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

  1. Yoshifumi Kawanabe1,2,
  2. Yasuo Okamoto2,
  3. Kazuhiko Nozaki1,
  4. Nobuo Hashimoto1,
  5. Soichi Miwa2 and
  6. Tomoh Masaki2
  1. 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
  1. 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
 
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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.

Endothelin-1 (ET-1) has a wide variety of biological effects on various tissues and cell types (Yanagisawa et al., 1988; Masaki, 1993) that are mediated by specific heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor subtypes, the endothelinA receptor (ETAR) and endothelinB receptor (ETBR) (Arai et al., 1990; Sakurai et al., 1990). The two receptors activate multiple subtypes of G proteins and can be distinguished by their selective coupling with specific G protein subtypes. When expressed in Chinese hamster ovary (CHO) cells, ETAR couples with members of the Gq and Gs families and stimulates phospholipase C (PLC) and adenylyl cyclase. ETBR couples with members of the Gq and Gi families, stimulates PLC, and inhibits adenylyl cyclase (Aramori and Nakanishi, 1992; Takagi et al., 1995).

ETAR and ETBR were shown to be palmitoylated at a cluster of cysteine residues located in the cytoplasmic tail (Horstmeyer et al., 1996; Okamoto et al., 1997). The functional role of palmitoylation and the cytoplasmic tail domain downstream of the palmitoylation site in coupling with G proteins has been studied for ETAR and ETBR (Horstmeyer et al., 1996; Okamoto et al., 1997). We found that in the case of ETBR, palmitoylation is necessary for coupling with both Gq and Gi, whereas the cytoplasmic tail downstream of the palmitoylation sites is also required for coupling with Gi (Okamoto et al., 1997). On the other hand, with ETAR, palmitoylation is reported to be essential for coupling with Gq but not with Gs, based solely on an experiment using an unpalmitoylated mutant ETAR (Horstmeyer et al., 1996). Thus, which domain of ETAR is necessary for coupling with Gs and which of the potential palmitoylation sites is necessary for coupling with Gq remains unknown. In this context, we first attempted to determine the structural basis essential for coupling ETAR with Gq and Gs by focusing on several potential palmitoylation sites and the cytoplasmic tail downstream of the palmitoylation sites. For this purpose, we constructed CHO cells that stably expressed an unpalmitoylated mutant (Cys383Cys385–388→Ser383Ser385–388) ETAR (CHO-SerETAR) and a series of truncated ETARs that lacked the cytoplasmic tail downstream of any of the five cysteine residues (Cys383Cys385–388).

ET receptors were demonstrated to couple with the G12 subfamily, consisting of G12 and G13, in NIH 3T3 cells (Mao et al., 1998). The G12 subfamily has been shown to mediate important signaling pathways such as for Rho/Rho-associated coiled-coil–forming protein kinase (ROCK)-dependent formation of actin stress fibers (Buhl et al., 1995) and vascular smooth muscle cell contraction (Gohla et al., 2000). These reports suggest that the G12 subfamily may play important roles in several ET-1–induced vascular disorders, such as stroke or vasospasm. Thus, the control of G12 subfamily activation may become a new treatment strategy for these conditions. Recently, it was shown that activation of ETAR induces actin stress-fiber formation via G12 but not G13 (Gohla et al., 1999). However, the domains in the ETAR that are necessary for coupling with G12 have not yet been elucidated. The second purpose of the present study is to reveal a functional coupling between ETAR and G12/G13 in CHO-ETAR and the functional roles of the palmitoylation site and cytoplasmic tail downstream of the palmitoylation site of ETAR in coupling of the receptor with G12 using mutated ETARs. Furthermore, the conclusion with regard to coupling of ETAR with G12is based on an experiment in which actin stress-fiber formation is lost after expression of a dominant negative mutant of G12 in fibroblast cell lines derived from Gq/G11-double deficient mice (Gohla et al., 1999). It remains unknown whether ET-1–induced actin stress-fiber formation requires other G proteins such as Gq and Gs in addition to G12. We have attempted to address this point using CHO cells expressing mutated ETARs. Previous reports demonstrated that CHO cells express both G12 and G13 (van de Westerlo et al., 1995; Malek et al., 2001).

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Materials and Methods

Mutagenesis.

