The results revealed that only the ETBR in the ground-state structure bound to caveolin-1, through the caveolin scaffolding and C-terminal domains, and that a fraction of ETBR was target
Trang 1Regulated interaction of endothelin B receptor with caveolin-1
Tomohiro Yamaguchi1, Yasunobu Murata1, Yoshinori Fujiyoshi1,2and Tomoko Doi1
1 Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Japan; 2 Japan Biological Information Research Centre, Tokyo, Japan
The peptide hormone endothelin transmits various signals
through G protein-coupled receptors, the endothelin type A
(ETAR) and B (ETBR) receptors Caveolae are specialized
lipid rafts containing polymerized caveolins We examined
the interaction of ETBR with caveolin-1, expressed in Sf9,
COS-1, and HEK293 cells, and its effects on the subcellular
distribution and the signal transduction of ETBR ETBR
formed a complex with caveolin-1 in cells in which these
two proteins were coexpressed and in the mixture after
purification and reconstitution (as examined by
immuno-precipitation) suggesting the direct binding of ETBR with
caveolin-1 The complex formed efficiently only when the
ETBR was ligand-free or bound to an antagonist,
RES-701-1, whereas the addition of ET-1 or another antagonist,
BQ788, dissociated the complex, suggesting the structural
recognition of ETBR by caveolin-1 In contrast, the
ETAR bound to caveolin-1 regardless of ligand binding Caveolin-1 utilized its scaffolding domain (residues 82–101) and the C-terminal domain (residues 136–178) to bind to
ETBR, as for other signalling molecules Furthermore, the amount of ETBR localized in caveolae increased significantly with the expression of caveolin-1 and decreased with the addition of ET-1 The disruption of caveolae by filipin reduced the ET-1-derived phosphorylation of ERK1/2 These results suggest the possibility that the binding to caveolin-1 retains the ligand-free ETBR in caveolae and regulates the ET signal
Keywords: caveolae; caveolin; endothelin; endothelin type B receptor; lipid raft
Endothelins (ETs) are 21-amino acid peptides that mediate
diverse physiological effects on vasoconstriction, cellular
development, differentiation, mitogenesis and other
func-tions, in various tissues via their G protein-coupled
recep-tors (GPCRs)) endothelin receptor type A (ETAR) and B
(ETBR) [1,2] For example, an ETAR mediates
vasocon-striction in vascular smooth muscle cells, whereas an ETBR
mediates the release of nitric oxide to stimulate
vasodilata-tion in vascular endothelial cells The ET ligand–receptor
complex exhibits a slow dissociation rate and almost
irreversible binding, which could explain the prolonged
vasoconstriction by ET [3] This property highlights the
importance of understanding ET signal regulation Both
ETAR and ETBR undergo rapid desensitization [4–6] and
internalize differently in transfected cells, as shown for many
GPCRs [7] Concerning the intracellular trafficking
path-ways, ETAR is internalized rapidly, either via caveolae or clathrin-coated pits, upon ligand binding [3,8,9] and follows
a recycling pathway, whereas ETBR follows a degradative pathway after internalization via coated-pits, implicated for clearance of plasma ET-1 [10] Moreover, the ETBR in the plasma membrane is constitutively transported to this pathway independently of ligand stimulation, indicating that ETBR is hardly retained in the plasma membrane at steady state [9] Differences in the final fates and subcellular localization of the ETRs are determined by the C-terminal sequences of the two receptors [9,11] However, the effects of the cell surface localization of the ETRs on the ET signals have not been studied carefully
Lipid rafts are microdomains of cell membranes that are rich in cholesterol and sphingolipid and serve as sites for gathering signalling molecules [12–14] Caveolae are vesi-cular invaginations of the plasma membrane, formed from lipid rafts with polymerized caveolins [15] The three caveolin genes have been cloned (caveolin-1, caveolin-2, and caveolin-3) The caveolin-1 and caveolin-2 show the same distribution pattern, whereas caveolin-3 is specific to muscle cells Caveolins act as scaffolding proteins to cluster and regulate signalling molecules targeted to the caveolae, such as Src-family tyrosine kinases, Ha-Ras, G protein
a subunits, endothelial nitric oxide synthase, protein kinase
C, and epidermal growth factor (EGF) receptor, among others [16] Caveolins are cholesterol-binding integral membrane proteins that are thought to form an unusual hairpin-like structure, with the central hydrophobic domain (residues 102–134 of caveolin-1) in the membrane and the N- (residues 1–101) and C-terminal (residues 135–178) domains inside the cell In this structure, the scaffolding domain of caveolin (residues 82–101) has been shown to
Correspondence to T Doi, Department of Biophysics,
Graduate School of Science, Kyoto University, Oiwake,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502 Japan.
