Abstract

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^385–388→Ser³⁸³Ser^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-{[17β-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.