The entire coding sequence of human ETAR was subcloned into pGEM-T. The truncated ETAR cDNAs shown in Fig.1 were created by polymerase chain reaction. The sequence of the oligonucleotide 5′-primers for all mutants was 5′-CTCGAGGTCGACGGTATCGATAAGCTTGATAT-3′. The sequences of the oligonucleotide 3′-primers for Δ388, Δ385, Δ383, and Δ382 were 5′-GCGGCCGCTCAACAGCAGCAGCAGAGGCAT-3′, 5′-GCGGCCGCTCA-GCAGAGGCATGACTGGAAA-3′, 5′-GCGGCCGCTCAGAGGCATGACTGGAAACAA-3′, and 5′-GCGGCCGCTCATGACTGGAAACAATTTTTA-3′, respectively. Each 3′-primer contained one nucleotide substitution to introduce a termination stop codon with a NotI restriction site, whereas the 5′-primer contained an XhoI restriction site. Fragments were amplified by the 5′-primer and each 3′-primer from ETAR cDNA as a template. The polymerase chain reaction amplification profiles were denaturation at 94°C for 1 min, primer annealing at 55°C for 30 s, and extension at 72°C for 1 min for 30 cycles. The mutations were confirmed by sequencing, and cDNA fragments were subcloned into a XhoI/NotI restriction site of a mammalian expression vector pME18Sf predigested by XhoI andNotI.

Figure 1
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Figure 1

Nomenclature of human ETAR mutants. Aligned are the amino acid sequences of the carboxyl-terminal tail of the wild-type human ETAR, unpalmitoylated mutant and four deletional mutants. The amino acid numbers of the three cysteine residues are indicated. TM VII, seventh transmembrane domain.

The entire coding sequence of human ETAR into pME18sf served as a template for unpalmitoylated mutagenesis using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following primers were used to substitute the cysteine residues in the cytoplasmic tail with serine residues: 5′-AATTGTTTCCAGTCATCCCTCTCCTCCTCCTCTTACCAGTCCAAA-3′ and 5′-TTTGGACTGGTAAGAGGAGGAGGAGAGGGATGACTGGAAACAATT-3′ to mutate Cys383Cys385–388. The mutations were confirmed by sequencing.

Wild-type G12 and G13 in pcDNA3(+) were kindly provided by Dr. Manabu Negishi (Kyoto University, Japan). G12G228A and G13G225A, which were shown to be the dominant negative types (Gohla et al., 1999), were generated by a QuickChange site-directed mutagenesis kit (Stratagene). Mutations were verified by sequencing.

Cell Culture and Transfection.

CHO cells were maintained in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS) under a humidified 5% CO2/95% air atmosphere. For stable expression, CHO cells were transfected with expression plasmids together with pSVbsrr using LipofectAMINE (Invitrogen, Tokyo, Japan). Cell populations expressing the bsrr gene product were selected in Ham's F-12 supplemented with 10% FCS containing blasticidine (10 μg/ml), and clonal cell lines were isolated by colony lifting and maintained in the same medium.

125I-ET-1 Binding Assay.

Assays using intact cells or membrane preparations were performed exactly as described previously (Sakamoto et al., 1993).

Cyclic AMP Formation and Inositol Phosphates Formation.

Cyclic AMP formation and inositol phosphate (IP) formation were determined as described previously (Okamoto et al., 1997).

Microinjection.

Microinjection was performed as described previously (Okazawa et al., 1998). Briefly, cells were seeded onto glass coverslips coated with fibronectin (Iwaki Glass, Chiba, Japan), which were marked with a cross to facilitate the localization of injected cells and incubated overnight in Ham's F-12 medium containing 1% FCS. Plasmids (100 ng/μl) encoding for G12G228A and G13G225A were microinjected into cell nuclei. As a control, expression plasmids without inserts were microinjected in an adjacent field on the same coverslip. Microinjection was performed using a manual microinjection system (Eppendorf–5 Prime, Inc., Hamburg, Germany) equipped with an Axiovert 100 inverted microscope (Carl-Zeiss GmbH, Frankfurt, Germany).

Stress-Fiber Formation.