Fax: + 81 75 753 4218, Tel.: + 81 75 753 4216,
E-mail: doi@em.biophys.kyoto-u.ac.jp
Abbreviations: ET, endothelin; ETR, endothelin receptor; ET A R and
ET B R, endothelin receptor type A and type B; GPCR, G
protein-coupled receptor; HEK293, human embryo kidney 293; CHO,
Chinese hamster ovary; EGF, epidermal growth factor; ERK1/2,
extracellular signal-regulated kinase 1 and 2; GST, glutathione
S-transferase; Cav.1-H6, His 6 -tagged caveolin-1; OG,
n-octyl-b-D-glucopyranoside; DRM, detergent-resistant membrane;
FL, full length.
(Received 27 November 2002, revised 11 February 2003,
accepted 27 February 2003)
Trang 2mediate the interactions with the signalling molecules
described above [17] On the other hand, the
caveolin-binding sequence motif (FXFXXXXF or FXXXXFXXF,
whereF is an aromatic amino acid, Trp, Phe, or Tyr) has
been suggested to exist in the signalling molecules, by which
they bind to the caveolin scaffolding domain [18]
Several GPCRs, including the B2 bradykinin,
b-adrener-gic, cholecystokinin, ETA, angiotensin II type 1, and
adenosine A1 receptors, are localized within caveolae
[19–25] Particularly, the B2 bradykinin, angiotensin II type
1, and m2 muscarinic acetylcholine receptors are
translo-cated to caveolae upon agonist binding, whereas adenosine
A1 and b2-adrenergic receptors are translocated out of
caveolae upon activation, and the binding of ETAR with
caveolins is not affected by agonist binding Furthermore,
the m2 muscarinic acetylcholine and ETAreceptors could be
internalized via caveolae [8,26] The mitogenic signal
through ETBR in primary astrocytes, where the ETBR is
relatively well expressed, originates from caveolae
micro-domains [27] In addition, a number of proteins mediating
intracellular Ca2+signalling are concentrated in caveolae
[15,16] Thus, the localization to caveolae and the
inter-action with caveolins appear to play fundamental roles in
the signal transduction by GPCRs However, the
inter-actions of GPCRs with caveolins are not fully understood
To examine the molecular mechanisms of the cell surface
localization, and its effects on signal transduction, we
studied the interactions of ETBR with caveolins in vivo and
in vitro, in terms of agonist-regulated localization on the cell
surface and signal transduction downstream The results
revealed that only the ETBR in the ground-state structure
bound to caveolin-1, through the caveolin scaffolding and
C-terminal domains, and that a fraction of ETBR was
targeted to caveolae by the expression of caveolin-1 and was
gradually released from caveolae by ET-1 stimulation In
addition, the disruption of caveolae impaired the
down-stream signals from ETBR These results suggest feasible
regulations for ETBR signal transduction by the interaction
with caveolin-1 The residence of ETBR in caveolae might
be a way of ensuring the cell surface localization of ETBR
against rapid internalization
Experimental procedures
Materials
The cyclic-peptide antagonist for ETBR, RES701-1, was
generously provided by M Yoshida (Kyowa Hakko Kogyo
Co., Ltd, Tokyo, Japan) The cDNAs encoding human
ETAR and ETBR were kindly provided by T Masaki
(National Cardiovascular Research Institute, Osaka, Japan)
and Y Furuichi (AGENE Research Institute, Kanazawa,
Japan), respectively The hybridoma producing the
anti-(bovine rhodopsin) mAb, 1D4, was generously provided by
R Molday (University of British Columbia, Vancouver,
Canada) The antagonist for ETBR, BQ788, was from
