ADP-Ribosylation Factors Modulate the Cell Surface Transport of G Protein-Coupled Receptors
ADP-Ribosylation Factors Modulate the Cell Surface Transport of G Protein-Coupled Receptors
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DOI：
10.1124/jpet.109.161489
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发表时间：
2010-04-01
影响因子：
3.5
通讯作者：
Wu, Guangyu
中科院分区：
文献类型：
--
作者：
Dong, Chunmin;Zhang, Xiaoping;Wu, Guangyu
adp核糖基化因子（ARFs）通过招募外壳蛋白调节囊泡交通。然而，它们在新生G蛋白偶联受体（gpcr）从内质网到质膜的顺行运输中的功能仍然知之甚少。本研究表明，brefeldin A （ARFs上的鸟嘌呤核苷酸交换抑制剂）显著降低了α (2B)-肾上腺素能受体（AR）、β (2)-AR、血管紧张素II型1受体和趋化因子（CXC基序）受体4的细胞表面数量。瞬时表达gtp结合突变体、gtp结合突变体和鸟嘌呤核苷酸缺陷突变体对单个ARF gtpase的功能性抑制表明，5种人ARF差异调节受体细胞表面表达，其中ARF1突变体产生最深刻的抑制作用。此外，ARF1 gtpase激活蛋白（GAP） ARFGAP1的表达显著阻断了受体的转运。有趣的是，结合GDP和gtp的ARF1突变体在不同的细胞内区室中抑制了受体。与受体细胞表面表达降低一致，受体激动剂对细胞外信号调节激酶1和2的激活被gdp结合突变体ARF1T31N显著减弱。此外，共免疫沉淀显示α (2B)-AR与ARF1相关，谷胱甘肽转移酶下拉试验表明α (2B)-AR C端直接与ARF1相互作用。这些数据表明，ARF1 GTPase在多个转运步骤中参与调控gpcr的细胞表面表达。
ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of alpha(2B)-adrenergic receptor (AR), beta(2)-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that alpha(2B)-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the alpha(2B)-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.