Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.
Biochem Biophys Res Commun. 2010 Jan 22;391(4):1616-22. doi: 10.1016/j.bbrc.2009.12.087. Epub 2009 Dec 23.
Endothelin type A receptor (ET(A)R) plays an important role in some cardiovascular disorders where ET(A)R levels are increased. However, regulatory mechanisms for ET(A)R levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET(A)R-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET(A)R as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET(A)R-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET(A)R and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET(A)R, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET(A)R after protein synthesis inhibition as an index of a degradation rate. ET(A)R was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET(A)R degradation and increased the amount of Jab1 bound to ET(A)R with a maximal level of 500% at 3h. In the absence of ET-1 stimulation, the level of ET(B)R was lower than that of ET(A)R and the degradation rate of ET(B)R was markedly faster than that of ET(A)R. Notably, the amount of Jab1 bound to ET(B)R and ubiquitination level of ET(B)R were markedly higher than those for ET(A)R. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET(A)R and ET(B)R by modulating ubiquitination of these receptors, leading to changes in ET(A)R and ET(B)R levels.
内皮素 A 型受体 (ET(A)R) 在一些心血管疾病中发挥着重要作用，这些疾病中 ET(A)R 水平升高。然而，ET(A)R 水平的调节机制尚不清楚。在这里，我们通过使用 ET(A)R 的羧基末端尾巴 (C-尾) 作为诱饵，从人心脏 cDNA 文库中进行酵母双杂交筛选，鉴定出 Jun 激活域结合蛋白 1 (Jab1) 作为 ET(A)R 的相互作用蛋白。通过谷胱甘肽 S-转移酶下拉测定、在表达 ET(A)R-myc 和 FLAG-Jab1 的 HEK293T 细胞中的共免疫沉淀以及共聚焦显微镜来确认相互作用。Jab1 敲低增加了表达 ET(A)R 的 HEK293T 细胞中的全细胞和细胞表面 ET(A)R 水平，以及 ET-1 诱导的 ERK1/2 磷酸化，而 Jab1 过表达则降低了它们。Jab1 过表达加速了蛋白合成抑制后 ET(A)R 的消失率，作为降解率的指标。ET(A)R 被持续泛素化，Jab1 过表达增强了泛素化水平。长期 ET-1 刺激显著加速了 ET(A)R 的降解速度，并增加了与 ET(A)R 结合的 Jab1 的量，在 3 小时时达到最大水平的 500%。在没有 ET-1 刺激的情况下，ET(B)R 的水平低于 ET(A)R，ET(B)R 的降解速度明显快于 ET(A)R。值得注意的是，与 ET(A)R 相比，与 ET(B)R 结合的 Jab1 的量和 ET(B)R 的泛素化水平显著更高。总之，这些结果表明，与 ETR 结合的 Jab1 的量通过调节这些受体的泛素化来调节 ET(A)R 和 ET(B)R 的降解率，从而导致 ET(A)R 和 ET(B)R 水平的变化。
