Localization of the sites mediating desensitization of the β2-adrenergic receptor by the GRK pathway
Localization of the sites mediating desensitization of the β2-adrenergic receptor by the GRK pathway
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DOI：
10.1124/mol.58.5.1162
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发表时间：
2000-11-01
影响因子：
3.6
通讯作者：
Clark, RB
中科院分区：
文献类型：
--
作者：
Seibold, A;Williams, B;Clark, RB
通过G蛋白偶联受体激酶（GRKs）和camp依赖性蛋白激酶A （PKA）磷酸化，人β(2)-肾上腺素能受体（β AR）在激动剂饱和浓度下迅速脱敏，随后是β -抑制素结合和受体内化。体内被GRK磷酸化的β AR位点尚未被发现。在这项研究中，我们研究了羧基末端丝氨酸355、356和364在grk介导的β AR脱敏中的作用。构建了这些丝氨酸残基的替代突变体，其中三个丝氨酸残基（S355,356,364A）、两个丝氨酸残基（S355,356A和S356,364A）或一个丝氨酸（S356A和S364A）被修饰。这些突变体是在β AR中构建的，其中PKA共识位点的丝氨酸被丙氨酸取代（称为PKA(-)），以消除PKA对脱敏的任何贡献，并将它们稳定地转染到人胚胎肾293细胞中。用10 μ M肾上腺素处理PKA（-）突变体5分钟后，肾上腺素刺激腺苷酸环化酶的EC50值增加3.5倍，V-max值降低42%。替换所有三个丝氨酸完全抑制肾上腺素诱导的EC50移位。双突变体s355356a和s356364a几乎完全丧失了EC50位移，而单突变体S356A和S364A仅引起脱敏性的轻微下降。这些突变都没有改变肾上腺素引起的V-max下降，这似乎是受体的下游。三突变导致肾上腺素诱导的内化减少45%，β - AR相对于PKA（-）的磷酸化减少90 - 95%（分别比基础磷酸化1.9 +/- 0.2和16.6 +/- 3.8倍）。双突变体导致内化（20-21%）和磷酸化（43-52%）的中度降低。丝氨酸突变都没有改变β - AR的再循环率。我们的数据表明，355至364 β - AR结构域内的丝氨酸簇赋予了β - AR的快速、grk介导的受体水平脱敏。
The human beta(2)-adrenergic receptor (beta AR) is rapidly desensitized in response to saturating concentrations of agonist by G protein-coupled receptor kinases (GRKs) and cAMP-dependent protein kinase A (PKA) phosphorylation of the beta AR, followed by beta-arrestin binding and receptor internalization. beta AR sites phosphorylated by GRK in vivo have not yet been identified. In this study, we examined the role of the carboxyl terminal serines, 355, 356, and 364, in the GRK-mediated desensitization of the beta AR. Substitution mutants of these serine residues were constructed in which either all three (S355,356,364A), two (S355,356A and S356,364A), or one of the serines (S356A and S364A) were modified. These mutants were constructed in a beta AR in which the serines of the PKA consensus site were substituted with alanines (designated PKA(-)) to eliminate any PKA contribution to desensitization, and they were stably transfected into human embryonic kidney 293 cells. Treatment of the PKA(-) mutant with 10 mu M epinephrine for 5 min caused a 3.5-fold increase in the EC50 value and a 42% decrease in the V-max value for epinephrine stimulation of adenylyl cyclase. Substitution of all three serines completely inhibited the epinephrine-induced shift in the EC50. Both double mutants, S355,356A and S356,364A, showed a nearly complete loss of the EC50 shift, whereas the single substitutions, S356A and S364A, caused only a slight decrease in desensitization. None of the mutations altered the epinephrine-induced decrease in V-max, which seems to be downstream of the receptor. The triple mutation caused a 45% decrease in epinephrine-induced internalization and a 90 to 95% reduction in phosphorylation of the beta AR relative to the PKA(-) (1.9 +/- 0.2- and 16.6 +/- 3.8- fold phosphorylation over basal, respectively). The double mutants caused an intermediate reduction in internalization (20-21%) and phosphorylation (43-52%). None of the serine mutations altered the rate of beta AR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 beta AR domain confer the rapid, GRK-mediated, receptor-level desensitization of the beta AR.