Kalant David, MacLaren Robin, Cui Wei, Samanta Ratna, Monk Peter N, Laporte Stephane A, Cianflone Katherine
Mike Rosenbloom Laboratory for Cardiovascular Research, Division of Cardiology, Department of Medicine, McGill University Health Centre, Montreal, Quebec H3A 1A1, Canada.
J Biol Chem. 2005 Jun 24;280(25):23936-44. doi: 10.1074/jbc.M406921200. Epub 2005 Apr 14.
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.
C5L2与酰化刺激蛋白（ASP）具有高亲和力结合，并在对ASP有反应的细胞中表达。C5L2的功能尚未得到证实。在此我们表明，C5L2在人前脂肪细胞和脂肪细胞的皮下及网膜脂肪组织中表达。在小鼠中，C5L2在所有脂肪组织中表达，其水平与其他组织相当。将人C5L2 cDNA稳定转染至HEK293细胞可导致ASP刺激甘油三酯合成（TGS）（193±33%，5μM ASP，p<0.001，基础水平=100%）和葡萄糖转运（168±21%，10μM ASP，p<0.001）。C3a同样刺激TGS（163±12%，p<0.001），但C5a和C5a去精氨酸对其无影响。ASP的作用机制是通过增加二酰甘油酰基转移酶活性（200%）来提高葡萄糖转运的Vmax（149%）和甘油三酯（TG）合成活性（165%）。人皮肤成纤维细胞中C5L2的反义寡核苷酸下调降低了细胞表面C5L2（降至对照的54±4%，p<0.001，与非免疫背景相当）。ASP反应也相应丧失（基础TGS=14.6±1.6，有ASP时=21.0±1.4（144%），有ASP+寡核苷酸时=11.0±0.8 pmol TG/mg细胞蛋白，p<0.001）。在小鼠3T3-L1前脂肪细胞中，反义寡核苷酸将C5L2表达降低至对照的69.5±0.5%，p<0.001（与非免疫相当），同时丧失了ASP刺激（基础TGS=22.4±2.9，有ASP时=39.6±8.8（177%），有ASP+寡核苷酸时=25.3±3.0 pmol TG/mg细胞蛋白，p<0.001）。C5L2下调与ASP反应降低相关（人皮肤成纤维细胞中r=0.761，p<0.0001；3T3-L1细胞中r=0.451，p<0.05）。在表达荧光标记β-抑制蛋白的HEK-hC5L2细胞中，ASP诱导β-抑制蛋白转位至质膜并形成内吞复合物，同时C5L2的磷酸化增加。这首次证明C5L2是一种功能性受体，介导ASP对甘油三酯的刺激作用。
