Palmitoylation of Human EndothelinB
ITS CRITICAL ROLE IN G PROTEIN COUPLING AND A DIFFERENTIAL REQUIREMENT FOR THE CYTOPLASMIC TAIL BY G PROTEIN SUBTYPES*

(Received for publication, January 27, 1997, and in revised form, May 19, 1997)

Yasuo Okamoto , Haruaki Ninomiya , Miki Tanioka , Aiji Sakamoto , Soichi Miwa and Tomoh Masaki Dagger

From the Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

By site-directed mutagenesis, three cysteine residues (amino acids 402, 403, and 405) in the carboxyl terminus of human endothelinB (ETB) were identified as potential palmitoylation sites. Substitutions of all of the three cysteine residues with serine gave an unpalmitoylated mutant, C2S/C3S/C5S. When expressed in Chinese hamster ovary cells, C2S/C3S/C5S was localized on the cell surface, retained high affinities to ET-1 and ET-3, and was rapidly internalized when bound to the ligand. However, unlike the wild-type ETB, C2S/C3S/C5S transmitted neither an inhibitory effect on adenylate cyclase nor a stimulatory effect on phospholipase C, indicating a critical role of palmitoylation in the coupling with G proteins, regardless of the G protein subtypes. Truncation of the carboxyl terminus including Cys403/Cys405 gave a deletion mutant Delta 403 that was palmitoylated on Cys402 and lacked the carboxyl terminus downstream to the palmitoylation site. Delta 403 did transmit a stimulatory effect on phospholipase C via a pertussis toxin-insensitive G protein but it failed to transmit an inhibitory effect on adenylate cyclase. These results indicated a differential requirement for the carboxyl terminus downstream to the palmitoylation site in the coupling with G protein subtypes, i.e. it is required for the coupling with Gi but not for that with Gq.


INTRODUCTION

One of the post-translational modifications of G protein-coupled receptors (GPCRs)1 is a covalent attachment of palmitic acid to one or more cysteine residues via a hydroxylamine-labile thioester bond. At least 10 GPCRs, including human endothelinA (ETA), have been experimentally shown to be palmitoylated (1-12). In every case in which the palmitoylation sites were determined, they were located in the carboxyl-terminal cytoplasmic tail (2-4, 9, 12).

Substitutions of the cysteine residues gave unpalmitoylated mutants of each GPCR and the role of the modification has been described, to a varying extent, on three aspects of the receptor functions; 1) ligand binding, 2) G protein activation, and 3) intracellular trafficking of the receptor molecule. To date, however, there appears to be no common rule applicable to all GPCRs on any of the three aspects. On ligand binding, the elimination of palmitoylation caused no changes in the binding characteristics of all the GPCRs examined (4, 9, 12-15) except for beta 2-adrenergic receptor (beta 2AR). The unpalmitoylated beta 2AR lacked the GTP-sensitive high affinity state for agonists and this lack was ascribed to its uncoupling from Gs (3, 16). On G protein coupling, no effects of the elimination have been found on the capacities of m2 cholinergic (13), thyrotropin-releasing hormone (14), and luteinizing hormone/human choriogonadotropin (9) receptors to activate G proteins whereas opposite effects of it, both enhancement and inhibition, were described for rhodopsin to activate Gt (17) and for beta 2AR to activate Gs (3), respectively. A differential requirement for the modification between G protein subtypes coupled to the same receptor has been highlighted in a study on human ETA (12). On intracellular trafficking of the receptor molecule, reduced cell surface expression was reported for unpalmitoylated mutants of thyrotropin-releasing hormone, luteinizing hormone/human choriogonadotropin, and vasopressin V2 receptors (9, 14, 15). Internalization of beta 2AR (18) or alpha 2AR (19) was not affected while that of luteinizing hormone/human choriogonadotropin receptors was enhanced by the elimination of palmitoylation (9). Because of these pleiotropic effects described, the functional role of palmitoylation in each GPCR is an open question.

The endothelins (ETs) are a family of potent vasoactive peptides that includes ET-1, -2, and -3 (20, 21). They have a wide variety of biological effects in various tissues and cell types (22) that are mediated by specific GPCR subtypes, ETA and ETB (23, 24). The two subtypes can be pharmacologically distinguished by different rank orders of affinity toward the three ET isopeptides; ETA is ET-1-selective, showing an affinity rank order of ET-1 >=  ET-2 >>  ET-3, whereas ETB exhibits similar affinities to all of the three isopeptides (23, 24). Both of them belong to a subfamily of GPCRs with a promiscuous nature that can activate multiple subtypes of G proteins and they can also be distinguished by selective coupling with G protein subtypes; when expressed in CHO cells, ETA couples with members of Gq and Gs families while ETB couples with those of Gq and Gi families (25, 26).

The purposes of the current study were to identify potential palmitoylation sites of ETB and to reveal a role of the modification in ETB functions including ligand binding, cell surface expression, internalization, and G protein activation. An additional objective was to reveal a functional role of the carboxyl-terminal tail downstream to the palmitoylation site in the receptor functions.


EXPERIMENTAL PROCEDURES

Materials

TransformerTM site-directed mutagenesis kit from CLONTECH Laboratories, Inc. (San Francisco, CA); Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, fetal calf serum (FCS), lipofectamine, and blasticidin from Life Technologies (Tokyo, Japan); synthetic human ET-1 and ET-3 from Peptide Institute (Osaka, Japan); [125I]ET-1 (74 TBq/mmol), myo-[3H]inositol (370 GBq/mmol), cAMP radioimmunoassay assay kit, and FluorolinkTM Cy5 reactive dye pack were from Amersham International (Buckinghamshire, UK); [3H]palmitic acid (1923 GBq/mmol) and EASYTAGTM Express Protein labeling mixture [35S] (43.5 TBq/mmol) from New England Nuclear (Tokyo, Japan); pertussis toxin (PTX) from Funakoshi Co. (Tokyo, Japan); fura-2 acetoxymethyl ester from Dojin Chemicals (Tokyo, Japan); BCA microprotein assay kit, ImmunoPureTM-immobilized avidin, and NHS-SS-biotin were from Pierce (Rockford, IL). All other chemicals were of reagent grade and were obtained commercially.

