Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant μ2 subunit and its effects on endocytosis
Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant μ2 subunit and its effects on endocytosis
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DOI：
10.1093/emboj/18.9.2489
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发表时间：
1999-05-04
期刊：
影响因子：
11.4
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Nesterov, A;Carter, RE;Sorkin, A
尽管网格蛋白接头蛋白复合物AP-2的mu 2亚基与酪氨酸内化基序之间的相互作用与选择性地将货物分子招募到包覆凹中有关，但这种相互作用对许多类型膜蛋白的内吞作用的功能意义尚不清楚。为了分析mu 2受体相互作用的功能，我们构建了一个表位标记的mu 2，它结合到AP-2中，并靶向涂膜坑。突变分析显示，mu 2的Asp176和Trp421参与了与TGN38和表皮生长因子（EGF）受体内化基序的相互作用，诱导突变体mu 2的过表达，这两个残基转变为丙氨酸，导致AP-2复合物中内源性mu 2的代谢性替代，并完全消除AP-2与酪氨酸内化基序的相互作用。结果，转铁蛋白受体的内吞作用严重受损，相反，EGF受体的内化不受影响。这些结果证明了显性干扰方法对接头蛋白家族功能分析的潜在有用性，并表明网格蛋白介导的内吞作用可能以依赖和独立的方式进行。
Although interactions between the mu 2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu 2-receptor interactions, we constructed an epitope-tagged mu 2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu 2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor, Inducible overexpression of mutant mu 2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu 2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired, In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu 2-dependent and -independent manner.