Phosphorylation of the beta2-adrenergic receptor in plasma membranes by intrinsic GRK5.
Phosphorylation of the beta2-adrenergic receptor in plasma membranes by intrinsic GRK5.
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DOI：
10.1021/bi700922h
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发表时间：
2007-11
期刊：
影响因子：
2.9
通讯作者：
T. Tran;Rasmus Jorgensen;R. B. Clark
中科院分区：
文献类型：
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作者：
T. Tran;Rasmus Jorgensen;R. B. Clark
参与β 2-肾上腺素能受体（β 2AR）磷酸化的GRKs的表征部分受到缺乏简单的膜结合β 2AR和GRKs的无细胞测定的限制。我们在此描述了一种无细胞测定法，用于测定β 2 AR在核后600 g组分中的GRK磷酸化，并使用识别pS（355，356）的GRK磷酸化位点特异性抗体通过固有GRK活性洗涤膜。用1.0 μ M异丙肾上腺素（ISO）处理这些无细胞制剂，导致β 2 AR的GRK位点磷酸化快速增加10-15倍（t1/2 = 1 min），ISO刺激的EC 50约为80 nM。广泛洗涤的质膜部分保留GRK位点磷酸化和GRK 5水平的10-15倍ISO刺激，同时耗尽GRK 2和GRK 6。一系列部分激动剂刺激GRK位点磷酸化与其刺激腺苷酸环化酶的内在功效密切相关。GRK磷酸化的β 2 AR在洗涤膜部分引起最小的脱敏ISO刺激腺苷酸环化酶活性。完整细胞中GRK 5与β 2 AR的关联通过使用β 2 AR-Rluc和GRK 5-GFP 2的高水平基础BRET 2来证明，其不被激动剂刺激减弱。激动剂增加了beta2 AR-Rluc和GFP 2-betaarrestin 2之间的BRET 2，而beta2 AR和GRK 2-GFP 2之间的BRET 2不显著。基于我们在分离的膜组分中观察到的GRK 5介导的磷酸化水平以及β 2AR和GRK 5的共定位，我们得出结论GRK 5在β 2AR的磷酸化中起着独特的作用。
Characterization of the GRKs participating in the phosphorylation of the beta2-adrenergic receptor (beta2AR) have in part been limited by the lack of a simple cell-free assay with membrane-bound beta2AR and GRKs. We describe here a cell-free assay for GRK phosphorylation of the beta2AR in a postnuclear 600g fraction and washed membranes by intrinsic GRK activity using the GRK phosphosite-specific antibody that recognizes pS(355,356). Treatment of these cell-free preparations with 1.0 microM isoproterenol (ISO) caused a rapid maximal 10-15-fold increase in GRK site phosphorylation of the beta2AR (t1/2 = 1 min) with an EC50 for ISO stimulation of approximately 80 nM. Extensively washed plasma membrane fractions retained the 10-15-fold ISO stimulation of GRK site phosphorylation and GRK5 levels while being depleted of GRK2 and GRK6. Stimulation of GRK site phosphorylation by a range of partial agonists correlated well with their intrinsic efficacy for stimulation of adenylyl cyclase. GRK phosphorylation of the beta2AR in the washed membrane fraction caused minimal desensitization of ISO stimulation of adenylyl cyclase activity. Association of GRK5 with the beta2AR in intact cells was demonstrated by a high level of basal BRET2 using beta2AR-Rluc and GRK5-GFP2 that was not diminished by agonist stimulation. BRET2 between the beta2AR-Rluc and GFP2-betaarrestin 2 was increased by agonist, whereas BRET2 between the beta2AR and GRK2-GFP2 was not significant. On the basis of the level of GRK5-mediated phosphorylation we observe in isolated membrane fractions and co-localization of the beta2AR and GRK5, we conclude that GRK5 plays a distinctive role in the phosphorylation of the beta2AR.