EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation
EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation
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DOI：
10.1091/mbc.e07-10-1058
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发表时间：
2008-07-01
影响因子：
3.3
通讯作者：
Ma, Lan
中科院分区：
文献类型：
--
作者：
Chen, Yuejun;Long, Hui;Ma, Lan
G蛋白偶联受体激酶(GRKs)是G蛋白偶联受体功能的关键调节因子。在这里，我们证明了表皮生长因子受体(EGFR)的激活，受体酪氨酸激酶家族的成员，刺激GRK2的活性和反式调节G蛋白偶联的阿片受体的功能。我们的数据表明，EGF处理促进了DOR激动剂诱导的DOR内化，这需要DOR中GRK2磷酸化位点的完整性。EGF刺激可诱导GRK2与激活的EGFR结合，并使GRK2移位至质膜。经EGF处理后，GRK2在酪氨酸残基上被磷酸化。突变分析表明，EGFR介导的磷酸化发生在以前被认为是c-Src磷酸化位点的GRK2 N末端酪氨酸残基。然而，c-Src活性不是EGFR介导的GRK2磷酸化所必需的。体外实验表明，GRK2是EGFR的直接相互作用和底物。在表达野生型GRK2的细胞中，EGF以依赖于EGFR酪氨酸激酶活性的方式显著增加DOR的磷酸化，而在表达缺乏EGFR酪氨酸激酶位点的突变型GRK2的细胞中，EGF刺激的DOR磷酸化显著降低。我们进一步表明，EGF还能刺激Mu-阿片受体内化，这种作用可被GRK2 siRNA抑制。这些数据表明，EGF通过EGFR介导的酪氨酸磷酸化和GRK2的激活来反式调节阿片受体，并提出GRK2是RTK到GPCR信号通路的串扰的中介。
G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.