Mutagenesis

The entire coding sequence of human ETB was subcloned into a BamHI restriction site of pUC19 and served as a template for mutagenesis using a TransformerTM site-directed mutagenesis kit (CLONTECH). The following primers were used to substitute the cysteine residue(s) with serine; 5'-CACCAGCAGCTTAAGCATGAC-3' to mutate Cys402, 5'-CTGGCACCAGCTGCATAAGCATG-3' to mutate Cys403, 5'-AATGACTGGCTCCAGCAGCAT-3' to mutate Cys405, 5'-CTGGCACCAGCTGCTTAAGCATGAC-3' to mutate Cys402/Cys403 and 5'-CTGGCTCCAGCTGCTTAAGCATGAC-3' to mutate Cys402Cys403Cys405. See Fig. 1 for nomenclature of the mutants. The mutations were confirmed by sequencing and the cDNA fragments were subcloned into a XhoI/NotI restriction site of a mammalian expression vector pME18Sf-. Procedures for construction of carboxyl-terminal deletion mutants (Delta 400, Delta 402, and Delta 403) were described (27).


Fig. 1. Nomenclature of the human ETB mutants. Aligned are the amino acids sequences of the carboxyl-terminal tail of the wild-type human ETB, five substitution mutants and three deletion mutants. The amino acid numbers of the four cysteine residues are indicated. Boxed are the serine residues substituted with the corresponding cysteine residues. TMVII, the seventh transmembrane domain.
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Cell Culture and Transfection

COS cells were routinely maintained in DMEM, 10% FCS at 37 °C in a humidified atmosphere containing 5% CO2. CHO cells were maintained under the same conditions except for the use of Ham's F-12 instead of DMEM. For transient expressions, COS cells in 60- or 100-mm dishes were transfected with pME18Sf- carrying the cDNA constructs using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. After 48 h the cells were subjected to the assays described below. For stable expressions, CHO cells were transfected with each expression plasmid together with pSVbsrr using Lipofectamine. Cell populations expressing the bsrr gene product were selected in Ham's F-12, 10% FCS containing blasticidin (10 µg/ml) and clonal cell lines were isolated by colony lifting and maintained in the same selection medium.

[125I]ET-1 Binding Assay

Assays using intact cells or membrane preparations were done exactly as described in Refs. 28 and 26, respectively.

Metabolic Labeling

For [35S]Methionine/cysteine (Met/Cys) labeling, COS cells in 60-mm dishes were washed with phosphate-buffered saline (PBS) and, after a preincubation in Met/Cys-free MEM for 20 min, pulse-labeled for 1.5 h with [35S]Met/Cys (50 µCi/ml) in the same medium supplemented with 10% FCS. The cells were then chased for 4.5 h in the same medium. The FCS were dialyzed against PBS prior to use. For [3H]palmitic acid labeling, COS cells in 100-mm dishes were washed with PBS and, after a preincubation in serum-free DMEM for 20 min, exposed for 6 h to the same medium containing [3H]palmitic acid (285 µCi/ml). Prior to use, [3H]palmitic acid was dried under vacuum and solubilized in serum-free DMEM containing 0.2% bovine serum albumin.

Biotinylation of ET-1

ET-1 was biotinylated using ImmunoPureTM NHS-SS-biotin (Pierce) according to the manufacturer's instructions. In brief, 48 nmol of ET-1 (in 480 µl of 50 mM NaHCO3, pH 8.5, containing 0.02% Triton X-100) was mixed with 480 nmol of NHS-SS-biotin (in 30 µl of the same buffer) and the reaction was let go for 3 h at room temperature. After the addition of another 480 nmol of NHS-SS-biotin, the reaction was further let go for 16 h. Biotinylated ET-1 was separated from naive ET-1 by high performance liquid chromatography using a C18 column (4.6 mm × 10 cm, Waters) as described (29). The biological activity of the biotinylated ET-1 was verified by its ability to induce a transient increase of [Ca2+]i in CHO cells expressing wild-type (wt) ETB (not shown).

Affinity Purification of Receptor Proteins

After the labeling with [35S]Cys/Met or [3H]palmitic acid, COS cells were harvested by incubation in PBS, 1 mM EGTA. The cells from each well were centrifuged and resuspended in 0.5 ml of PBS containing biotinylated ET-1 (100 nM) and incubated for 60 min at 25 °C. The cells were centrifuged and then lysed by incubation for 2 h at 4 °C in 0.5 ml of the lysis buffer (20 mM sodium phosphate buffer, pH 7.4, 130 mM NaCl, 1 mM EDTA, 0.2 mM phenymethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 0.25% CHAPS, and 0.4% digitonin). After removing insoluble materials by centrifugation at 100,000 × g for 1 h at 4 °C, 30 µl of avidin-agarose (50% (v/v) slurry in the lysis buffer) was added to the supernatant and the reaction was let go at 4 °C for 16 h. The agarose-avidin-biotin-ET-1-receptor complex was recovered by centrifugation and then extensively washed with ice-cold lysis buffer containing high (0.5 M) or low (0.03 M) concentrations of NaCl. The receptor protein was eluted from the resulting pellet by incubation in 25 µl of 0.2 M 2-mercaptoethanol at room temperature for 20 min. The recovered proteins were subjected to 12% SDS-PAGE under reducing conditions. The dried gels were exposed to the imaging plates for 2 days for [35S]Cys/Met-labeled proteins or for 14 days for [3H]palmitic acid-labeled proteins and the autoradiographs were developed with a BAS2000 image analyzer (Fujitsu, Tokyo, Japan).

Cyclic AMP Formation

Cells at ~50% confluence in 48-well plates were incubated for 16 h with or without PTX (50 ng/ml). The cells were washed with PBS and then incubated at 37 °C for 10 min with 0.3 ml of PBS containing 3-isobuthyl-1-methylxanthine (1 mM). They were then stimulated for 10 min with forskolin (100 µM) alone or simultaneously with forskolin and ET-1. The reaction was halted by addition of 10% (w/v) trichloroacetic acid, and the cAMP content in the trichloroacetic acid-soluble cell extract was measured using a radioimmunoassay kit (Amersham).

