McDermott-Roe Chris, Martin Juliette, Collot-Teixeira Sophie, McGregor John L
Functional Genomics and Atherothrombosis Unit, Thrombosis Research Institute, London SW3 6LR, UK.
Biosci Rep. 2008 Jun;28(3):145-51. doi: 10.1042/BSR20080045.
The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.
动脉内膜中表达CD36的巨噬细胞对氧化型低密度脂蛋白（OxLDLs）的摄取以及随后的“泡沫细胞”形成是动脉粥样硬化斑块起始和发展的关键步骤。本研究探讨了CD36 N端胞质结构域在OxLDL结合和内化中的作用。构建了一系列CD36 N端胞质结构域突变体，并在人胚肾（HEK-293）细胞中稳定表达。然后使用免疫荧光显微镜和定量荧光法研究了三个突变体[CD36_C3/7-A（CD36-C3A/C7A）、CD36_D4/R5-A（CD36-D4A/R5A）和CD36_nCPD(-)（缺失N端胞质结构域的CD36）]结合和内吞OxLDL的能力。通过流式细胞术和蛋白质印迹法检测，每个CD36构建体在细胞表面的表达水平不同。用碘化1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青（DiI）标记的OxLDL孵育后，携带CD36_wt（野生型CD36）、CD36_C3/7-A、CD36_D4/R5-A和CD36_nCPD(-)构建体的细胞均将DiI-OxLDL内吞至内体结构中，而空载体转染的细胞则不能，这表明与C端胞质结构域不同，N端胞质结构域对于OxLDL的内吞并非必需。总之，CD36对OxLDL的摄取不依赖于CD36 N端胞质结构域的存在。然而，可以想象N端胞质结构域可能与CD36的成熟有关。
