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Involvement of the 90-kDa Heat Shock Protein (Hsp-90) in CB2 Cannabinoid Receptor-Mediated Cell Migration: A New Role of Hsp-90 in Migration Signaling of a G Protein-Coupled Receptor

Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky (F.H., Z.-H.Q., J.C., W.P., Z.-H.S.); and Institute of Cell Biology, College of Life Science, Beijing Normal University, Beijing, People's Republic of China (F.H., D.-C.H.)

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/G_i2 interaction. As a whole, our data indicate that Hsp90 may serve as scaffold to keep the CB2 receptor and its signaling components, including G_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.

Address correspondence to: Dr. Zhao-Hui Song, Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292. E-mail: zhsong{at}louisville.edu