Ugur Ozlem, Oner Sükrü Sadik, Molinari Paola, Ambrosio Caterina, Sayar Kemal, Onaran H Ongun
Ankara Universitesi Tip Fakültesi, Farmakoloji ve Klinik Farmakoloji Ab.D. Sihhiye 06100, Ankara, Turkey.
Mol Pharmacol. 2005 Sep;68(3):720-8. doi: 10.1124/mol.104.010306. Epub 2005 Jun 2.
beta2-adrenoceptor-mediated activation of Gs and adenylyl cyclase or other receptor-mediated G protein activations is believed to occur by receptor-catalyzed replacement of GDP with GTP on the alpha-subunit of the G protein. Here we showed that a beta2-adrenoceptor-Gs system, heterologously expressed in cyc- or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). The potency and maximal ability of GDP to activate Gs and adenylyl cyclase were identical to those of GTP. GDP-mediated activation of adenylyl cyclase, similar to that mediated by GTP, was concentration-dependent, required high magnesium concentrations, was inhibited by inverse agonists, and was correlated with the efficacy of receptor ligands used to stimulate the receptor. UDP did not block the GDP-mediated activation, although it completely blocked the formation of a small amount of GTP ( approximately 5% GDP) from GDP. Moreover, the activation of Gs in the presence of GDP was insensitive to cholera toxin treatment of the cells, whereas that observed in the presence of GTP was amplified by the treatment, which showed that the activation observed in the presence of GDP was not mediated by GTP. Therefore, we concluded that GDP itself could mediate beta-adrenoceptor-induced activation of Gs-adenylyl cyclase system as much as GTP. We discuss the results in the context of the current paradigm of receptor-mediated G protein activation and propose an additional mode of activation for beta2-adrenoceptor-G(s) adenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptor-induced Gs protein activation.
β2肾上腺素能受体介导的Gs和腺苷酸环化酶激活或其他受体介导的G蛋白激活被认为是通过受体催化G蛋白α亚基上的GDP被GTP取代而发生的。在这里我们表明，在爪蟾卵母细胞或人胚肾（HEK）-293细胞中异源表达的β2肾上腺素能受体-Gs系统，在存在GDP或其磷酸化抗性类似物鸟苷5'-O-（2-硫代二磷酸）（GDPβS）的情况下可以被激活。GDP激活Gs和腺苷酸环化酶的效力和最大能力与GTP相同。GDP介导的腺苷酸环化酶激活与GTP介导的类似，呈浓度依赖性，需要高镁浓度，被反向激动剂抑制，并且与用于刺激受体的受体配体的效力相关。UDP没有阻断GDP介导的激活，尽管它完全阻断了从GDP形成少量GTP（约5%GDP）。此外，在存在GDP的情况下Gs的激活对细胞的霍乱毒素处理不敏感，而在存在GTP的情况下观察到的激活则被该处理放大，这表明在存在GDP的情况下观察到的激活不是由GTP介导的。因此，我们得出结论，GDP本身可以像GTP一样介导β肾上腺素能受体诱导的Gs-腺苷酸环化酶系统激活。我们在受体介导的G蛋白激活的当前范式背景下讨论了这些结果，并提出了β2肾上腺素能受体-G（s）腺苷酸环化酶系统的另一种激活模式，其中核苷酸交换不是必需的，并且GDP和GTP在受体诱导的Gs蛋白激活中发挥相同作用。