Phosphoinositide Breakdown

CHO cells in 24-well plates were incubated for 24 h in Ham's F-12, 10% FCS containing myo-[3H]inositol (5 µCi/ml). Where indicated, PTX (50 ng/ml) was added to the labeling medium for the last 16 h. The cells were washed with Krebs-Hensleit buffer with LiCl (110 mM NaCl, 4.5 mM KCl, 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11.7 mM glucose, 5 mM HEPES, pH 7.4, and 10 mM LiCl) equilibrated with 5% CO2 and then incubated in 250 µl of the same buffer. ET-1 (50-µl solutions in Krebs-Hensleit) was added at various concentrations and the plates were kept in a CO2 incubator for 30 min. The reactions were terminated by adding ice-cold 10% perchloric acid (100 µl/well). The following procedures including neutralization of the extracts and separation of [3H]inositol phosphates ([3H]IPs) by anion-exchange chromatography were done exactly as described (30).

Measurement of [Ca2+]i

CHO cells in 100-mm dishes were incubated for 16 h with or without PTX (50 ng/ml) and dispersed by incubation in PBS, 1 mM EGTA. The following procedures including fura-2-loading and measurement of [Ca2+]i with a CAF-110 spectrofluorometer (Japan Spectroscopy Inc., Tokyo, Japan) were exactly as described (30).

Cy5 Labeling of ET-1

ET-1 was labeled with a fluorescent dye Cy5 using a FluorolinkTM Cy5 reactive dye pack (Amersham) according to the manufacturer's instructions. In brief, 48 nmol of ET-1 in 1 ml of 0.1 M sodium carbonate buffer, pH 9.3, was applied to a vial containing the dye. The reaction was let go for 3 h at room temperature and the Cy5-labeled ET-1 was separated from naive ET-1 by high performance liquid chromatography using a C18 column as described above for the purification of biotinylated ET-1. The biological activity of Cy5-labeled ET-1 was verified by its ability to induce a transient increase of [Ca2+]i in CHO cells expressing wtETB (not shown).

In Situ Binding and Internalization of Cy5-labeled ET-1

CHO cells grown on poly-L-lysine-coated glass coverslips were washed with ice-cold binding buffer (140 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 1 mM MgCl2, 25 mM HEPES, pH 7.4, 11.7 mM glucose, 0.1% bovine serum albumin) and then incubated at 4 °C for 2 h in the same buffer containing 10 nM Cy5-labeled ET-1. After washing with ice-cold binding buffer, the fluorescent images of the cells were obtained with a MRC1024 laser-scanning confocal microscope (Bio-Rad, Osaka, Japan). To facilitate internalization of the bound ligand, the cells were then incubated in the binding buffer at 37 °C and the images were obtained at the time indicated.

Statistical Analysis

Student's t test was used for the statistical analysis of the results. p values of <0.05 were considered to be significant.


RESULTS

Identification of the Potential Palmitoylation Sites of ETB

When COS cells were transfected with pME/wtETB and then metabolically labeled with [35S]Cys/Met, two radioactive proteins with the average molecular sizes of 52 and 34 kDa were affinity-purified from the cell lysate with biotinylated ET-1 (Fig. 2a). The specificity of either band was verified by their absence in the lysate from vector-transfected cells and by their disappearance in the presence of excess unlabeled ET-1 in the binding step. Kozuka et al. (29) purified endogenous ETB from bovine lung membrane preparations in essentially the same way and proved that the 52- and 34-kDa species correspond to a full-length intact receptor and a proteolytic derivative with amino-terminal truncation, respectively. When the cells were labeled with [3H]palmitic acid, the radioactivity was incorporated into both bands (Fig. 2b), indicating the palmitoylation of ETB.


Fig. 2. Transient expression and palmitoylation of the wild-type or mutant ETB in COS cells. a, wild-type or mutant receptor proteins were affinity purified from cell lysates using biotinylated ET-1. The purified proteins were resolved by SDS-PAGE followed by autoradiography. Lysates were prepared from cells transfected with empty vector or pMEsf- carrying each cDNA construct and metabolically labeled with [35S]Met/Cys. In a displacement experiment, excess unlabeled ET-1 (5 µM) was included with biotinylated ET-1 (100 nM) in the binding step of the purification procedures (third lane from left). b, the receptor proteins were affinity-purified from cell lysates as in a, except that the cells were metabolically labeled with [3H]palmitic acid. Molecular sizes are indicated on the left (kDa).
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Potential palmitoylation sites of ETB were then determined by expression and affinity purification of a series of mutant receptors with substitutions of cysteine residues. All the substitution mutant receptors were successfully expressed in and purified from the transfected cells as judged by the recovery of [35S]Cys/Met-labeled proteins (Fig. 2a). Of the four cysteine residues at the carboxyl-terminal juxtamembrane portion of ETB, Cys402 is highly conserved among GPCRs and corresponds to the site that was proven to be palmitoylated in some of them (1). However, a single substitution of Cys402 with serine did not inhibit the [3H]palmitic acid incorporation. Neither that of Cys403 nor Cys405 affected the incorporation. In contrast, simultaneous substitutions of Cys402 and Cys403 resulted in an apparent decrease and further substitution of Cys405 resulted in a complete disappearance. Thus, we concluded that Cys402, Cys403, and Cys405, but not Cys400 are the potential palmitoylation sites of ETB.

Ligand-binding Properties of the Wild-type and Mutant Receptors Expressed in CHO Cells

That all the mutant receptors with cysteine substitutions were successfully affinity-purified by binding of biotinylated ET-1 to intact cells suggested that palmitoylation is required neither for the ligand binding nor the cell surface expression of ETB. [125I]ET-1 binding assays on intact COS cells expressing the wild-type or mutant receptors failed to reveal any differences in the binding characteristics (data not shown), giving a supportive evidence to the notions. To further confirm these and to explore a functional significance of palmitoylation in the receptor trafficking and signal transduction, the mutant receptors were stably expressed in CHO cells. This cell line was adopted because we had already shown that wtETB, when stably expressed in CHO cells, directly couples with members of both Gi and Gq families to inhibit adenylate cyclase and activate phospholipase C (PLC), respectively (26).

