Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts
Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts
复制标题
DOI：
10.1042/0264-6021:3510607
复制
发表时间：
2000-11-01
影响因子：
4.1
通讯作者：
Williams, KJ
中科院分区：
文献类型：
--
作者：
Fuki, IV;Meyer, ME;Williams, KJ
Syndecan硫酸乙酰肝素蛋白多糖直接介导一种新的内吞途径。利用表达人Syndecan 1核心蛋白或嵌合受体FCR-Synd的中国仓鼠卵巢细胞，FCR-Synd由连接到Syndecan 1跨膜区和细胞质结构域的Ig G Fc受体Ia的外区组成，我们以前报道过有效的内化是由配体簇触发的，需要完整的肌动蛋白微丝和酪氨酸激酶，进行的t(1/2)约为1。1h，有别于包衣坑道。我们现在已经研究了富含胆固醇、不溶于洗涤剂的膜筏的参与。在聚集时，结合到FCR-Synd上的I-125标记的Ig G在冷的Triton X-100中迅速变得不溶，很好地在内吞之前，聚集的FCR-Synd配体的不溶性不需要Syndecan的胞外结构域，细胞质尾部与细胞骨架的连接，或依赖于能量的细胞代谢。用环糊精预处理细胞以去除木筏上的胆固醇，消除了簇状配体的不溶性，并以剂量反应的方式抑制了内吞作用。用I-125标记的脂蛋白脂肪酶结合到真正的细胞表面突触素上也得到了类似的结果。相反，39 kDa受体相关蛋白(RAP)是一种包被的凹坑配体，即使在内化后也有80%以上的可溶于冷Triton；细胞内胆固醇的消耗并没有实质性地影响I-125-RAP的内化。总体而言，我们的结果表明，这是一个多步骤的内吞过程，包括配体结合、聚集、能量无关的横向移动到不溶于洗涤剂的膜筏中，最后重新招募肌动蛋白和酪氨酸激酶将配体带入细胞。
Syndecan heparan sulphate proteoglycans directly mediate a novel endocytic pathway. Using Chinese hamster ovary cells expressing the human syndecan 1 core protein or a chimaeric receptor, FcR-Synd, consisting of the ectodomain of the IgG Fc receptor Ia linked to the transmembrane and cytoplasmic domains of syndecan 1, we previously reported that efficient internalization is triggered by ligand clustering, requires intact actin microfilaments and tyrosine kinases, proceeds with a t(1/2) of approx. 1 h and is distinct from coated-pit pathways. We have now examined the involvement of cholesterol-rich, detergent-insoluble membrane rafts. On clustering, I-125-labelled IgG bound to FcR-Synd rapidly became insoluble in cold Triton X-100, well before endocytosis, Insolubility of clustered FcR-Synd ligand did not require the syndecan ectodomain, linkage of the cytoplasmic tail to the cytoskeleton, or energy-dependent cellular metabolism. Pretreatment of cells with cyclodextrin to deplete cholesterol from rafts abolished insolubility of the clustered ligand and inhibited endocytosis in a dose-responsive fashion. Similar results were obtained with I-125-labelled lipoprotein lipase bound to authentic cell-surface syndecan. In contrast, the 39 kDa receptor-associated protein (RAP), a coated-pit ligand, was more than 80% soluble in cold Triton even after internalization; cellular cholesterol depletion failed to substantially affect the internalization of I-125-RAP. Overall, our results indicate a multi-step endocytic process consisting of ligand binding, clustering, energy-independent lateral movement into detergent-insoluble membrane rafts and finally recruitment of actin and tyrosine kinases to bring the ligands into the cell.