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

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Abstract
The purposes of the present study were to clarify the significance of the palmitoylation site and the cytoplasmic tail of the endothelinA receptor (ETAR) 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 ETAR (CHO-ETAR). For these purposes, we constructed CHO cells stably expressing an unpalmitoylated (Cys383Cys385–388→Ser383Ser385–388) ETAR (CHOSerETAR) and a series of truncated ETARs that lacked the cytoplasmic tail downstream of either of the five cysteine residues (Cys383Cys385–388). All truncated ETARs but not SerETAR failed to stimulate adenylyl cyclase. With the truncated ETARs holding Cys385, ET-1 stimulated formation of inositol phosphates, but such stimulation failed with truncated ETARs lacking Cys385. With wild-type ETARs, 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 ETARs, the formation was sensitive to G12G228A or U73122, respectively. These results indicate that 1) Cys385 of ETAR is critical for coupling with Gq, 2) the cytoplasmic tail downstream of the palmitoylation sites of ETAR is essential for coupling with Gs and G12, and 3) the signal for ET-1–induced stress-fiber formation is transmitted through the Gq/PLC- and G12-dependent pathway to the Rho/ROCK system.