Molecular mechanism for endothelin-1-induced stress-fiber formation: analysis of G proteins using a mutant endothelin(A) receptor.
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(A)R) 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(A)R (CHO-ET(A)R). For these purposes, we constructed CHO cells stably expressing an unpalmitoylated (Cys(383)Cys(385-388)-->Ser(383)Ser(385-388)) ET(A)R (CHOSerET(A)R) and a series of truncated ET(A)Rs that lacked the cytoplasmic tail downstream of either of the five cysteine residues (Cys(383)Cys(385-388)). All truncated ET(A)Rs but not SerET(A)R failed to stimulate adenylyl cyclase. With the truncated ET(A)Rs holding Cys(385), ET-1 stimulated formation of inositol phosphates, but such stimulation failed with truncated ET(A)Rs lacking Cys(385). With wild-type ET(A)Rs, 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-[[17beta-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 G(12) (G(12)G228A) or G(13) (G(13)G225A), whereas it was inhibited by U73122 in combination with G(12)G228A but not G(13)G225A. Dibutyryl cAMP alone did not induce stress-fiber formation. With unpalmitoylated or truncated ET(A)Rs, the formation was sensitive to G(12)G228A or U73122, respectively. These results indicate that 1) Cys(385) of ET(A)R is critical for coupling with G(q), 2) the cytoplasmic tail downstream of the palmitoylation sites of ET(A)R is essential for coupling with G(s) and G(12), and 3) the signal for ET-1-induced stress-fiber formation is transmitted through the G(q)/PLC- and G(12)-dependent pathway to the Rho/ROCK system.
Kawanabe
Yoshifumi
Y
Department of Neurosurgery, Kyoto University Faculty of Medicine, Kyoto, Japan. kawanabe@kuhp.kyoto-u.ac.jp
Okamoto
Yasuo
Y
Nozaki
Kazuhiko
K
Hashimoto
Nobuo
N
Miwa
Soichi
S
Masaki
Tomoh
T
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D022502:Stress Fibers / Q000378:metabolism*; D014162:Transfection
D019332:Endothelin-1; D007295:Inositol Phosphates; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins