Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA.
Department of Physics, University of California-San Diego, La Jolla, CA 92093, USA.
Cell Signal. 2018 Aug;48:25-37. doi: 10.1016/j.cellsig.2018.04.005. Epub 2018 Apr 24.
To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.
为了研究在受到趋化因子环腺苷酸（cAMP）刺激时，Dictyostelium 中异三聚体 G 蛋白 Gα2βγ的激活动力学和控制机制，我们使用生物发光共振能量转移（BRET）在活细胞中监测 G 蛋白亚基相互作用。我们发现 cAMP 以相似的程度诱导 cAR1 介导的 G 蛋白亚基解聚，无论是在未分化细胞还是分化细胞中，这表明只有少量的 cAR1（如在未分化细胞中表达）足以诱导 Gα2βγ的完全激活。此外，我们发现用咖啡因处理细胞会增加 cAMP 诱导的 Gα2βγ激活的效力；破坏微管网络而不是 F-肌动蛋白会抑制 cAMP 诱导的 Gα2βγ解聚。因此，微管对于 cAR1 介导的异三聚体 G 蛋白的有效激活是必要的。最后，不同 cAMP 浓度诱导的 Gα2βγ 亚基解离的动力学分析表明，当细胞受到高于 500 nM 的 cAMP 浓度刺激时，异三聚体 G 蛋白亚基解离有两种不同的速率，而在较低的 cAMP 浓度下只有一种速率。定量建模表明，Gα2βγ 亚基解离的动力学特征源于存在对 cAMP 具有不同亲和力的未偶联和 G 蛋白预偶联的 cAR1，因此通过不同的速率诱导 G 蛋白亚基解离。我们认为，这些不同的信号转导动力学特征可能在初始趋化因子梯度感应中发挥重要作用。