After incubation of cells with serum-free Ham's F-12 medium for 24 h, ET-1 was added at 37°C for 5 min. Cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. After being washed five times with PBS containing 0.1% Triton X-100 (PBS-Tx), the cells were incubated with fluorescein rhodamine-phalloidin (Molecular Probes, Eugene, OR) in PBS-Tx (1:200) at room temperature for 10 min. After being washed five times with PBS-Tx, the labeled cells were mounted on glass slides and examined with an MRC 1024 laser-scanning confocal microscope (Bio-Rad, Hercules, CA) equipped with an Axiovert 135 M inverted microscope (Carl-Zeiss GmbH).

Images were converted to PICT files in Adobe Photoshop (Adobe Systems Inc., San Jose, CA) and analyzed using NIH Image software (http://rsb.info.nih.gov/nih-image/) by quantifying the average pixel intensities as described previously (Barnett et al., 1997).

Drugs.

Y-27632 was kindly provided by Welfide Corporation (Osaka, Japan). Chemicals were obtained from the following sources: ET-1 from the Peptide Institute (Osaka, Japan),125I-ET-1 and myo-[3H]inositol from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK), rhodamine-phalloidin from Molecular Probes, U73122 from Funakoshi (Tokyo, Japan), and dibutyryl cAMP from Sigma (St. Louis, MO). All other chemicals were of reagent grade and were obtained commercially.

Statistical Analysis.

All results were expressed as mean ± S.E.M. The data were subjected to a two-way analysis of variance, and when a significant F value was encountered, the Newman-Keuls multiple range test was used to test for significant differences between treatment groups. A probability level ofP < 0.05 was considered statistically significant.

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Results

Stable Expression of Truncated or Unpalmitoylated Mutant ETARs in CHO Cells.

By cotransfecting CHO cells with each expression plasmid and pSVbsrr and then selecting for resistance against blasticidine, we obtained more than five individual clonal cell lines that stably expressed each receptor construct. In CHO cells expressing truncated mutant ETAR, 125I-ET-1 binding assays on membrane preparations from various clones gaveKd values of 30 to 120 pM andBmax values of 0.7 to 1.4 pmol/mg of protein. On the other hand, in CHO-SerETAR,125I-ET-1 binding assays on membrane preparations from various clones gave Kd values of 50 to 140 pM and Bmax values of 0.8 to 1.7 pmol/mg of protein. Cell clones showing similar levels of receptor densities were used in the subsequent study. TheKd and Bmaxvalues for the receptors expressed on each clone adopted are listed in Table 1.

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Table 1

Densities and affinities of wild-type and mutant ETA receptors expressed on CHO cells

Formation of IPs and cAMP in CHO Cells Expressing Truncated or Unpalmitoylated Mutant ETARs after Stimulation with ET-1.

To reveal the functional significance of the palmitoylation site and the cytoplasmic tail downstream of the palmitoylation site in coupling with Gq and Gs, we tested the abilities of the mutant receptors to stimulate accumulation of [3H]IPs and cAMP, respectively. [3H]Palmitic acid was metabolically incorporated into CHO-ETARΔ388 and CHO-ETARΔ385 but not into CHO-ETARΔ383, CHO-ETARΔ382, or CHO-SerETAR (data not shown).

In CHO-ETAR, ET-1 caused a concentration-dependent stimulation of [3H]IP accumulation with an EC50 value of 2.7 ± 0.3 nM, and the maximal effect of a ∼15-fold increase was obtained at concentrations ≥10 nM (Fig. 2A). In CHO-ETARΔ388 or CHO-ETARΔ385, ET-1 caused a concentration-dependent stimulation of [3H]IP accumulation with an EC50 value and a maximum increase that were comparable with those of CHO-ETAR (Fig. 2A). In contrast, ET-1 failed to stimulate [3H]IP accumulation in CHO-ETARΔ383, CHO-ETARΔ382, or CHO-SerETAR (Fig. 2A).

Figure 2
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Figure 2

Effects of ET-1 on IP (A) or cAMP (B) accumulation in CHO cells expressing wild-type or mutant ETARs. Formation of total IPs or cAMP after stimulation with varying concentrations of ET-1 in CHO-ETAR (■), CHO-ETARΔ388 (closed triangle), CHO-ETARΔ385 (○), CHO-ETARΔ383 (▵), CHO-ETARΔ382 (●), and CHO-SerETAR (▪). A, cells that had been incubated withmyo-[3H]inositol for 18 h were stimulated by various concentrations of ET-1 for 30 min. B, cells that were stimulated by various concentrations of ET-1 for 10 min in the presence of 3-isobutyl-1-methylxanthine. Total IPs and cAMP in the cell extract were measured as described under Materials and Methods. Values are expressed as -fold increases above basal values. Each data point represents mean ± S.E.M. of five experiments.