Phoenix Pharmaceuticals, Inc The anti-ETBR mAb, 2A5,
was generated against Sf9-expressed human ETBR (T
Yamaguchi, I Arimoto, Y Fujiyoshi & T Doi, unpublished
data) Goat anti-mouse and anti-(rabbit IgG)–alkaline
phosphatase and –horseradish peroxidase conjugates were
from Promega Biotech
DNA construction The caveolin-1 cDNA was produced from human lung mRNA by RT-PCR using appropriate oligonucleotide primers, and was subsequently subcloned into the BamHI– NotI sites of the mammalian expression vector pcDNA3.1 (Invitrogen) The ETBR cDNA was subcloned into the BamHI–NotI sites of pcDNA3.1 Mutagenesis of the ETBR cDNA was carried out with appropriate oligonucleo-tide primers by a PCR-based site-directed mutagenesis approach In each ETBR mutant, the aromatic amino acid residue (Tyr127, Tyr200, Trp206, Trp217, Phe326, Tyr387, Phe393, Phe397, Trp404, or Phe408) was replaced with alanine These cDNAs were subcloned into the BamHI– NotI sites of pcDNA3.1
In the C av.1-H6 cDNA, a His6-tag was attached to the Cterminus of caveolin-1 for affinity purification using
Ni2+–NTA agarose (Qiagen) The 1D4 epitope sequence (KTETSQVAPA, an epitope for the anti-rhodopsin mAB) was fused to the Cterminus of ETAR For expression in insect cells, the cDNAs encoding caveolin-1, Cav.1-H6,
ETBR, and ETAR-1D4 were subcloned into a transfer vector, pVL1393 (BD Biosciences), and the isolation of recombinant viruses was carried out with linearized Bacu-logold DNA according to the manufacture’s instructions (BD Biosciences)
To generate glutathione S-transferase (GST)-fused caveolin-1 proteins, selected regions of caveolin-1 [amino acids 1–101, 1–81, 61–101, 136–178, 101–136, and 1–136 (full length; FL)] were amplified by PCR GST-Cav.1 (1–101), GST-Cav.1(1–81), GST-Cav.1(61–101), and GST-Cav.1(101–136) were subcloned into the BamHI– XhoI sites of pGEX-4T-3 (Amersham Biosciences Inc.), whereas GST-Cav.1 (136–178) and GST-Cav.1-FL were subcloned into the BamHI–NotI sites After expression
in Escherichia coli (BL21 strain), the GST fusion pro-teins were affinity-purified on glutathione–agarose beads (Amersham Biosciences Inc.) according to the manufac-turer’s instructions
Cell culture COS-1, HEK293, and CHO-K1 cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma) containing 10% foetal bovine serum (Invitrogen) and penicillin/strep-tomycin at 37Cin a 5% CO2atmosphere For transient expression, LipofectAmine Plus reagent (Invitrogen) was used to transfect COS, HEK293, and CHO cells in a 100-mm diameter plate or in a 6-well plate with 1–8 lg pcDNA3.1 encoding the wild type or mutant ETBRs, caveolin-1, or vector only These cells were subjected to each study 36 h after transfection
For the isolation of stably transfected HEK293 cell lines expressing ETBR, the cells were transfected with the plasmid pcDNA3.1 containing the ETBR cDNA, using the calcium phosphate precipitation technique (Invitrogen) Two days after transfection, the cells were plated in selective medium containing 500 lgÆmL)1 G418 (Invitrogen) The G418-resistant colonies were selected, and the single colonies were purified further The ETBR expression level by the HEK293 cell lines isolated for further studies was 1–2 pmol per mg membrane protein
Trang 3The culture of Sf9 insect cells and the expression of ETBR
in Sf9 cells were performed as described previously [28] The
expression of caveolin-1 and His6-tagged caveolin-1
(Cav.1-H6) was also performed similarly Co-expression of ETBR
and caveolin-1 in Sf9 cells was carried out by a double
