Lombardi Maria Stella, Vroon Anne, Sodaar Peter, van Muiswinkel Freek L, Heijnen Cobi J, Kavelaars Annemieke
Laboratory for Psycho-neuro-immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
J Neurochem. 2007 Aug;102(3):731-40. doi: 10.1111/j.1471-4159.2007.04576.x. Epub 2007 Apr 16.
G protein-coupled receptor kinase 2 (GRK2) modulates G protein-coupled receptor desensitization and signaling. We previously described down-regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia-ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down-regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post-OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post-transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD-induced decrease of GRK2. It has been shown that extracellular signal-regulated kinase-dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670-GRK2 after OGD, inhibition of the extracellular signal-regulated kinase pathway by PD98059 did neither prevent the hypoxia-ischemia-induced increase in pSer670-GRK2 nor the down-regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide-3-kinase with wortmannin inhibits both OGD-induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD-induced phosphoinositide-3-kinase-dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down-regulation of GRK2 protein via a proteasome-dependent pathway.
G蛋白偶联受体激酶2（GRK2）可调节G蛋白偶联受体的脱敏和信号传导。我们之前描述过，在新生大鼠脑内，缺氧缺血后GRK2表达在体内会下调。在本研究中，我们使用暴露于氧糖剥夺（OGD）的新生大鼠海马脑片的器官型培养物，研究了GRK2下调所涉及的分子机制。我们观察到OGD后4小时GRK2表达下降了40%。仅将海马脑片暴露于糖剥夺后，GRK2蛋白没有变化。未检测到GRK2 mRNA表达有显著改变，这表明OGD对GRK2表达有转录后效应。MG132阻断蛋白酶体途径可防止OGD诱导的GRK2减少。已经表明，细胞外信号调节激酶依赖性的GRK2在Ser670位点的磷酸化会通过蛋白酶体途径触发其周转。然而，尽管OGD后pSer670 - GRK2显著增加，但用PD98059抑制细胞外信号调节激酶途径既不能阻止缺氧缺血诱导的pSer670 - GRK2增加，也不能阻止GRK2蛋白的下调。有趣的是，渥曼青霉素抑制磷酸肌醇-3-激酶既抑制OGD诱导的GRK2在Ser670位点的磷酸化，也抑制GRK2的减少。总之，OGD诱导的GRK2在Ser670位点的磷酸肌醇-3-激酶依赖性磷酸化是一种通过蛋白酶体依赖性途径导致GRK2蛋白下调的新机制。
