G蛋白偶联受体激酶2对EGF诱导的cAMP生成的调控

Regulation of EGF-induced cAMP Accumulation by G Protein-coupled Receptor Kinase 2

目的 研究G蛋白偶联受体激酶2(G protein-coupled receptor kinase 2,GRK2)对表皮生长因子(Epidermal growth factor,EGF)诱导cAMP生成的调控作用及其可能的机制。方法 用放射免疫竞争法测定EGF刺激前后细 胞内cAMP浓度。用免疫共沉淀的方法检测GRK2与EGF受体(EGF receptor,EGFR)的相互作用。结果 (1) EGF刺激使HEK293细胞内第二信使cAMP的浓度显著升高,约是Forskolin刺激诱导的cAMP浓度升高的30％～ 40％。而过表达GRK2后,EGF刺激诱导的cAMP的浓度升高仅为Forskolin刺激诱导的cAMP浓度升高的不足 10％(P<0．01),说明过表达GRK2能显著抑制EGF刺激的cAMP的生成。(2)免疫共沉淀实验的结果显示EGF 刺激后,EGFR受体免疫沉淀复合物中检测到大量GRK2,说明EGF刺激能诱导EGFR-GRK2复合物的形成。结论 GRK2对EGF刺激诱导的第二信使cAMP生成有负反馈调控作用,这种调控作用可能是通过EGF刺激的GRK2 与EGFR的相互作用来实现的。

Objective To investigate the role of G protein-coupled receptor kinase 2 （GRK2） in epidermal growth factor （EGF）-induced cAMP accumulation and the underlying mechanisms. Methods cAMP assay was used to measure cAMP accumulation in HEK293 cells transfected with GRK2 or β-Gal as control upon EGF stimulation. Coimmunoprecipitation method was used to study the interaction between GRK2 and EGFR. Results First, intracellular second messenger cAMP accumulation was enhanced upon EGF stimulation in HEK293 cells. The cAMP accumulation induced by EGF stimulation was about 30%-40% of forskolin-induced cAMP accumulation. EGF-induced cAMP accumulation was greatly inhibited when the cells were transfected with GRK2 compared with control cells transfected with （-Gal （P 〈 0.05）, suggesting the negative regulation of EGF-induced cAMP accumulation by GRK2. Second, immunoprecipitation experiments showed that EGF stimulated GRK2 binding to EGFR complex in HEK293 cells in a time-dependent manner, suggesting EGF-induced GRK2-EGFR complex formation. Conclusion Overall, these data indicate that in HEK293 cells overexpressions of GRK2 and EGFR exist, GRK2 plays a negative modulatory role in EGF-induced cAMP accumulation, and this may involve the interaction between GRK2 and EGFR.