infection with ETBR and caveolin-1 recombinant viruses
Purification of ETBR and caveolin-1 from Sf9 cells
All operations were carried out at 4C Purification of
ETBR by ligand-affinity chromatography using biotinylated
ET-1, and reconstitution of the purified ligand-free ETBR
into phospholipid vesicles were performed as described
previously [28,29]
Cav.1-H6 was purified from infected Sf9 cells using
Ni2+–NTA-agarose beads The membranes were prepared
as described ( 100 mg protein from a 500-mL suspension
culture) and were solubilized in 20 mL NaCl/Tris (20 mM
Tris/HCl pH 7.5, 150 mMNaCl) with protease inhibitors
(1 mM phenylmethylsulfonyl fluoride, 10 lgÆmL)1
aproti-nin, 10 lgÆmL)1leupeptin, 10 lgÆmL)1pepstatin), 60 mM
n-octyl-b-D-glucopyranoside (OG; Dojindo), and 1%
Tri-ton X-100 (Wako) After 30 min solubilization, the lysates
were ultracentrifuged for 1 h at 100 000 g The
super-natants were adjusted to 10 mM imidazole and were
incubated with 200 lL Ni2+-nitrilotriacetic acid-agarose
beads overnight After the incubation, the beads were
washed extensively with washing buffer (NaCl/Tris
con-taining 20 mMimidazole, 30 mMOG, and 0.03% Triton
X-100) four times by rotating for 5 min After washing, the
bound proteins were eluted with 400 lL elution buffer
(NaCl/Tris containing 100 mMimidazole, 30 mMOG, and
0.03% Triton X-100) by rotating for 1 h
Immunoprecipitation
All preparations were carried out at 4Cunless stated
otherwise
ETRs expressed with caveolin-1 in Sf9 membranes Sf9
membranes (10 mg membrane proteins) were prepared as
described above [28,29], resuspended in NaCl/Tris/EDTA
(20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA)
containing protease inhibitors (1 mM
phenylmethanesulfo-nyl fluoride, 10 lgÆmL)1aprotinin, 10 lgÆmL)1leupeptin,
10 lgÆmL)1pepstatin), and incubated with or without 2 lM
ET-1 for 1 h at room temperature After the incubation, the
membranes were solubilized with 60 mM OG and 1%
Triton X-100 for 30 min, and were centrifuged at 100 000 g
for 30 min The supernatants were incubated with 30 lL of
either 1D4 or 2A5 monoclonal antibody-immobilized resin
(1 mg antibody per mL resin) overnight After this
incubation, the resins were washed extensively at least four
times with NaCl/Tris/EDTA containing the
aforemen-tioned detergents, and then the bound proteins were eluted
with 100 lMof either the 1D4 or 2A5 epitope peptides for
1 h at room temperature
Purified ETBR and Cav.1-H6 Reconstituted
phospho-lipid vesicles with or without ETBR (1–2 pmol) were
incubated with 1 lM ET-1 for 1 h at room temperature,
and then were incubated with 10–20 lg purified Cav.1-H6
overnight In these mixtures, the concentrations of OG and Triton X-100 derived from the Cav.1-H6 solution were far below the critical micelle concentrations (OG, 2–3 mM; Triton X-100, 0.002–0.003%) so that the membranes were not solubilized After incubation, the membranes were solubilized with 60 mM OG and 1% Triton X-100, incubated with 2A5-immobilized resin, washed, and eluted with 2A5-epitope peptides
GST–Cav.1 fusion proteins Sf9 membranes containing
ETBR (2.5 mg protein per sample) were incubated with the respective GST–Cav.1 fusion proteins (50 lg) overnight and then centrifuged The concentrations of detergents in these mixtures derived from the GST–Cav.1 fusion proteins were far below the critical micelle concentration The membranes were solubilized in 1 mL NaCl/Tris containing 1% n-decyl-b-D-maltopyranoside (Dojindo), and were centrifuged again After ultracentrifugation, the superna-tants were incubated with 2A5-immobilized resin, washed, and eluted with 2A5-epitope peptides
