Lichtenstein Drew L, Krajcsi Peter, Esteban David J, Tollefson Ann E, Wold William S M
Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104, USA.
J Virol. 2002 Nov;76(22):11329-42. doi: 10.1128/jvi.76.22.11329-11342.2002.
The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.
腺病毒编码的受体内化与降解（RID）蛋白（以前称为E3-10.4K/14.5K）由RIDα和RIDβ亚基组成，可下调肿瘤坏死因子（TNF）受体超家族中的一些细胞表面受体，即Fas、TRAIL受体1和TRAIL受体2。这些“死亡”受体的下调可保护腺病毒感染的细胞免受死亡受体配体Fas配体和TRAIL诱导的凋亡。RID还可下调某些酪氨酸激酶细胞表面受体，尤其是表皮生长因子受体（EGFR）。RID介导的Fas和EGFR下调是通过受体被内吞进入内体，随后转运至溶酶体并在其中降解来实现的。然而，RID此功能背后的分子相互作用尚不清楚。为了研究参与受体下调的RIDβ的分子决定因素，构建了RIDβ胞质尾内的突变体，并分析了突变蛋白内化和降解Fas和EGFR以及保护细胞免受死亡受体配体诱导凋亡的能力。结果表明，RIDβ C末端附近的一个酪氨酸残基至关重要；该残基突变为丙氨酸会消除RID功能。将酪氨酸突变为苯丙氨酸不会消除RID功能，这表明该功能不需要酪氨酸磷酸化。这些数据表明，这个酪氨酸残基构成了基于酪氨酸的分选信号（Yxxphi）的一部分。针对另一个潜在分选基序和几个可能的蛋白质-蛋白质相互作用基序的其他突变对RID功能没有明显影响。还证明，将丝氨酸116突变为丙氨酸可消除RIDβ的磷酸化，但不影响所检测的RID的任何功能。这些结果提示了一个模型，其中RID中基于酪氨酸的分选信号在RID下调受体的能力中发挥作用。
