Vol. 61, Issue 2, 277-284, February 2002

Molecular Mechanism for Endothelin-1-Induced Stress-Fiber Formation: Analysis of G Proteins Using a Mutant Endothelin_A Receptor

Yoshifumi Kawanabe, Yasuo Okamoto, Kazuhiko Nozaki, Nobuo Hashimoto, Soichi Miwa, and Tomoh Masaki

Departments of Neurosurgery (Y.K., K.N., N.H.) and Pharmacology (Y.K., Y.O., S.M., T.M.), Kyoto University Faculty of Medicine, Kyoto, Japan

The purposes of the present study were to clarify the significance of the palmitoylation site and the cytoplasmic tail of the endothelin_A receptor (ET_AR) in coupling with G proteins and to determine the subtypes of G protein that are involved in actin stress-fiber formation in Chinese hamster ovary cells that stably express ET_AR (CHO-ET_AR). For these purposes, we constructed CHO cells stably expressing an unpalmitoylated (Cys³⁸³Cys^385-388Ser³⁸³Ser^385-388) ET_AR (CHOSerET_AR) and a series of truncated ET_ARs that lacked the cytoplasmic tail downstream of either of the five cysteine residues (Cys³⁸³Cys^385-388). All truncated ET_ARs but not SerET_AR failed to stimulate adenylyl cyclase. With the truncated ET_ARs holding Cys³⁸⁵, ET-1 stimulated formation of inositol phosphates, but such stimulation failed with truncated ET_ARs lacking Cys³⁸⁵. With wild-type ET_ARs, ET-1 induced actin stress-fiber formation, which was inhibited by (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), a Rho-associated coiled-coil-forming protein kinase (ROCK) inhibitor. The formation was unaffected by 1-(6-{[17-3-methoxyestra-1,3.5(10)-trien-17-yl] amino}hexyl)-1Hpyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor, or dominant negative mutants of G₁₂ (G₁₂G228A) or G₁₃ (G₁₃G225A), whereas it was inhibited by U73122 in combination with G₁₂G228A but not G₁₃G225A. Dibutyryl cAMP alone did not induce stress-fiber formation. With unpalmitoylated or truncated ET_ARs, the formation was sensitive to G₁₂G228A or U73122, respectively. These results indicate that 1) Cys³⁸⁵ of ET_AR is critical for coupling with G_q, 2) the cytoplasmic tail downstream of the palmitoylation sites of ET_AR is essential for coupling with G_s and G₁₂, and 3) the signal for ET-1-induced stress-fiber formation is transmitted through the G_q/PLC- and G₁₂-dependent pathway to the Rho/ROCK system.