Iyer Varsha, Tran Tuan M, Foster Estrella, Dai Wenping, Clark Richard B, Knoll Brian J
Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Science and Research Bldg 2, Houston, TX 77204, USA.
Br J Pharmacol. 2006 Feb;147(3):249-59. doi: 10.1038/sj.bjp.0706551.
Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.
蛋白激酶 A（PKA）使丝氨酸 262 磷酸化以及 G 蛋白偶联受体激酶（GRK）使丝氨酸 355、356 磷酸化，可使激活的β2 - 肾上腺素能受体迅速脱敏。我们试图确定这些位点的磷酸化及随后的去磷酸化是否具有相似的动力学以及对受体内吞作用的要求。使用识别磷酸化丝氨酸 262 和磷酸化丝氨酸 355、356 的抗体来检测 PKA 和 GRK 位点的磷酸化情况。在稳定转染的 HEK293 细胞中，通过诱导表达显性负性发动蛋白 -1 K44A 或用高渗蔗糖处理来阻断内吞作用。在 10μM 异丙肾上腺素处理期间，GRK 位点丝氨酸 355、356 的磷酸化迅速达到稳态，磷酸化动力学程度不受发动蛋白 -1 K44A 表达的影响，仅受高渗蔗糖轻微影响。相比之下，在 10μM 异丙肾上腺素处理期间，PKA 位点丝氨酸 262 的磷酸化在 2 分钟后达到峰值，然后迅速下降，而内吞作用的抑制增强并延长了磷酸化。用 300 pM 异丙肾上腺素处理，该浓度过低不足以引发内吞作用，也导致 PKA 位点磷酸化延长。去除激动剂后检测这些位点的去磷酸化情况。在极低内吞作用条件下观察到磷酸化丝氨酸 262 和 355、356 有显著去磷酸化，然而，如果去除激动剂后存在拮抗剂，GRK 位点的去磷酸化程度更大。结果表明，β2 - 肾上腺素能受体 GRK 和 PKA 位点磷酸化的动力学不同，且受内吞作用的影响也不同，并且受体去磷酸化可发生在质膜或内部隔室中。
