Molecular Mechanism for Endothelin-1–Induced Stress-Fiber Formation: Analysis of G Proteins Using a Mutant EndothelinA Receptor
- Yoshifumi Kawanabe1,2,
- Yasuo Okamoto2,
- Kazuhiko Nozaki1,
- Nobuo Hashimoto1,
- Soichi Miwa2 and
- Tomoh Masaki2
- Departments of 1Neurosurgery (Y.K., K.N., N.H.) and 2Pharmacology (Y.K., Y.O., S.M., T.M.), Kyoto University Faculty of Medicine, Kyoto, Japan
- Yoshifumi Kawanabe, M.D., Department of Neurosurgery, Kyoto University Faculty of Medicine, 54 Shougoin-Kawaharachou, Sakyo-ku, Kyoto 6060-8507, Japan. E-mail:kawanabe{at}kuhp.kyoto-u.ac.jp
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.
Footnotes
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This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan; by Special Coordination Funds for Science and Technology from the Science and Technology Agency; by a Research Grant for Cardiovascular Disease (11C-1) from the Ministry of Health and Welfare; and by a grant from the Smoking Research Foundation, Japan.
- Abbreviations:
- ET-1
- endothelin-1
- ETAR
- endothelinA receptor
- ETBR
- endothelinB receptor
- CHO
- Chinese hamster ovary
- PLC
- phospholipase C
- ROCK
- Rho-associated coiled-coil–forming protein kinase
- CHO-ETAR
- Chinese hamster ovary cells that stably express human endothelinA receptor
- CHO-ETARΔCys x
- Chinese hamster ovary cells that express human endothelinA receptor truncated at the carboxyl-terminal downstream of Cys x (in whichx is 382, 383, 385, or 388)
- CHO-SerETAR
- Chinese hamster ovary cells that express an unpalmitoylated (Cys383Cys385–388→Ser383Ser385–388) human endothelinA receptor
- G12G228A
- dominant negative mutant of G12
- G13G225A
- dominant negative mutant of G13
- FCS
- fetal calf serum
- IP
- inositol phosphate
- PBS
- phosphate-buffered saline
- PBS-Tx
- phosphate-buffered saline containing 0.1% Triton X-100
- Y-27632
- (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide
- U73122
- 1-(6-{[17β-3-methoxyestra-1,3.5(10)-trien-17-yl] amino}hexyl)-1H-pyrrole-2,5-dione
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- Received July 31, 2001.
- Accepted October 11, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
