Received for publication March 29, 2007.
Revised August 13, 2007.
Accepted for publication August 14, 2007.

INVOLVEMENT OF HSP-90 IN CB2 CANNABINOID RECEPTOR-MEDIATED CELL MIGRATION ----A NEW ROLE OF HSP-90 IN MIGRATION SIGNALING OF A G PROTEIN-COUPLED RECEPTOR

Abstract

The endocannabinoid 2-arachidonoylglycerol (2-AG) enhances cell migration through the CB2 receptor. In this study, using an immunoprecipitation and mass spectrometry based proteomic approach; we first identified Hsp90, a chaperone protein with novel signaling functions, as a CB2 interacting protein. The CB2/Hsp90 interaction was confirmed in HEK293 cells expressing transfected CB2 and in differentiated HL-60 cells expressing endogenous CB2, by co-immunoprecipitation 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. Importantly, expression of the dominant negative form of Rac1 reduced 2-AG-induced cell migration. These data demonstrate that 2-AG-induced activation of Rac1 activity 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. Collectively, our data indicate that Hsp90 serves as scaffold to keep the CB2 receptor and its signaling components, including G_i2, in close 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.

Key words: Cannabinoid, Cdc42, rho, rac, other small G proteins