Dong J F, Li C Q, Sae-Tung G, Hyun W, Afshar-Kharghan V, López J A
Department of Internal Medicine, Baylor College of Medicine and Veterans' Affairs Medical Center, Houston, Texas 77030, USA.
Biochemistry. 1997 Oct 14;36(41):12421-7. doi: 10.1021/bi970636b.
To study the role of the glycoprotein (GP) Ibalpha cytoplasmic domain in the mobility of the GP Ib-IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines expressing GP Ib-IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibalpha or one of a series of GP Ibalpha truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibalpha monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibalpha was negligible. However, when only six amino acids were removed from the GP Ibalpha carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibalpha), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55-67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibalpha cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibalpha fixes the position of the GP Ib-IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.
为了研究糖蛋白（GP）Ibalpha胞质结构域在GP Ib-IX复合物在质膜内的流动性及其结合血管性血友病因子（vWf）能力中的作用，我们建立了8个表达GP Ib-IX复合物（这些复合物缺乏GP V，但作为vWf的受体功能正常）的细胞系，这些复合物包含野生型GP Ibalpha或一系列缺失不同长度胞质结构域的GP Ibalpha截短突变体之一。为了测试这些复合物在质膜内的流动性，我们在用荧光素偶联的抗GP Ibalpha单克隆抗体标记它们后，使用了光漂白后荧光恢复技术。通过在漂白后用低强度激光扫描细胞表面3分钟，然后将恢复值外推到无限时间，来评估细胞表面漂白区域内的荧光恢复情况。在表达野生型GP Ibalpha的细胞中，荧光恢复可以忽略不计。然而，当从GP Ibalpha羧基末端仅去除6个氨基酸（t604突变体，多肽长度为604个残基，而野生型GP Ibalpha为610个残基）时，复合物的流动性大大增加，这通过漂白区域中荧光的更快恢复来判断（48%的恢复率）。在t594突变体中流动性进一步增加，并且在t534突变体中保持大致相同（55-67%的恢复率）。在t518突变体中观察到流动性进一步增加（>80%的恢复率），该突变体几乎缺乏所有的GP Ibalpha胞质结构域。还评估了突变细胞系的瑞斯托菌素依赖性结合。vWf与表达任何突变复合物的细胞的结合明显低于与表达野生型复合物的细胞的结合。这些研究表明，GP Ibalpha的胞质结构域固定了GP Ib-IX复合物在血小板表面的位置，并且这种取向是复合物结合vWf能力的重要决定因素。
