The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor
The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor
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DOI：
10.1161/circresaha.108.176594
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发表时间：
2008-06-20
影响因子：
20.1
通讯作者：
Alam, Jawed
中科院分区：
文献类型：
--
作者：
Cook, Julia L.;Re, Richard N.;Alam, Jawed
与七螺旋整合膜受体的细胞内扩展区（特别是细胞质尾区）结合的蛋白质特别令人感兴趣，因为它们可以介导或调节运输或细胞内信号传导。为了区分可能促进血管紧张素II 1型（AT（1））受体细胞内事件的新蛋白质，我们筛选了具有大鼠AT（1A）受体（AT（1）R）羧基末端的酵母双杂交小鼠脑文库，并鉴定了GABARAP，一种参与GABA（A）受体细胞内运输的蛋白质，作为AT（1）R的结合伴侣。GABARAP与AT（1）R羧基末端的相互作用用GST下拉法进一步证实，全长标记的AT（1）R与GABARAP的结合用免疫共沉淀法验证。生物发光共振能量转移实验进一步证实了GABARAP与AT（1）R的特异性相互作用。此外，GABARAP明显增加PC-12细胞中质膜相关AT（1）R的稳态水平。GABARAP与AT（1）R荧光融合蛋白共转染使PC-12细胞表面AT（1）R的表达增加了6倍以上，当标准化为细胞内表达水平时。此外，GABARAP在CHO-K1细胞中的过表达使血管紧张素II结合位点增加了3.7倍，血管紧张素II诱导的磷酸化细胞外信号调节激酶1/2和细胞增殖显著高于单独过表达AT（1）R的水平。此外，小干扰RNA介导的GABARAP敲低使AT 1 R荧光融合蛋白的稳态水平降低了43%，其细胞表面表达降低了84%。免疫印迹分析证实了定量图像数据。我们的结论是GABARAP结合并促进AT（1）R向质膜的运输。
Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT1R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.