Fingerhut A, von Figura K, Honing S
Institute for Biochemistry II, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany.
J Biol Chem. 2001 Feb 23;276(8):5476-82. doi: 10.1074/jbc.M009516200. Epub 2000 Oct 23.
The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15-33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis.
已知两种网格蛋白相关衔接复合物AP1和AP2参与反式高尔基体网络和质膜处网格蛋白包被囊泡的形成。在此过程中，衔接蛋白通过与货物蛋白胞质结构域内的分选信号结合参与囊泡货物的隔离，并参与网格蛋白包被的募集。网格蛋白包被囊泡出芽后，网格蛋白和衔接蛋白从囊泡上解离。我们在此表明，受体介导的内吞作用的初始步骤之一，即AP2与分选信号的体外结合，受衔接蛋白磷酸化的调节。通过在ATP存在下孵育纯化的AP2使AP2磷酸化，并通过与碱性磷酸酶孵育使其去磷酸化。磷酸化的AP2对酪氨酸、亮氨酸基序和非经典分选基序的亲和力比对去磷酸化的AP2高15 - 33倍。AP2与膜的结合也受衔接蛋白磷酸化/去磷酸化的调节，磷酸化的AP2比去磷酸化的AP2高约8倍。此外，从胞质溶胶中分离的AP2比从膜中提取的磷酸化程度更高，并且其结合亲和力比从膜中提取的AP2高5倍。综上所述，这些数据表明磷酸化/去磷酸化循环是在受体介导的内吞作用过程中调节AP2与膜和分选信号可逆结合的一种机制。
