Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2.
Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2.
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发表时间：
1991-03
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通讯作者：
Kenneth A Beck;J. H. Keen
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作者：
Kenneth A Beck;J. H. Keen
已发现磷酸肌醇循环的几种组分在生理浓度下与质膜相关网格蛋白组装（衔接子）蛋白AP-2特异性相互作用。这些包括磷脂酰肌醇4，5-二磷酸和肌醇1，4，5-三磷酸，它们存在于质膜上，以及其他聚磷酸肌醇。然而，ATP和其他聚磷酸分子与聚磷酸肌醇一起完成，它们的效力至少低80倍。此外，与聚磷酸肌醇不同，ATP的作用被生理浓度的Mg 2+阻断。通过[α-32 P]8-叠氮腺苷5 '-三磷酸对AP-2的光亲和标记及其与聚磷酸肌醇的竞争，已用于鉴定AP-2复合物的α亚基作为与聚磷酸肌醇特异性相互作用的位点，并确认直接超滤结合实验。标记的AP-2的蛋白水解解剖表明，结合只发生在α亚基的N-末端部分。纯化的AP-2与亚微摩尔浓度的聚磷酸肌醇的相互作用对随附论文中描述的新型AP-2自缔合具有抑制作用（Beck，K.一、和基恩，J.H.，J.Biol.Chem.266，4437-4441），以及在较高浓度下对AP-2与解离的网格蛋白三聚体的结合以及AP-2介导的网格蛋白外壳组装的影响。文献综述表明，已知导致完整细胞中毛坑形成增加的几种生理刺激与磷酸肌醇周转增加相关。这些在体内的相关性和体外观察报告表明，包被膜和磷脂酰肌醇周期可能是相互依赖的细胞内。
Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated clathrin assembly (adaptor) protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [alpha-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the alpha subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the alpha subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated clathrin trimers as well as AP-2-mediated clathrin coat assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.