Desensitization of β2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites
Desensitization of β2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites
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DOI：
10.1074/jbc.273.13.7637
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发表时间：
1998-03-27
影响因子：
4.8
通讯作者：
Clark, RB
中科院分区：
文献类型：
--
作者：
Seibold, A;January, BG;Clark, RB
G蛋白偶联受体激酶2和5的初步鉴定最近，基于重组受体的体外磷酸化，报道了β（2）-肾上腺素能受体（β AR）的磷酸化位点（GRK 2和GRK 5）。（Fredericks，Z，L，Pitcher，J，A，和Lefkowitz，R，J，（1996）J，Biol Chem.271，13796-13803），GRK 2鉴定的磷酸化残基是苏氨酸384和丝氨酸396、401和407，GRK 5磷酸化这四个残基以及苏氨酸393和丝氨酸411。为了确定这些位点的突变是否改变脱敏，我们构建了β AR，其中推定的GRK 2和GRK 5位点的苏氨酸和丝氨酸被丙氨酸取代。这些构建体进一步修饰以消除cAMP依赖性蛋白激酶（PKA）共有位点，将突变体β AR转染到HEH 293细胞中，并在10 μ M肾上腺素处理细胞后测量标准动力学参数。突变体和野生型（WT）受体在10 μ M肾上腺素刺激5分钟后均脱敏89-94%，在30分钟预处理后脱敏96-98%。在10 μ M肾上腺素诱导的内化程度方面，相对于WT，没有观察到任何突变体β AR的显著变化（30分钟后77-82%），肾上腺素处理1分钟诱导GRK 5和PKA（-）突变体β AR以及WT的磷酸化快速增加，我们的结论是，在体外磷酸化的GRK 2和GRK 5网站以外的网站参与介导的主要影响，在体内GRK依赖脱敏的β AR。
Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta(2)-adrenergic receptor (beta AR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z, L,, Pitcher, J, A, and Lefkowitz, R, J, (1996) J, Biol Chem. 271, 13796-13803), Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407, GRK5 phosphorylated these four residues as well as threonine 393 and serine 411, To determine if mutation of these sites altered desensitization, we have constructed beta ARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines, These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites, Mutants beta ARs were transfected into HEH 293 cells, and standard kinetic parameters were measured following 10 mu M epinephrine treatment of cells, The mutant and wild type (WT) receptors were all desensitized 89-94% after 5 min of 10 mu M epinephrine stimulation and 96-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant beta ARs relative to the WT in the extent of 10 mu M epinephrine-induced internalization (77-82% after 30 min), Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA(-) mutant beta ARs as well as the WT, We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the beta AR.