Liu C H, Thangada S, Lee M J, Van Brocklyn J R, Spiegel S, Hla T
Center for Vascular Biology, Department of Physiology, University of Connecticut School of Medicine, Farmington, Connecticut 06030, USA.
Mol Biol Cell. 1999 Apr;10(4):1179-90. doi: 10.1091/mbc.10.4.1179.
The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.
内皮细胞衍生的G蛋白偶联受体EDG-1是生物活性脂质介质鞘氨醇-1-磷酸（SPP）的高亲和力受体。在本研究中，我们构建了EDG-1-绿色荧光蛋白（GFP）嵌合体，以研究SPP-EDG-1相互作用的动力学和亚细胞定位。SPP与EDG-1-GFP结合，并以与野生型受体所见方式无法区分的方式转导细胞内信号。稳定转染EDG-1-GFP cDNA的人胚肾293细胞主要在质膜上表达该受体。外源性SPP处理以剂量依赖性方式诱导受体向核周囊泡转位，半衰期约为15分钟。含有EDG-1-GFP的囊泡与线粒体不同，但部分与内吞囊泡和溶酶体共定位。低亲和力激动剂溶血磷脂酸以及其他鞘脂类，神经酰胺、神经酰胺-1-磷酸或鞘氨醇磷酸胆碱，均不影响受体运输。C末端截短可完全抑制受体内化。SPP洗脱后，EDG-1-GFP以约30分钟的半衰期循环回到质膜。我们得出结论，高亲和力配体SPP通过内体途径特异性诱导EDG-1的可逆运输，并且受体的C末端细胞内结构域对该过程至关重要。
