C5L2 Is a Functional Receptor for Acylation-stimulating Protein

Katherine Cianflone,David Kalant,Peter N Monk,Wei Cui,Stephane A Laporte,Robin Maclaren,Ratna Samanta

doi:10.1074/jbc.m406921200

Abstract

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.

Highlights

The function of acylation-stimulating protein (ASP,1 known as C3a des-Arg) as a stimulator of triglyceride synthesis (TGS) has been well documented in human adipocytes, 3T3-L1 preadipocytes, and human skin fibroblasts (HSF) [2,3,4,5]
We recently have reported that the orphan G protein-coupled receptor (GPCR) C5L2 binds ASP and C3a with high affinity in transfected RBL cells and exhibits saturable binding of ASP in transfected HEK293 cells [1]
We demonstrated the expression of C5L2 mRNA in human adipose tissue, HSF, and 3T3-L1 by reverse transcription-PCR and expression of the protein on the cell surface of HSF using anti-C5L2 polyclonal antiserum

Summary

EXPERIMENTAL PROCEDURES

Analysis of Murine Receptor Expression by Real Time PCR—Wild type C57BL6 mice were sacrificed at 9 –10 weeks of age and dissected, and ϳ50 –100 mg of tissue was stored in 500 ␮l of RNAlater stabilization reagent (Qiagen, Mississauga, Canada) at Ϫ80 °C. Cells were incubated with vehicle (phosphate-buffered saline), ASP, or insulin (Sigma) at the indicated concentrations in the presence of 100 ␮M [3H]oleate complexed to albumin (molar ratio 5:1, specific activity 50 –70 dpm/pmol oleate) diluted in serum-free medium for 4 h at 37 °C. The reaction was stopped with rapid washing of the cells with cold phosphate-buffered saline, and cell protein was dissolved in 0.1 N NaOH. Cell Surface C5L2 Quantitation Assay—Following incubation in serum-free medium for 2 h, HSF and 3T3-L1 cells were incubated with anti-human or anti-mouse polyclonal C5L2 antiserum, respectively, at a 1:100 dilution in buffer containing 20 mM Hepes, 0.25% albumin, 6.5 mM glucose, 1 mM calcium, 1 mM magnesium, 125 mM NaCl, 5 mM KCl, pH 7.2 (HAG-CM), for 30 min at room temperature. Significance was determined by t test or by ANOVA (with Bonferroni post hoc test) where significance was set at p Ͻ 0.05

RESULTS AND DISCUSSION

NS NS NS

NS NS

With insulin p

Antisense experiments were also carried out in mouse