He Fang, Qiao Zhuan-Hong, Cai Jian, Pierce William, He Da-Cheng, Song Zhao-Hui
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
Mol Pharmacol. 2007 Nov;72(5):1289-300. doi: 10.1124/mol.107.036566. Epub 2007 Aug 14.
The endocannabinoid 2-arachidonoylglycerol (2-AG) enhances cell migration through the CB2 cannabinoid receptor. In this study, using an immunoprecipitation and mass spectrometry-based proteomic approach, we first identified the 90-kDa heat shock protein (Hsp90), a chaperone protein with novel signaling functions, as a CB2-interacting protein. The CB2/Hsp90 interaction was confirmed in human embryonic kidney 293 cells expressing transfected CB2 and in differentiated HL-60 cells expressing endogenous CB2, by coimmunoprecipitation and Western blot experiments, as well as by treatment with geldanamycin (GA), a specific Hsp90 inhibitor. Disruption of the CB2/Hsp90 interaction by treatment with GA or reducing Hsp90 levels with specific short interfering RNAs markedly inhibited 2-AG-induced cell migration, demonstrating that Hsp90 is crucial for 2-AG-induced cell migration. 2-AG treatment resulted in a CB2-mediated stimulation of Rac1 activity, and treatment with GA blocked 2AG-induced activation of Rac1. It is noteworthy that expression of the dominant-negative form of Rac1 reduced 2-AG-induced cell migration. These data demonstrate that 2-AG-induced activation of Rac1 is essential for 2-AG-induced cell migration, and the CB2/Hsp90 interaction is needed for 2-AG-induced activation of Rac1. Furthermore, 2-AG-induced Rac1 activation was sensitive to pertussis toxin treatment, hence involving G(i) proteins. In addition, treatment with GA significantly inhibited the CB2/Galpha(i2) interaction. As a whole, our data indicate that Hsp90 may serve as scaffold to keep the CB2 receptor and its signaling components, including Galpha(i2), in proximity, thus facilitating CB2-mediated signaling to cell migration through the G(i)-Rac1 pathway. By demonstrating that Hsp90 is essential for CB2-mediated signaling to cell migration, this study reveals a novel role of Hsp90 in the signaling events mediated by a G protein-coupled receptor.
内源性大麻素2-花生四烯酸甘油酯（2-AG）通过CB2大麻素受体增强细胞迁移。在本研究中，我们首先使用基于免疫沉淀和质谱的蛋白质组学方法，鉴定出90-kDa热休克蛋白（Hsp90），一种具有新型信号传导功能的伴侣蛋白，作为与CB2相互作用的蛋白。通过共免疫沉淀和蛋白质印迹实验，以及用特异性Hsp90抑制剂格尔德霉素（GA）处理，在表达转染CB2的人胚肾293细胞和表达内源性CB2的分化HL-60细胞中证实了CB2/Hsp90相互作用。用GA处理破坏CB2/Hsp90相互作用或用特异性短发夹RNA降低Hsp90水平，显著抑制2-AG诱导的细胞迁移，表明Hsp90对2-AG诱导的细胞迁移至关重要。2-AG处理导致CB2介导的Rac1活性刺激，而GA处理阻断2-AG诱导的Rac1激活。值得注意的是，Rac1显性负性形式的表达降低了2-AG诱导的细胞迁移。这些数据表明,2-AG诱导的Rac1激活对2-AG诱导的细胞迁移至关重要，而CB2/Hsp90相互作用是2-AG诱导的Rac1激活所必需的。此外，2-AG诱导的Rac1激活对百日咳毒素处理敏感，因此涉及G(i)蛋白。此外，GA处理显著抑制CB2/Galpha(i2)相互作用。总体而言，我们的数据表明，Hsp90可能作为支架，使CB2受体及其信号传导成分（包括Galpha(i2)）保持接近，从而促进CB2介导的通过G(i)-Rac1途径向细胞迁移的信号传导。通过证明Hsp90对CB2介导的细胞迁移信号传导至关重要，本研究揭示了Hsp90在G蛋白偶联受体介导的信号传导事件中的新作用。