In CHO-ETAR, ET-1 stimulated cAMP formation with an EC50 of 2.7 ± 0.3 nM, and a maximal effect of an ∼8-fold increase was obtained at concentrations ≥10 nM (Fig. 2B). ET-1 also stimulated cAMP accumulation in a concentration-dependent manner in CHO-SerETAR (Fig. 2B). The EC50 value and the maximal effect of cAMP accumulation in CHO-SerETAR were similar to those in CHO-ETAR (Fig. 2B). In contrast, ET-1 failed to stimulate cAMP formation in CHO cells expressing all truncated ETAR (Fig. 2B).

ET-1–Induced Actin Stress-Fiber Formation in CHO-ETAR.

We attempted to determine the structural basis for coupling of ETAR with G12/G13 and subtypes of G proteins involved in ET-1–induced stress-fiber formation. For these purposes, we examined the effects of inhibition of either one of the G protein-mediated signaling cascades by blockers and dominant negative mutants of G12 or G13(G12G228A or G13G225A, respectively) on ET-1–induced actin stress-fiber formation in CHO-ETARΔ385, CHO-SerETAR, and CHO-ETAR. Subsequently, we deduced the domains of ETAR that were critical for coupling with G12, based on the structure of the mutant ETARs that did not have the ability to couple to G12.

As reported for NIH 3T3 cells and fibroblasts (Mao et al., 1998; Gohla et al., 1999), ET-1 induced actin stress-fiber formation in CHO-ETAR (Fig. 3B). In contrast, ET-1 failed to induce stress-fiber formation in CHO-ETAR that had been preincubated with 10 μM Y-27632, a selective ROCK inhibitor (Fig. 3C). Stress-fiber formation was not affected by pretreatment with 5 μM U73122, a PLC inhibitor, or microinjection of G12G228A or G13G225A in CHO-ETAR (Fig.3F). This concentration (5 μM) of U73122 abolished ET-1–induced IP accumulation in CHO-ETAR (data not shown). Notably, when the cells were subjected to microinjection of G12G228A followed by pretreatment with U73122, ET-1 failed to induce stress-fiber formation (Fig. 3D). In contrast, microinjection of G13G225A in combination with pretreatment by U73122 had no effect on ET-1–induced stress-fiber formation (Fig. 3F).

Figure 3
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Figure 3

A-D, effects of Y-27632, U73122, and G12G228A on the ET-1–induced actin stress-fiber formation in CHO-ETAR. Cells were stimulated with (B) or without (A) 10 nM ET-1. The effects of preincubation with 10 μM Y27632 (C) and combination treatment of G12G228A microinjection and 5 μM U73122 preincubation (D) on ET-1–induced stress-fiber formation are shown. Y-27632 and U73122 were added 15 min before stimulation with ET-1. Expression plasmids encoding for G12G228A and G13G225A were microinjected into the cell nuclei 24 h before stimulation with ET-1. E, effects of dibutyryl cAMP on actin stress-fiber formation in CHO-ETAR. Cells were incubated for 5 min with 10 μM dibutyryl cAMP alone. Actin stress fibers were visualized as described under Materials and Methods. Representative examples of stress fibers in individual cells are shown. F, pixel intensity of images was quantified using NIH Image software. Values are expressed as -fold increases above the values in the absence of ET-1. Each data point represents mean ± S.E.M. of at least 20 cells.

To clarify the role of Gs in actin stress-fiber formation, we examined the effect of dibutyryl cAMP in quiescent CHO-ETAR. Treatment with dibutyryl cAMP up to 10 μM alone failed to induce actin stress-fiber formation in CHO-ETAR (Fig. 3E).

ET-1–Induced Actin Stress-Fiber Formation in CHO-SerETAR and CHO-ETARΔ385.

