Ganguly Sourav, Pucadyil Thomas J, Chattopadhyay Amitabha
Centre for Cellular and Molecular Biology, Hyderabad, India.
Biophys J. 2008 Jul;95(1):451-63. doi: 10.1529/biophysj.107.125732. Epub 2008 Mar 13.
Analyzing the dynamics of membrane proteins in the context of cellular signaling represents a challenging problem in contemporary cell biology. Lateral diffusion of lipids and proteins in the cell membrane is known to be influenced by the cytoskeleton. In this work, we explored the role of the actin cytoskeleton on the mobility of the serotonin(1A) (5-HT(1A)) receptor, stably expressed in CHO cells, and its implications in signaling. FRAP analysis of 5-HT(1A)R-EYFP shows that destabilization of the actin cytoskeleton induced by either CD or elevation of cAMP levels mediated by forskolin results in an increase in the mobile fraction of the receptor. The increase in the mobile fraction is accompanied by a corresponding increase in the signaling efficiency of the receptor. Interestingly, with increasing concentrations of CD used, the increase in the mobile fraction exhibited a correlation of approximately 0.95 with the efficiency in ligand-mediated signaling of the receptor. Radioligand binding and G-protein coupling of the receptor were found to be unaffected upon treatment with CD. Our results suggest that signaling by the serotonin(1A) receptor is correlated with receptor mobility, implying thereby that the actin cytoskeleton could play a regulatory role in receptor signaling. These results may have potential significance in the context of signaling by GPCRs in general and in the understanding of GPCR-cytoskeleton interactions with respect to receptor signaling in particular.
在细胞信号传导的背景下分析膜蛋白的动态变化是当代细胞生物学中的一个具有挑战性的问题。已知细胞膜中脂质和蛋白质的横向扩散受细胞骨架的影响。在这项工作中，我们探讨了肌动蛋白细胞骨架对稳定表达于CHO细胞中的5-羟色胺（1A）（5-HT（1A））受体流动性的作用及其在信号传导中的意义。对5-HT（1A）R-EYFP进行的荧光恢复后光漂白（FRAP）分析表明，细胞松弛素D（CD）诱导的肌动蛋白细胞骨架破坏或福斯可林介导的cAMP水平升高均导致受体可移动部分增加。可移动部分的增加伴随着受体信号传导效率的相应增加。有趣的是，随着所用CD浓度的增加，可移动部分的增加与受体配体介导信号传导效率呈现出约0.95的相关性。发现用CD处理后受体的放射性配体结合和G蛋白偶联不受影响。我们的结果表明，5-羟色胺（1A）受体的信号传导与受体流动性相关，从而意味着肌动蛋白细胞骨架可能在受体信号传导中发挥调节作用。这些结果可能在一般G蛋白偶联受体（GPCR）信号传导的背景下，特别是在理解GPCR与细胞骨架在受体信号传导方面的相互作用方面具有潜在意义。
