Transforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit.
Transforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit.
复制标题
DOI：
10.1091/mbc.02-07-0104
复制
发表时间：
2002-11
影响因子：
3.3
通讯作者：
Diying Yao;M. Ehrlich;Y. Henis;E. Leof
中科院分区：
文献类型：
--
作者：
Diying Yao;M. Ehrlich;Y. Henis;E. Leof
转化生长因子- β (tgf - β)超家族成员通过结合两种跨膜丝氨酸/苏氨酸激酶受体I型和II型来调节广泛的生物过程。我们之前的研究表明，这些受体的内化受到K（+）耗竭、细胞质酸化或高渗介质的抑制，这表明网格蛋白包被的凹坑参与其中。然而，网格蛋白相关接头复合物AP2的参与以及与受体结合的AP2亚基的身份尚不清楚。在这里，我们通过结合对完整细胞的研究和体外实验来研究这些问题。使用荧光光漂白恢复来测量活细胞上受体的横向迁移率（未经处理或处理以改变其涂层凹坑结构），我们证明了它们的迁移率受到与涂层凹坑相互作用的限制。这些相互作用是短暂的，通过受体的细胞质尾部介导。为了测量受体与特定AP2亚基的直接结合，我们使用酵母双杂交筛选和体外生化分析。与大多数其他通过mu2亚基与AP2结合的质膜受体不同，AP2/ tgf - β受体的结合是通过β 2-适应蛋白n端主干结构域和受体的细胞质尾部之间的直接相互作用介导的；未观察到与AP2的mu2、alpha或sigma2亚基或AP1的mu1结合。这些数据在体内和体外都独特地证明了β 2-适应蛋白直接将tgf - β受体偶联到AP2和网格蛋白包被的凹处的能力，为跨膜受体与β 2-适应蛋白的相互作用提供了第一个体内证据。
Transforming growth factor-beta (TGF-beta) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K(+) depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the mu2 subunit, AP2/TGF-beta receptor binding was mediated by a direct interaction between the beta2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the mu2, alpha, or sigma2 subunits of AP2 or to mu1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of beta2-adaptin to directly couple TGF-beta receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with beta2-adaptin.