ET-1 induced stress-fiber formation in CHO-SerETAR, in which coupling of the receptor with Gs but not Gq was retained (Fig.4B). Like CHO-ETAR, ET-1–induced stress-fiber formation was inhibited by preincubation of CHO-SerETAR with Y-27632 (Fig. 4C) but was not affected by preincubation with U73122 or microinjection of G13G225A (Fig. 4, E and F). Notably, unlike CHO-ETAR, it was inhibited by microinjection of G12G228A (Fig. 4D).

Figure 4
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Figure 4

Effects of Y27632, U73122, G12G228A, and G13G225A on ET-1–induced actin stress-fiber formation in CHO-SerETAR. Cells were stimulated with (B) or without (A) 10 nM ET-1. C, Y-27632 at 10 μM was added 15 min before stimulation with ET-1. Expression plasmids encoding for G12G228A (D) and G13G225A (E) were microinjected into cell nuclei 24 h before stimulation with ET-1. Actin stress fibers were visualized as described under Materials and Methods. Representative examples of stress fibers in individual cells are shown. F, pixel intensity of images was quantified using NIH Image software. Values are expressed as -fold increases above the values in the absence of ET-1. Each data point represents mean ± S.E.M. of at least 20 cells.

ET-1 induced stress-fiber formation in CHO-ETARΔ385, in which coupling of the receptor with Gq but not Gs was retained (Fig. 5B). Like CHO-ETAR, ET-1–induced stress-fiber formation was inhibited by preincubation of CHO-ETARΔ385 with Y-27632 (Fig. 5C) but was not affected by microinjection of G12G228A or G13G225A (Fig.5, E-G). Notably, unlike CHO-ETAR, it was inhibited by preincubation with U73122 (Fig. 5D).

Figure 5
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Figure 5

Effects of Y27632, U73122, G12G228A, and G13G225A on ET-1–induced actin stress-fiber formation in CHO-ETARΔ385. Cells were stimulated with (B) or without (A) 10 nM ET-1. Y-27632 at 10 μM (C) and U73122 at 5 μM (D) were added 15 min before stimulation with ET-1. Expression plasmids encoding G12G228A (E) and G13G225A (F) were microinjected into the cell nuclei 24 h before stimulation with ET-1. Actin stress fibers were visualized with fluorescein rhodamine-phalloidin as described under Materials and Methods. Representative examples of stress fibers in individual cells are shown. G, pixel intensity of images was quantified using NIH Image software. Values are expressed as -fold increases above the values in the absence of ET-1. Each data point represents mean ± S.E.M. of at least 20 cells.

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Discussion

125I-ET-1 binding assays on intact CHO cells expressing the wild-type or truncated ETARs yielded Kd andBmax values within similar ranges (Table1). These results were consistent with previous data (Hashido et al., 1993) and suggest that truncation of the receptor is not essential for cell surface expression and ligand binding of ETAR. High affinity binding of ET-1 by the mutant receptors is a good indication that the overall structure of the receptor is unchanged by truncation as described earlier (Hashido et al., 1993).

As reported previously (Horstmeyer et al., 1996), with SerETAR in which a cluster of five cysteine residues in the cytoplasmic tail as potential palmitoylation sites were substituted with serine, ET-1 failed to stimulate formation of IPs (Fig. 2A). In the present study, we extended this finding using truncated ETARs. The truncated ETARs holding Cys385(CHO-ETARΔ385 and CHO-ETARΔ388) retained the ability to stimulate IP formation, whereas those lacking Cys385(CHO-ETARΔ383 and CHO-ETARΔ382) lost such ability (Fig. 2A). These results taken together strongly indicate that Cys385 in ETAR is critical for coupling of ETAR with Gq and that the cytoplasmic tail downstream of the palmitoylation site is not necessary for this coupling (Fig.6).

Figure 6
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Figure 6

Schematic representation of signaling pathways for actin stress-fiber formation activated by ET-1 in CHO-ETAR (A), CHO-SerETAR (B), and CHO-ETARΔ385 (C). Wild-type ETAR couple to Gq, Gs, and G12, whereas SerETAR or ETARΔ385 couple to Gs/G12 or Gq, respectively. Actin stress-fiber formation is stimulated by ET-1 via Gq- and G12-dependent pathways in CHO-ETAR, whereas via G12- or Gq-dependent pathway in CHO-SerETAR or CHO-ETARΔ385, respectively. See Results and Discussion for details.

