Characterization of β2-adrenergic receptor dephosphorylation:: Comparison with the rate of resensitization
Characterization of β2-adrenergic receptor dephosphorylation:: Comparison with the rate of resensitization
复制标题
DOI：
10.1124/mol.106.028456
复制
发表时间：
2007-01-01
影响因子：
3.6
通讯作者：
Clark, Richard B.
中科院分区：
文献类型：
--
作者：
Tran, Tuan M.;Friedman, Jacqueline;Clark, Richard B.
β(2)肾上腺素能受体（β (2)AR）的环amp依赖性蛋白激酶（PKA）位点磷酸丝氨酸262和G蛋白偶联受体激酶（GRK）位点磷酸丝氨酸355和356的去磷酸化在完整的人胚胎肾293细胞和亚细胞组分中都有表征，并且与异丙肾上腺素刺激腺苷酸环化酶在异丙肾上腺素治疗和拮抗剂阻断后的再敏化率相关。在没有内化的完整细胞中，300 pM异丙肾上腺素刺激后PKA位点的去磷酸化发生在t（1/2）为9分钟（k = 0.08 +/- 0.016/min）。在1.0 μ M异丙肾上腺素作用5分钟后，完整细胞中GRK位点的去磷酸化表现出大约5分钟的滞后期，之后去磷酸化缓慢进行，t（1/2）为18分钟（k = 0.039 +/- 0.006/min）。与GRK位点去磷酸化的缓慢速率一致，在持续激动剂刺激下，磷酸酶抑制剂calyculin A和冈田酸未能增强完整细胞的磷酸化，这表明GRK位点的去磷酸化是最小的。然而，在加入拮抗剂后，两者都抑制了GRK位点的去磷酸化。通过使用磷酸化位点特异性抗体的免疫荧光显微镜观察到细胞内磷酸化β (2)AR的相对稳定性，以及通过对蔗糖梯度上GRK磷酸化β (2)AR的亚细胞定位研究发现，在质膜和囊泡部分中GRK位点磷酸化水平几乎相等，也证明了拮抗剂处理后GRK位点的缓慢去磷酸化。此外，内源性磷酸酶活性对GRK位点的去磷酸化作用仅发生在重囊泡部分。与缓慢的去磷酸化速度相比，异丙肾上腺素刺激腺苷酸环化酶的再敏化速度分别比PKA和GRK位点去磷酸化快5倍和10倍（k = 0.43 +/- 0.009/min; t(1/2) = 1.6 min），明显地将再敏化的快速阶段（0-5 min）与去磷酸化分离开来。
Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the beta(2)-adrenergic receptor (beta(2)AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t(1/2) of 9 min (k = 0.08 +/- 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 mu M isoproterenol for 5 min exhibited a lag phase of approximate to 5 min, after which dephosphorylation proceeded slowly with a t(1/2) of 18 min (k = 0.039 +/- 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated beta(2)AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated beta(2)AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5-and 10-fold faster (k = 0.43 +/- 0.009/min; t(1/2) = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.