Chemotaxis Signal Section, National Institutes of Health, Rockville, MD 20852, USA.
Sci Signal. 2010 Sep 28;3(141):ra71. doi: 10.1126/scisignal.2000980.
The coupling of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) with G proteins is fundamental for GPCR signaling; however, the mechanism of coupling is still debated. Moreover, how the proposed mechanisms affect the dynamics of downstream signaling remains unclear. Here, through experiments involving fluorescence recovery after photobleaching and single-molecule imaging, we directly measured the mobilities of cyclic adenosine monophosphate (cAMP) receptor 1 (cAR1), a chemoattractant receptor, and a G protein βγ subunit in live cells. We found that cAR1 diffused more slowly in the plasma membrane than did Gβγ. Upon binding of ligand to the receptor, the mobility of cAR1 was unchanged, whereas the speed of a fraction of the faster-moving Gβγ subunits decreased. Our measurements showed that cAR1 was relatively immobile and Gβγ diffused freely, suggesting that chemoattractant-bound cAR1 transiently interacted with G proteins. Using models of possible coupling mechanisms, we computed the temporal kinetics of G protein activation. Our fluorescence resonance energy transfer imaging data showed that fully activated cAR1 induced the sustained dissociation of G protein α and βγ subunits, which indicated that ligand-bound cAR1 activated G proteins continuously. Finally, simulations indicated that a high-affinity coupling of ligand-bound receptors and G proteins was essential for cAR1 to translate extracellular gradient signals into directional cellular responses. We suggest that chemoattractant receptors use a ligand-induced coupling rather than a precoupled mechanism to control the activation of G proteins during chemotaxis.
异三聚体鸟苷酸结合蛋白 (G 蛋白)-偶联受体 (GPCR) 与 G 蛋白的偶联是 GPCR 信号转导的基础；然而，偶联的机制仍存在争议。此外，所提出的机制如何影响下游信号转导的动力学尚不清楚。在这里，通过涉及光漂白后荧光恢复和单分子成像的实验，我们直接测量了活细胞中环磷酸腺苷 (cAMP) 受体 1 (cAR1)、趋化因子受体和 G 蛋白βγ亚基的流动性。我们发现 cAR1 在质膜中的扩散速度比 Gβγ慢。在配体与受体结合后，cAR1 的迁移率保持不变，而一部分更快迁移的 Gβγ亚基的速度降低。我们的测量结果表明，cAR1 相对不移动，而 Gβγ 自由扩散，这表明与趋化剂结合的 cAR1 与 G 蛋白短暂相互作用。使用可能的偶联机制模型，我们计算了 G 蛋白激活的时间动力学。我们的荧光共振能量转移成像数据表明，完全激活的 cAR1 诱导 G 蛋白α和βγ亚基的持续解离，这表明配体结合的 cAR1 持续激活 G 蛋白。最后，模拟表明，配体结合的受体和 G 蛋白的高亲和力偶联对于 cAR1 将细胞外梯度信号转化为定向细胞反应至关重要。我们认为，趋化因子受体在趋化过程中使用配体诱导的偶联而不是预偶联机制来控制 G 蛋白的激活。