By co-transfecting CHO cells with each expression plasmid and pSVbsrr and then selecting for resistance against blasticidin, we obtained more than three individual clonal cell lines that stably expressed each receptor construct. [125I]ET-1 binding assays on membrane preparations from various clones gave Kd values of 30-150 pM and Bmax values of 0.5-1.7 pmol/mg protein. Representative clones were used for the subsequent study because of the similar receptor densities as listed in Table I. On the selected clones, we also performed competition binding experiments of [125I]ET-1 with ET-1 or ET-3. The IC50 values for ET-1 and ET-3 to inhibit the binding of [125I]ET-1 (25 pM) were similar in the wild-type and all mutated ETB, being in the range from 63 to 110 pM for ET-1 and from 99 to 141 pM for ET-3.

Table I. Densities and affinities of the wild-type and mutant ETB expressed on CHO cells

Clonal cell lines expressing each receptor construct were isolated as described under "Experimental Procedures." Binding parameters were determined by saturation isotherms of [125I]ET-1 binding assay using membrane preparations. These clones were selected for use in the present study because of the similar receptor densities as listed. Substitutions and deletions of amino acids in mutant receptors are given in Fig. 1. Shown are the means ± S.E. values from at least three independent experiments each done in duplicate.

Receptor construct KD Bmax

pM pmol/mg protein
Wild-type 43  ± 3 0.98  ± 0.11
C2S 40  ± 2 0.95  ± 0.25
C3S 104  ± 9 0.68  ± 0.02
C5S 32  ± 3 1.16  ± 0.07
C2S/C3S 108  ± 7 0.82  ± 0.07
C2S/C3S/C5S 38  ± 3 1.29  ± 0.10
 Delta 400 33  ± 2 0.99  ± 0.19
 Delta 402 95  ± 7 1.11  ± 0.14
 Delta 403 122  ± 10 1.66  ± 0.12

Cell Surface Expression and Internalization of the Wild-type and Mutant Receptors Expressed in CHO Cells

To examine the cell surface expression and internalization, the localization of Cy5-labeled ET-1 in CHO cells expressing wtETB or C2S/C3S/C5S was visualized by confocal microscopy. When CHO/wtETB cells were incubated with the ligand at 4 °C, Cy5-ET-1 showed a homogenous distribution on the plasma membrane. The specificity of the binding was verified by its disappearance in the presence of excess unlabeled ET-1 and also by its absence in native CHO cells (Fig. 3). Subsequent incubation at 37 °C elicited internalization of the surface-bound ligand within minutes and, after 30 min, the ligand showed a patchy distribution both below the plasma membrane and around the nucleus, presumably being localized in lysozomes. As shown in Fig. 4, there were no apparent differences between wtETB and C2S/C3S/C5S in the localization of Cy5-ET-1.


Fig. 3. Specificity of Cy5-ET-1 binding to CHO/wtETB cells. CHO/wtETB cells (a, b, d, and e) were incubated with Cy5-ET-1 (10 nM) in the presence (b and e) or absence (a and d) of unlabeled ET-1 (1 µM) at 4 °C for 2 h. Native CHO cells (c and f) were processed in the same way in the absence of unlabeled ET-1. The cells were washed with ice-cold PBS and fluorescent (d-f) or differential interference contrast (a-c) images of the cells were obtained by confocal microscopy.
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Fig. 4. Localization of Cy5-ET-1 in cells expressing the wild-type or unpalmitoylated mutant ETB. CHO cells expressing wtETB (a and c) or C2S/C3S/C5S (b and d) were incubated with Cy5-ET-1 (10 nM) at 4 °C for 2 h, washed, and then further incubated at 37 °C for 30 min. Fluorescent images of the cells were obtained by confocal microscopy at the end of the 4 °C incubation (a and b) or the 37 °C incubation (c and d).
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Failure of an Unpalmitoylated Mutant to Activate Gi

To reveal a functional significance of palmitoylation in the coupling with Gi, we tested the abilities of the mutant receptors to transmit an inhibitory effect on adenylate cyclase (Fig. 5). In CHO/wtETB cells, ET-1 caused a dose-dependent inhibition of forskolin-stimulated cAMP formation with EC50 values of 58 ± 1 pM (mean ± S.E., n = 3) and the maximum inhibition to ~40% of control. This effect was abolished by pretreatment of the cells with PTX (50 ng/ml for 16 h) as reported previously (26). An unpalmitoylated mutant C2S/C3S/C5S totally failed to transmit this effect while C2S/C3S did transmit the effect with EC50 values of 63 ± 6 pM (n = 3) and the maximum inhibition to ~35%, both of which were comparable with those obtained for wtETB. Also comparable with the effect transmitted by wtETB were those transmitted by C2S, C3S, or C5S (data not shown). These results suggested a critical role of palmitoylation of ETB in the coupling with Gi.


