related subjects expressing only wildtype C5L2(+/+), suggesting altered C5L2 function (Marcil et al., 2006).

Following ligand activation and internalization, GPCRs are recycled back to cell-surface membrane or targeted for degradation. To study trafficking of internalized receptors, Rab proteins, Ras-like small G-proteins, are commonly used. Rab proteins are involved in exocytosis, endocytosis, endosome fusion, and trafficking (Seachrist and Ferguson, 2003). Rab5 is involved in formation and endocytosis of plasma membrane clathrin-coated vesicles and fusion with early endosomes, and subsequent sorting. Rab11 is usually present in perinuclear recycling endosomes and is involved in regulation of slow receptor recycling traffic from early endosomes to recycling endosomes or the trans-golgi network. Rab7 is localized to late endosomes and lysosomes, regulating trafficking of receptors to late endosomes and lysosomes for degradation.

In the present study, our aims were: (i) to compare ASP- and C5a-mediated C5L2 internalization using colocalization of receptor with β-arrestin-2-GFP, (ii) to study trafficking of internalized C5L2 with Rab5, Rab7, and Rab11 and (iii) to characterize the effects of the previously identified naturally occurring S323I mutation on C5L2 activation and internalization.

Section snippets

Recombinant ASP and C5a

Recombinant ASP (rASP) was purified based on a modification of a previously published method (Murray et al., 1997), using a His-Tag labelled recombinant ASP, with purification using Ni+ affinity chromatography (GE Healthcare, Piscataway, NJ) followed by HPLC (Murray et al., 1997). Fluorescently labelled Fluos-ASP was prepared as described previously (Kalant et al., 2003). Recombinant C5a (rC5a) was purchased from EMD Biosciences (Gibbstown, NJ). Note, as normal human physiological plasma

rASP induces β-arrestin-2-mediated C5L2 internalization

Previously, we demonstrated that ASP stimulation of C5L2-HEK cells activated β-arrestin-2 translocation to the cell membrane followed by formation of endosomes (Kalant et al., 2005), while non-transfected HEK cells were non-responsive. Further, in the absence of ligand, there was no change in β-arrestin-2 distribution (Kalant et al., 2005). To demonstrate: (i) that ASP indeed activates C5L2 and (ii) that C5L2 is subsequently internalized we evaluated β-arrestin-2-GFP translocation, C5L2

Discussion

In the present study we address directly two controversial issues: (i) is ASP/C3adesArg a ligand for C5L2? and (ii) is C5L2 a receptor that is activated, internalized and recycled following ligand stimulation? Our data suggest that both ASP and C5a bind and activate C5L2, and that ASP stimulates a functional response. Further support for this is demonstrated by the lack of activation by C5a and ASP of the C5L2 S323I mutation. Specifically, we clearly demonstrate that with both C5a and

Conclusion

In conclusion, the present paper demonstrated that both ASP and C5a are cognate ligands of C5L2. ASP as well as C5a induces β-arrestin-2 recruitment, C5L2 endocytosis, and recycling. As ASP is an anabolic hormone and C5a is an inflammatory factor, C5L2 may be one more bridge between the immune and the adipose systems (MacLaren et al., 2008), integrating multiple physiological systems.

Acknowledgements

This study was supported by a grant from CIHR (#64446 to KC). K. Cianflone holds a Canada Research Chair in Adipose Tissue.

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