(Received for publication, January 27, 1997, and in revised form, May 19, 1997)
From the Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
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 403
that was palmitoylated on Cys402 and lacked the carboxyl
terminus downstream to the palmitoylation site.
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.
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 2-adrenergic receptor (
2AR). The
unpalmitoylated
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
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
2AR (18) or
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.
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.
MutagenesisThe 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 (
400,
402, and
403) were
described (27).
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.
Assays using intact cells or membrane preparations were done exactly as described in Refs. 28 and 26, respectively.
Metabolic LabelingFor [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-1ET-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 ProteinsAfter 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 FormationCells 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 BreakdownCHO 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+]iCHO 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-1ET-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-1CHO 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 AnalysisStudent's t test was used for the statistical analysis of the results. p values of <0.05 were considered to be significant.
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.
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 CellsThat 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.
|
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.
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.
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.
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
403 but not in cells
expressing
402 or
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 403 but not into
402 or
400 (Fig.
8b), as expected from the notion that Cys402,
Cys403, and Cys405, but not Cys400
are the potential palmitoylation sites of ETB.
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).
ET-1-induced signaling was examined
in cells expressing an unpalmitoylated mutant 402 or a palmitoylated
mutant
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
402 while it did elicit both responses in cells
expressing
403 (Figs. 10 and
11). The EC50 values and
the maximum effect for ET-1 to stimulate [3H]IPs
accumulation in cells expressing
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
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
403 were
mediated solely by a member(s) of the Gq family.
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 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 2AR. In the case of
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 GTP
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 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 2AR (16) and ETA (12).
In the case of
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
402 and
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.