Fig. 5. Inhibitory effects of ET-1 on cAMP formation in CHO cells expressing the wild-type or mutant ETB. Cells expressing wtETB (circles), C2S/C3S (triangles), or C2S/C3S/C5S (squares) were stimulated either with forskolin (100 µM) alone or with forskolin and increasing concentrations of ET-1 for 10 min in the presence of 3-isobutyl-1-methylxanthine (1 mM). The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. The values were expressed as relative to the forskolin-stimulated formation (100%). There were no significant differences in the absolute values of forskolin-stimulated formations in the cell clones examined. Shown are the means ± S.E. of three determinations each done in duplicate. *, p < 0.01; significantly different from the values of cAMP formation stimulated with forskolin alone.
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Failure of an Unpalmitoylated Mutant to Activate Gq

To reveal a functional significance of palmitoylation in the coupling with Gq, we tested the abilities of the mutant receptors to transmit a stimulatory effect on PLC (Fig. 6). In CHO/wtETB cells, ET-1 caused a dose-dependent stimulation of [3H]IPs accumulation with EC50 values of 2.5 ± 0.9 nM (n = 3) and the maximum effect of ~2.5-fold increase. PTX treatment of the cells partially inhibited the ET-1-induced accumulation, suggesting an involvement of both PTX-sensitive and -insensitive G-proteins, most likely the members of Gi and Gq families, respectively. Neither PTX-sensitive nor -insensitive increase in the accumulations was observed in cells expressing C2S/C3S/C5S while both were observed in cells expressing C2S/C3S. [Ca2+]i measurement with fura-2-loaded cells reproduced the findings (Fig. 7). Both PTX-sensitive and -insensitive increases were detected in cells expressing wtETB or C2S/C3S while neither was detected in cells expressing C2S/C3S/C5S. Both of the effects of ET-1 on [3H]IPs accumulation and [Ca2+]i in cells expressing C2S, C3S, or C5S were indistinguishable from those in cells expressing wtETB (data not shown). These results suggested a critical role of palmitoylation in the coupling with Gq as in the case of Gi.


Fig. 6. ET-1-induced [3H]IPs accumulation in CHO cells expressing the wild-type or mutant ETB. Cells expressing wtETB (a), C2S/C3S (b), or C2S/C3S/C5S (c) were labeled with [3H]inositol and then stimulated for 30 min with increasing concentrations of ET-1 in the presence of 10 mM LiCl. The cells were untreated (open circles) or treated (closed circles) with PTX (50 ng/ml for 16 h) before the stimulation. [3H]IPs accumulations were determined as described under "Experimental Procedures" and expressed as fold increases above the basal values. Shown are the means ± S.E. of three determinations each done in duplicate. *, p < 0.01; and #, p < 0.05; significantly different from the values from the PTX-untreated cells.
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Fig. 7. ET-1-induced increase of [Ca2+]i in CHO cells expressing the wild-type or mutant ETB. Cells expressing wtETB (a), C2S/C3S (b), or C2S/C3S/C5S (c) were loaded with fura-2 and then stimulated with 5 nM ET-1 (arrow). The cells had been untreated or treated (*) with PTX (50 ng/ml for 16 h) before the stimulation. The changes in [Ca2+]i were monitored as described under "Experimental Procedures." Shown are the representative results from more than three independent determinations.
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Expression and Palmitoylation of the Carboxyl-terminal Deletion Mutants of ETB in COS Cells

We have constructed a series of carboxyl-terminal deletion mutants of ETB and showed that ET-1 induced a [Ca2+]i response in Ltk- cells expressing a mutant Delta 403 but not in cells expressing Delta 402 or Delta 400 (27). It was, however, left unknown whether the lack of response was due to a lack of palmitoylation or due to that of the carboxyl terminus per se. To resolve the issue and further explore a functional role of the carboxyl terminus, the deletion mutants were expressed and subjected to the same assays as described.

All the three deletion mutants were successfully expressed in and purified from the transfected COS cells as judged by the recovery of [35S]Cys/Met-labeled proteins (Fig. 8a). [3H]Palmitic acid was metabolically incorporated into Delta 403 but not into Delta 402 or Delta 400 (Fig. 8b), as expected from the notion that Cys402, Cys403, and Cys405, but not Cys400 are the potential palmitoylation sites of ETB.


Fig. 8. Transient expression and palmitoylation of the carboxyl-terminal deletion mutants of ETB in COS cells. Transfection and metabolic labeling of cells and affinity purification of receptor proteins were done exactly as described in the legend to Fig. 2. The cells were labeled with [35S]Met/Cys (a) or [3H]palmitic acid (b). Molecular sizes are indicated on the left (kDa).
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Ligand Binding and Intracellular Trafficking of the Carboxyl-terminal Deletion Mutants Expressed in CHO Cells

Binding parameters obtained from saturation isotherms with [125I]ET-1 on representative CHO cell clones stably expressing the deletion mutants are listed in Table I. Consistent with our previous results on Ltk- cells expressing these mutant receptors (27), all of them retained a high affinity to ET-1 and also that to ET-3 as judged from the competition binding experiments of [125I]ET-1 with ET-3 (data not shown). In situ binding and internalization assays with Cy5-ET-1 on these mutant receptors also failed to reveal any apparent differences from the behavior of wtETB (data not shown).

A Palmitoylated Deletion Mutant Delta 403 Coupled with Gq but Not with Gi

ET-1-induced signaling was examined in cells expressing an unpalmitoylated mutant Delta 402 or a palmitoylated mutant Delta 403. ET-1 failed to inhibit forskolin-induced cAMP formation in CHO cells expressing either receptor (Fig. 9), suggesting a lack of coupling with Gi. ET-1 also failed to stimulate [3H]IPs accumulation and to induce a [Ca2+]i increase in cells expressing Delta 402 while it did elicit both responses in cells expressing Delta 403 (Figs. 10 and 11). The EC50 values and the maximum effect for ET-1 to stimulate [3H]IPs accumulation in cells expressing Delta 403 were 8.7 ± 1 nM (n = 3) and ~2.5-fold increase, both of which were comparable with those obtained in cells expressing wtETB. A distinct difference in the responses elicited by Delta 403 from those elicited by wtETB was the lack of PTX-sensitive components both in [3H]IPs accumulation (Fig. 10) and [Ca2+]i response (Fig. 11), suggesting that the responses of the cells expressing Delta 403 were mediated solely by a member(s) of the Gq family.


