Tran Tuan M, Friedman Jackie, Qunaibi Eyad, Baameur Faiza, Moore Robert H, Clark Richard B
The University of Texas, Houston, Medical School, Houston, TX 77225, USA.
Mol Pharmacol. 2004 Jan;65(1):196-206. doi: 10.1124/mol.65.1.196.
Agonist-stimulated desensitization of the beta2-adrenergic receptor (beta2AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to beta-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human beta2AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E1, and forskolin at maximum concentrations stimulated phosphorylation of the beta2AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still approximately 200-fold greater than for the PKA site. The EC50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the beta2AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agonist-stimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine > or = formoterol = fenoterol > terbutaline = zinterol = albuterol > salmeterol > dobutamine > or = ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous beta2AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.
激动剂刺激引起的β2-肾上腺素能受体（β2AR）脱敏是由一种强效的cAMP依赖性蛋白激酶（PKA）介导的磷酸化以及一种较弱的、占据依赖性的G蛋白偶联受体激酶（GRK）介导的磷酸化共同导致的，后者会引起β-抑制蛋白结合和内化。在本研究中，使用磷酸丝氨酸特异性抗体对在人胚肾（HEK）293细胞中过表达的人β2AR的第三个细胞内环PKA位点Ser262以及假定的C末端GRK位点Ser355、Ser356的磷酸化动力学进行了表征。抗体的特异性通过其与缺乏相应位点的突变β2AR无反应性得以证明。此外，GRK2和GRK5的过表达增加了COS-7和HEK 293细胞中GRK位点Ser355、Ser356的基础磷酸化水平。肾上腺素、前列腺素E1和福斯可林在最大浓度时可使β2AR PKA位点（Ser262）的磷酸化增加4倍，而佛波酯可使其增加2倍。肾上腺素刺激PKA位点磷酸化的EC50为20至40 pM。相比之下，肾上腺素刺激GRK位点磷酸化（Ser355、Ser356）的EC50为200 nM（1分钟处理），相对于PKA位点磷酸化高出4000多倍，这与一个占据驱动的过程一致。10至30分钟后，肾上腺素刺激GRK位点磷酸化的EC50降至10至2 nM，但仍比PKA位点大约高200倍。内化的EC50与GRK位点磷酸化相关，并且随着肾上腺素刺激时间呈现类似的变化。在缺乏PKA共有位点的β2AR突变体中，肾上腺素刺激的GRK位点磷酸化动力学未改变。一系列激动剂刺激的GRK位点磷酸化的初始水平（2分钟）与它们激活腺苷酸环化酶的效力相关，即肾上腺素≥福莫特罗 = 非诺特罗＞特布他林 = 齐帕特罗 = 沙丁胺醇＞沙美特罗＞多巴酚丁胺≥麻黄碱。然而，处理20至30分钟后，中等强度的激动剂，如沙丁胺醇和沙美特罗，刺激的GRK位点磷酸化与肾上腺素产生的磷酸化大致相等，这种相关性消失。GRK和PKA位点抗体也可有效检测A431人表皮样癌细胞中内源性β2AR的磷酸化。总之，我们的结果显示相对于假定的GRK位点磷酸化，PKA位点磷酸化有显著放大，PKA位点磷酸化的异源刺激，GRK位点磷酸化不依赖于PKA位点，以及GRK位点磷酸化的初始水平与一系列激动剂强度之间存在合理的相关性。
