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.
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.
He
Fang
F
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
Qiao
Zhuan-Hong
ZH
Cai
Jian
J
Pierce
William
W
He
Da-Cheng
DC
Song
Zhao-Hui
ZH
D001095:Arachidonic Acids / Q000494:pharmacology*; D016227:Benzoquinones / Q000494:pharmacology; D063385:Cannabinoid Receptor Modulators / Q000494:pharmacology*; D002460:Cell Line; D002465:Cell Movement; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000378:metabolism; D005989:Glycerides / Q000494:pharmacology*; D018922:HL-60 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D001095:Arachidonic Acids; D016227:Benzoquinones; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C107953:RAC1 protein, human; D034741:RNA, Small Interfering; D043885:Receptor, Cannabinoid, CB2; D043562:Receptors, G-Protein-Coupled; C094503:glyceryl 2-arachidonate; D051960:GTP-Binding Protein alpha Subunit, Gi2; D020830:rac1 GTP-Binding Protein; C001277:geldanamycin