Fig. 9. Inhibitory effects of ET-1 on cAMP formation in CHO cells expressing the carboxyl-terminal deletion mutants of ETB. Cells expressing wtETB (circles), Delta 402 (squares), or Delta 403 (triangles) were stimulated either with forskolin (100 µM) alone or with forskolin and increasing concentrations of ET-1 for 10 min. The contents of cAMP in the trichloroacetic acid-soluble cell extracts were determined by radioimmunoassay. The values were expressed as relative to the forskolin-stimulated formation (100%). Shown are the means ± S.E. of three determinations each done in duplicate. *, p < 0.01; significantly different from the values of cAMP formation stimulated with forskolin alone.
[View Larger Version of this Image (18K GIF file)]


Fig. 10. ET-1-induced [3H]IPs accumulation in CHO cells expressing the carboxyl-terminal deletion mutants of ETB. Cells expressing Delta 402 (a) or Delta 403 (b) were labeled with [3H]inositol and stimulated for 30 min with increasing concentrations of ET-1. The cells were untreated (open circles) or treated (closed circles) with PTX (50 ng/ml for 16 h) before the stimulation. [3H]IPs accumulations were determined as described under "Experimental Procedures." Shown are the means ± S.E. of three determinations each done in duplicate.
[View Larger Version of this Image (13K GIF file)]


Fig. 11. ET-1-induced increase in [Ca2+]i of CHO cells expressing the carboxyl-terminal deletion mutants of ETB. Cells expressing Delta 402 (a) or Delta 403 (b) were loaded with fura-2 and then stimulated with 5 nM ET-1 (arrow). The cells were untreated or treated (*) with PTX (50 ng/ml for 16 h) before the stimulation. The changes in [Ca2+]i were monitored as described under "Experimental Procedures." Shown are the representative results from more than three independent determinations.
[View Larger Version of this Image (16K GIF file)]


DISCUSSION

We have demonstrated that human ETB is covalently modified by thioesterification of palmitic acid and that the potential palmitoylation sites are the cysteine residues at amino acids 402, 403, and 405. The results obtained in the present study, however, did not indicate which of the three potential sites were actually palmitoylated in wtETB but suggested that palmitoylation of the individual cysteines was not an independent event but both alternative and hierarchical modifications of the three residues were taking place. Mutation of individual cysteines did not affect the level of [3H]palmitic acid incorporation but that of two caused a significant reduction (Fig. 2b), suggesting that, in the wild-type receptor, not all of the three but two of them are palmitoylated and that, in the single-substitution mutants (C2S, C3S, and C5S), the remaining two cysteines were alternatively palmitoylated. The reduction of [3H]palmitic acid incorporation in the double mutant C2S/C3S was more than 80% on densitometry suggesting the presence of a hierarchical order between Cys402/Cys403 and Cys405, i.e. palmitoylation of either Cys402 or Cys403 may be a prerequisite for the efficient palmitoylation of Cys405. Hierarchical modification of two potential palmitoylation sites has been demonstrated so far only for rhodopsin (5). Identification of the actual palmitoylation sites in wtETB as well as verification of the alternative/hierarchical palmitoylation must await further studies that employ chemical or enzymatic methods to detect the modification of individual cysteines.

Also left unaddressed in the present study was the possible regulation of the palmitoylation level by receptor activation as has been described for beta 2AR (31) or D1 dopaminergic receptor (7). Because the expressed receptors were recovered after [3H]palmitic acid labeling and washing the cells, we could at least conclude that wtETB as well as the various mutants were constitutively palmitoylated, without agonist-stimulation. However, because of the use of biotin-labeled agonist in the purification step, we did not assess the effect of receptor activation on the palmitoylation level. Alternative purification procedures including epitope-tagged receptors or specific antibodies are required to pursuit the issue.

[125I]ET-1 binding assays on the wild-type and various mutant receptors gave Kd and Bmax values within similar ranges regardless of the presence or absence of palmitoylation, suggesting that the overall integrity of the ligand-binding surface did not depend on the modification. These results are in line with the data obtained in many GPCRs (4, 9, 12-15) except for beta 2AR. In the case of beta 2AR, the lack of palmitoylation eliminated the GTP-sensitive high affinity state of the receptor, secondary to its uncoupling from Gs (3, 16). There was indeed a ~4-fold difference in the Kd values of [125I]ET-1 binding to various mutant receptors used in the present study (Table I). It is, however, unlikely that the apparent difference in the affinities was due to the presence or absence of a GTP-sensitive high affinity state of the receptor, because GTPgamma S failed to affect the [125I]ET-1 binding to wtETB as well as the various mutant receptors (data not shown). The absence of a GTP-sensitive high affinity state of wtETB may imply that the receptor does not couple to G proteins prior to agonist binding as has been suggested for ETA (32). In the present study, a precise reason for the apparent difference in the binding affinities between the various mutants was left unknown. It is, however, at least clear that the differences in the G protein coupling capacities of the various mutants cannot be ascribed to the differences in the ligand binding affinities, because of the all or none feature of the coupling (discussed below).

In situ binding assays with Cy5-ET-1 (Fig. 4) indicated that palmitoylation was not required for the overall sequestration (cell surface expression and internalization) of wtETB. The binding assay used, however, is not quantitative and the intracellular traffic of the receptor molecule from the site of synthesis (endoplasmic reticulum) to the plasma membrane could not be assessed with this assay. Therefore, it is still an open question whether the intracellular traffic of wtETB is actively regulated by palmitoylation of the receptor molecule.

The most distinct finding of the present study was the failure of unpalmitoylated mutant receptors to activate the G protein-dependent signaling pathways. The unpalmitoylated mutant C2S/C3S/C5S totally failed to transmit an inhibitory effect on adenylate cyclase (Fig. 5) and a stimulatory effect on PLC (Figs. 6 and 7) while C2S/C3S retained the signaling activities comparable with those of wtETB. These results indicated that either the presence of Cys405 per se or the palmitoylation of the same residue in C2S/C3S was required for the G protein coupling. Because the specific requirement for the presence of Cys405 can be excluded by the unaltered signaling activities of the mutant C5S, the signaling activities of C2S/C3S must be ascribed to the palmitoylation of Cys405. The unaltered signaling activities of C5S can in turn be ascribed to the palmitoylation of Cys402 and Cys403. Therefore, we conclude that palmitoylation of at least one of the three potential sites is required for the G protein coupling, regardless of the G protein subtypes.

