Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins.
Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins.
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DOI：
10.1016/s0021-9258(18)43930-0
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发表时间：
1994-11
期刊：
影响因子：
--
通讯作者：
A. Gao;W. Frazier
中科院分区：
文献类型：
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作者：
A. Gao;W. Frazier
血小板反应蛋白-1（TS 1）的羧基末端细胞结合结构域（CBD）包含两个细胞附着肽，4 N1（RFYVVMWK）和7 N3（FIRVVMY-EGKK），它们共享序列VVM。这些肽和更可溶的衍生物已用125 I放射性标记，并与各种膜不渗透交联剂结合使用，以鉴定和表征几种细胞类型上的受体候选物。用五种不同的交联试剂测试的所有含有VVM的肽特异性标记52-kDa蛋白质，其也被重组TS 1 CBD亲和标记。交联肽K562细胞，以阻止52 kDa的蛋白质后，细胞粘附和亲和标记的VVM肽抑制浓度依赖性的方式。肽标记，如细胞粘附，部分抑制肝素和刺激EDTA。52-kDa蛋白似乎不含硫酸化聚糖链，并且对胰蛋白酶敏感。它被回收在膜部分，并很容易与Triton X-100和X-114溶解。在Triton X-114的相分离后，52-kDa的蛋白质分配到疏水洗涤剂相中。洗涤剂溶解的受体候选者选择性地结合到麦胚凝集素-琼脂糖凝胶，并在细胞表面标记膜不渗透的生物素化试剂后，结合到链霉亲和素-琼脂糖凝胶。此外，荧光珠共价衍生肽特异性装饰完整的K562细胞。因此，52-kDa蛋白质的性质与TS 1和其他TS同种型的CBD受体的性质一致。
The carboxyl-terminal cell binding domain (CBD) of thrombospondin-1 (TS1) contains two cell attachment peptides, 4N1s (RFYVVMWK) and 7N3 (FIRVVMY-EGKK), which share the sequence VVM. These peptides, and more soluble derivatives have been radiolabeled with 125I and used in conjunction with a variety of membrane impermeant cross-linking reagents to identify and characterize receptor candidates on several cell types. All of the VVM containing peptides tested with five different cross-linking reagents specifically labeled a 52-kDa protein, which was also affinity labeled by the recombinant TS1 CBD. After cross-linking peptide to K562 cells to block the 52-kDa protein, both cell adhesion to and affinity labeling by VVM peptides were inhibited in a concentration-dependent manner. Peptide labeling, like cell adhesion, was partially inhibited by heparin and stimulated by EDTA. The 52-kDa protein did not appear to contain sulfated glycan chains and was trypsin sensitive. It was recovered in a membrane fraction and was readily solubilized with Triton X-100 and X-114. Upon phase separation of the Triton X-114, the 52-kDa protein partitioned into the hydrophobic detergent phase. The detergent-solubilized receptor candidate bound selectively to wheat germ agglutinin-Sepharose, and after cell surface labeling with a membrane impermeant biotinylating reagent, bound to streptavidin-Sepharose. Furthermore, fluorescent beads covalently derivatized with peptide specifically decorated intact K562 cells. Thus the properties of the 52-kDa protein are consistent with those of a receptor for the CBD of TS1 and other TS isoforms.