He Guocheng, Gupta Sarita, Yi Ming, Michaely Peter, Hobbs Helen H, Cohen Jonathan C
McDermott Center for Human Growth and Development, the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, 75290, USA.
J Biol Chem. 2002 Nov 15;277(46):44044-9. doi: 10.1074/jbc.M208539200. Epub 2002 Sep 8.
Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K(D), approximately 44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the beta(2)-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of beta(2)-adaptin that is required for interaction with the adapter protein beta-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.
磷酸酪氨酸结合结构域蛋白ARH的突变会导致常染色体隐性高胆固醇血症，这是一种由肝脏中低密度脂蛋白受体（LDLR）内化缺陷引起的疾病。为了研究ARH的功能，我们使用下拉实验来检测ARH、LDLR以及参与网格蛋白介导的内吞作用的蛋白质之间的相互作用。ARH的磷酸酪氨酸结合结构域以序列特异性方式与LDLR细胞质尾巴中的内化序列（NPVY）相互作用。先前已证明NPVY序列中的突变会降低LDLR内化，这些突变消除了其在体外与ARH的结合。重组ARH以高亲和力（K(D)，约44 nm）结合纯化的牛网格蛋白。ARH与网格蛋白之间的相互作用定位于ARH中的一个典型网格蛋白盒序列（LLDLE）以及网格蛋白重链的N端结构域。ARH C端区域中一个高度保守的20个氨基酸的序列与AP-2的β(2)-衔接蛋白亚基结合。β(2)-衔接蛋白附属结构域中与衔接蛋白β-抑制蛋白相互作用所需的一个谷氨酸残基发生突变，会显著降低其与ARH的结合。这些数据与ARH作为一种衔接蛋白将LDLR与内吞机制偶联的假说一致。