The activation of signaling pathways to adenylate cyclase and PLC by various receptors was an all or none phenomenon and the quantitative relationship between the palmitoylation level and the signaling effects was not detected in the present study. Both the inhibition of cAMP formation and the stimulation of [3H]IPs accumulations caused by C2S/C3S were comparable with those by wtETB (and other single-substitution mutants) (Figs. 5 and 6) despite the apparently reduced palmitoylation level of C2S/C3S (Fig. 2b). A precise mechanism for C2S/C3S to cause the maximum effects is left unknown, however, possible explanations include the intracellular amplification of the signal by sequential interactions of receptor-G protein-effector molecules.

In addition to the critical role of palmitoylation in the G protein coupling, the present study also revealed a differential requirement for the carboxyl-terminal tail downstream to the palmitoylation site by G protein subtypes. The palmitoylated deletion mutant Delta 403 failed to transmit an inhibitory effect on adenylate cyclase (Fig. 9) but did transmit a stimulatory effect on PLC (Figs. 10 and 11) indicating that the carboxyl-terminal tail downstream to the palmitoylation site was required for the coupling with Gi but not for that with Gq. To reveal a role of the carboxyl terminus in ETB signaling, Aquilla et al. (33) constructed a deletion mutant which terminates within the seventh transmembrane domain and showed a lack of a capacity of this mutant to activate various cellular kinases. The critical role of palmitoylation and that of the carboxyl-terminal tail in the G protein coupling described here are consistent with their findings.

The requirement for palmitoylation in the G protein coupling has so far been documented for beta 2AR (16) and ETA (12). In the case of beta 2AR, the decreased coupling of unpalmitoylated mutant receptors was linked to an increased phosphorylation of the carboxyl tail of the receptor and not to the formation of a fourth intracellular loop (ICLIV) (16). Indeed there are as many as 10 putative phosphorylation sites in the carboxyl-terminal tail of ETB (34). However, both deletion mutants Delta 402 and Delta 403 lacked the potential phosphorylation sites in the carboxyl terminus and the difference of their abilities to activate Gq depended on the presence or absence of the potential palmitoylation site Cys402. Therefore, it is unlikely that the lack of a capacity of unpalmitoylated mutants was secondary to an altered phosphorylation state of the carboxyl terminus. Although the data presented does not exclude the possible regulation of G protein coupling by phosphorylation, it favors a structural requirement for the formation of ICLIV in the G protein coupling of ETB.

Comparison of the data obtained here on ETB and that reported on ETA (12) revealed some features shared by these receptor subtypes. In both cases, the receptors appeared to be constitutively palmitoylated and palmitoylation was not essential for the ligand binding capacities. Another feature shared by ETA and ETB is the absolute requirement for palmitoylation in the coupling with G proteins of the Gq family. This is, at present, a feature unique to these receptor subtypes; whether it is shared by any other GPCRs coupled with Gq is a subject for the future study. A distinct difference between ETA and ETB lies in the structural basis for the coupling of ETA with Gs and that for the coupling of ETB with Gi. Palmitoylation was required for ETB-Gi interaction but not for ETA-Gs interaction. Using chimeric receptors between ETA and ETB, we have shown that ICLII of ETA and ICLIII of ETB are the major determinants for the selective coupling of each receptor subtype with Gs and Gi, respectively (26). The requirement for palmitoylation in ETB-Gi interaction suggested an involvement of ICLVI either in selection or activation of Gi. Also suggested from the data obtained from deletion mutants was an involvement of the cytoplasmic free tail. Thus, in the case of ETB-Gi interaction, all of the three intracellular domains of the receptor, ICLIII, VI, and the cytoplasmic free tail appear to be involved.

Recently, a splice variant of human ETB was identified by molecular cloning (35). It is formed by a substitution of a large part of the carboxyl-terminal tail and the newly identified carboxyl-terminal sequence lacks any potential palmitoylation sites. When expressed in cultured cells, the splice variant retained ligand binding capacities but apparently lacked a capacity to activate G proteins, giving rise to a hypothesis that it may represent the "spare" ETB, the presence of which has been predicted by some functional studies (36, 37). An obvious explanation for the failure of this splice variant to activate G proteins is a lack of palmitoylation. If this is the case, it raises a possibility of a novel mechanism to adjust cells' responses by alternative expression of palmitoylation-positive and -negative GPCR variants.

In conclusion, we have identified the potential palmitoylation sites of human ETB and revealed a critical role of the modification in the coupling with G proteins. The relevance of these findings to the functional defects of ETB variant will be clarified in the future study.


FOOTNOTES

*   This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. Tel.: 81-75-753-4477; Fax: 81-75-753-4402; E-mail: masaki{at}mfour.med.kyoto-u.ac.jp.
1   The abbreviations used are: GPCR, guanyl nucleotide-binding regulatory protein-coupled receptor; beta 2AR, beta 2-adrenergic receptor; cAMP, cyclic adenosine monophosphate; CHO, Chinese hamster ovary; ET, endothelin; FCS, fetal calf serum; G protein, guanyl nucleotide-binding regulatory protein; ICL, intracellular loop; PBS, phosphate-buffered saline; PLC, phospholipase C; PTX, pertussis toxin; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylamminio]-1-propanesulfonic acid; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