In the present study, ET-1 stimulated adenylyl cyclase in CHO-SerETAR, which lacked potential palmitoylation sites but retained the cytoplasmic tail (Fig. 2B). These results are consistent with a previous report (Horstmeyer et al., 1996). In contrast, ET-1 failed to stimulate adenylyl cyclase in all truncated ETARs lacking the cytoplasmic tail, regardless of the absence or presence of palmitoylation sites of ETAR (Fig. 2B). These results, taken together, strongly demonstrate that the cytoplasmic tail of ETAR is critical for coupling with Gs, although it is not necessary for coupling with Gq (Fig. 6). Moreover, it was previously demonstrated that the second and third intracellular loops of ETAR were major determinants of the selective coupling of ETAR with Gs(Takagi et al., 1995). Therefore, we conclude that both the cytoplasmic tail and the second and third intracellular loops of ETAR are necessary for coupling of ETAR with Gs.

Next, we attempted to identify the subtypes of G proteins that are involved in ET-1–induced stress-fiber formation using CHO-ETARΔ385, CHO-SerETAR, and CHO-ETAR. Based on sensitivity to Y-27632, the Rho/ROCK pathway plays important roles in ET-1–induced stress-fiber formation in CHO-ETAR (Fig. 5C) as in NIH 3T3 cells and fibroblasts (Mao et al., 1998; Gohla et al., 1999). ET-1–induced stress-fiber formation in CHO-ETAR was affected by neither pretreatment with U73122 nor microinjection of G12G228A or G13G225A (Fig.5F) but was inhibited by combined treatment with U73122 and G12G228A microinjection (Fig. 3D). These results indicate that ET-1–induced stress-fiber formation is mediated via two signaling pathways (i.e., the Gq/PLC- and G12-dependent pathways in CHO-ETAR) (Fig. 6) and also that only one of the two is sufficient for actin stress-fiber formation. Moreover, the present study indicates that Gs is not involved in ET-1–induced stress-fiber formation, because dibutyryl cAMP failed to induce actin stress-fiber formation in CHO-ETAR (Fig. 3E).

These conclusions are supported by findings obtained with SerETAR. That is, because SerETAR does not couple with Gq, which is one of the two signaling pathways necessary for ET-1–induced stress-fiber formation, blockade of another signaling pathway with G12G228A leads to inhibition of actin stress-fiber formation. Furthermore, these results indicate that SerETAR retains the ability to couple with G12.

In CHO-ETARΔ385, in which coupling of the receptor with Gq but not Gsis retained, ET-1–induced stress-fiber formation was inhibited by U73122 but not G12G228A. Based on the conclusion obtained from wild-type ETAR, these data can be interpreted to mean that because ETARΔ385 lacks coupling with G12, which is one of the two signaling pathways necessary for ET-1–induced actin stress-fiber formation, blockade of another signaling pathway with U73122 leads to inhibition of actin stress-fiber formation. Therefore, these results indicate that ETARΔ385 has lost the ability to couple with G12, although it can still induce stress-fiber formation via the Gq-dependent pathway.

Finally, we deduced the structural determinant for coupling of ETAR with G12 based on data from experiments using mutated ETARs. That is, loss of coupling of ETARΔ385 with G12 and retention of coupling of SerETAR with G12 clearly show that the cytoplasmic tail downstream of Cys385 but not the palmitoylation site of ETAR is essential for coupling with G12.

In conclusion, the present study showed that 1) the cytoplasmic tail downstream of the palmitoylation site of ETAR is essential for coupling with Gs and G12, 2) Cys385 of ETAR is critical for coupling with Gq, and 3) the signal for ET-1–induced stress-fiber formation is mediated via the Gq/PLC- and G12-dependent pathway to Rho/ROCK system in CHO-ETAR. Thus, the presence of one of the two pathways is sufficient for stress-fiber formation.

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Acknowledgments

We thank Mitsubishi Pharma Corporation for the kind donation of Y-27632.

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Footnotes

  • 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
    • Received July 31, 2001.
    • Accepted October 11, 2001.
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  1. doi: 10.1124/mol.61.2.277 Molecular Pharmacology February 1, 2002 vol. 61 no. 2 277-284
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