REFERENCES

  1. Morello, J. P., and Bouvier, M. (1996) Biochem. Cell Biol. 74, 449-457 [Medline] [Order article via Infotrieve]
  2. Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A. S. (1988) FEBS Lett. 230, 1-5 [CrossRef][Medline] [Order article via Infotrieve]
  3. O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569 [Abstract/Free Full Text]
  4. Kennedy, M. E., and Limbird, L. E. (1993) J. Biol. Chem. 268, 8003-8011 [Abstract/Free Full Text]
  5. Karnik, S. S., Ridge, K. D., Bhattacharya, S., and Khorana, H. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 40-44 [Abstract]
  6. Ng, G. Y. K., George, S. R., Zastawny, R. L., Caron, M., Bouvier, M., Dennis, M., and O'Dowd, B. F. (1993) Biochemistry 32, 11727-11733 [Medline] [Order article via Infotrieve]
  7. Ng, G. Y. K., Mouillac, B., George, S. R., Caron, M., Dennis, M., Bouvier, M., and O'Dowd, B. F. (1994) Eur. J. Pharmacol. 267, 7-19 [CrossRef][Medline] [Order article via Infotrieve]
  8. Ng, G. Y. K., O'Dowd, B. F., Caron, M., Dennis, M., Brann, M. R., and George, S. R. (1994) J. Neurochem. 63, 1589-1595 [Medline] [Order article via Infotrieve]
  9. Kawate, N., and Menon, K. M. J. (1994) J. Biol. Chem. 269, 30651-30658 [Abstract/Free Full Text]
  10. Butkerait, P., Zheng, Y., Hallak, H., Graham, T. E., Miller, H. A., Burris, K. D., Molinoff, P. B., and Manning, D. R. (1995) J. Biol. Chem. 270, 18691-18699 [Abstract/Free Full Text]
  11. Alaluf, S., Mulvihill, E. R., and McIlhinney, R. A. J. (1995) J. Neurochem. 64, 1548-1555 [Medline] [Order article via Infotrieve]
  12. Horstmeyer, A., Cramer, H., Sauer, T., Müler-Esterl, W., and Schroeder, C. (1996) J. Biol. Chem. 271, 20811-20819 [Abstract/Free Full Text]
  13. van Koppen, C. J., and Nathanson, N. M. (1991) J. Neurochem. 57, 1873-1877 [Medline] [Order article via Infotrieve]
  14. Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392 [Abstract/Free Full Text]
  15. Schülein, R., Liebenhoff, U., Müller, H., Birnbaumer, M., and Rosenthal, W. (1996) Biochem. J. 313, 611-616 [Medline] [Order article via Infotrieve]
  16. Moffett, S., Mouillac, B., Bonin, H., and Bouvier, M. (1993) EMBO J. 2, 349-356
  17. Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123 [Abstract/Free Full Text]
  18. Campbell, P. T., Hnatowich, M., O'Dowd, B. F., Caron, M. G., Lefkowitz, R. J., and Hausdorff, W. P. (1991) Mol. Pharmacol. 39, 192-198 [Abstract]
  19. Eason, M. G., Jacinto, M. T., Theiss, C. T., and Liggett, S. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11178-11182 [Abstract/Free Full Text]
  20. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., and Masaki, T. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2863-2867 [Abstract]
  21. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-415 [CrossRef][Medline] [Order article via Infotrieve]
  22. Masaki, T. (1993) Endocr. Rev. 14, 256-268 [Medline] [Order article via Infotrieve]
  23. Arai, H., Hori, S., Aramori, S., Ohkubo, H., and Nakanishi, S. (1990) Nature 348, 730-732 [CrossRef][Medline] [Order article via Infotrieve]
  24. Sakurai, T., Yanagisawa, M., Takuwa, H., Miyazaki, H., Kimura, S., Goto, K., and Masaki, T. (1990) Nature 348, 732-735 [CrossRef][Medline] [Order article via Infotrieve]
  25. Aramori, I., and Nakanishi, S. (1992) J. Biol. Chem. 267, 12468-12474 [Abstract/Free Full Text]
  26. Takagi, Y., Ninomiya, H., Sakamoto, A., Miwa, S., and Masaki, T. (1995) J. Biol. Chem. 270, 10072-10078 [Abstract/Free Full Text]
  27. Koshimizu, T., Tsujimoto, G., Ono, K., Masaki, T., and Sakamoto, A. (1995) Biochem. Biophys. Res. Commun. 217, 354-362 [CrossRef][Medline] [Order article via Infotrieve]
  28. Sakamoto, A., Yanagisawa, M., Sawamura, T., Enoki, T., Ohtani, T., Sakurai, T., Nakao, K., Toyo-oka, T., and Masaki, T. (1993) J. Biol. Chem. 268, 8547-8553 [Abstract/Free Full Text]
  29. Kozuka, M., Ito, T., Hirose, S., Lodhi, K. M., and Hagiwara, H. (1991) J. Biol. Chem. 266, 16892-16896 [Abstract/Free Full Text]
  30. Sugawara, F., Ninomiya, H., Okamoto, Y., Miwa, S., Mazda, O., Katsura, Y., and Masaki, T. (1996) Mol. Pharmacol. 49, 447-457 [Abstract]
  31. Mouillac, B., Caron, M., Bonin, H., Dennis, M., and Bouvier, M. (1992) J. Biol. Chem. 267, 21733-21737 [Abstract/Free Full Text]
  32. Rose, P. M., Krystek, S. R., Jr., Patel, P. S., Liu, E. C., Lynch, J. S., Lach, D. A., Fisher, S. M., and Webb, M. L. (1995) FEBS Lett. 361, 243-249 [CrossRef][Medline] [Order article via Infotrieve]
  33. Aquilla, E., Whelchel, A., Knot, H. J., Nelson, M., and Posada, J. (1996) J. Biol. Chem. 271, 31572-31579 [Abstract/Free Full Text]
  34. Sakamoto, A., Yanagisawa, M., Sakurai, T., Takuwa, Y., Yanagisawa, H., and Masaki, T. (1991) Biochem. Biophys. Res. Commun. 178, 656-663 [Medline] [Order article via Infotrieve]
  35. Elshourbagy, N. A., Adamou, J. E., Gagnon, A. W., Wu, H.-L., Pullen, M., and Nambi, P. (1996) J. Biol. Chem. 271, 25300-25307 [Abstract/Free Full Text]
  36. Fukuroda, T., Fujikawa, T., Ozaki, S., Ishikawa, K., and Nishikibe, M. (1994) Biochem. Biophys. Res. Commun. 199, 1461-1465 [CrossRef][Medline] [Order article via Infotrieve]
  37. Ozaki, S., Ohwaki, K., Ihara, M., Fukuroda, T., Ishikawa, K., and Yano, M. (1995) Biochem. Biophys. Res. Commun. 209, 483-489 [CrossRef][Medline] [Order article via Infotrieve]

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