Mutant ETBRs expressed in COS cells Thirty six hours after transfection with the wild type ETBR cDNA, each mutant ETBR cDNA or the vector only, COS cells (one 100-mm diameter plate per sample) were washed twice with NaCl/Pi, lysed with 1 mL NaCl/Tris/EDTA containing
60 mM OG and 1% Triton X-100, and centrifuged at
100 000 g (Fig 3B) In addition, the washed cells were collected in 1 mL hypotonic buffer (20 mMTris/HCl pH 7.5,
1 mM EDTA, and protease inhibitors) to burst the cells, incubated with 1 lM ET-1 for 1 h at room temperature, solubilized with 60 mM OG and 1% Triton X-100, and centrifuged at 100 000 g (Fig 3C) The supernatants were incubated with 2A5-immobilized resin, washed, and eluted with 2A5-epitope peptides
Effects of antagonists on the interaction The membranes prepared from HEK293 cells, which expressed ETBR stably and caveolin-1 transiently (1–2 mgÆprotein per sample), were resuspended in NaCl/Tris/EDTA containing protease inhibitors, and incubated with 1 lM ET-1, 10 lM RES-701-1, or 10 lMBQ788 for 1 h at room temperature After this incubation, the samples were solubilized with 60 mM
OG and 1% Triton X-100, incubated with 2A5-immobilized resin, washed, and eluted with 2A5-epitope peptides Electrophoresis and immunoblotting
Samples prepared in Laemmli sample buffer were subjected
to SDS/PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell) The nitrocellulose membranes were blocked with 2.5% BSA and 0.5% gelatin for 30 min, and were then incubated with the 2A5 mAb (1 lgÆmL)1), anti-GST (1 lgÆmL)1, Amersham Biosciences Inc.), anti-caveolin-1 mAb (2297) (1 : 1000, Transduction Laborator-ies), anti-caveolin polyclonal Ig (1 : 10 000, Transduction Laboratories), anti-(transferrin receptor) mAb (1 lgÆmL)1, Zymed Laboratories Inc.), anti-flotillin mAb (1 : 250, Transduction Laboratories), anti-(extracellular signal-regu-lated kinase) (ERK)1 mAb (1 : 2000, Transduction Labor-atories), or anti-phosphoERK1/2 mAb (E10) (1 : 2000; Cell Signaling Technology) overnight at 4C The membranes
Trang 4were washed with buffer (5 mMTris/HCl pH 7.5, 150 mM
NaCl, 0.05% Tween 20), incubated with secondary
anti-bodies for 1 h, washed, and then visualized by the BCIP/
NBT colour development substrates or the ECL+ plus
Western blotting detection system (Promega Biotech and
Amersham Biosciences Inc., respectively)
Preparation of caveolin-enriched, low buoyant
membrane fractions
Thirty-six hours after the transfection with the Cav.1 gene,
the HEK293 cells stably expressing ETBR were cultured in
serum-free medium for 6 h, and were then incubated with
or without 100 nM ET-1 for 30 min at 37C The
following operations, except for the elution, were carried
out at 4C After the incubation, the confluent HEK293
cells of two 100-mm diameter plates ( 8 mg total protein)
per sample were washed twice with NaCl/Pi, scraped into
2 mL buffer (25 mM Mes pH 6.5, 150 mM NaCl and
protease inhibitors) containing 1% Triton X-100, and
homogenized using a loose-fitting Dounce homogenizer
(10 strokes) The homogenates were then adjusted to 40%
sucrose by the addition of an equal volume of an 80%
sucrose solution prepared in the above buffer, but lacking
Triton X-100, placed in the bottom of ultracentrifuge
tubes, and then overlaid with a discontinuous sucrose
gradient of 5 mL 30% (w/v) sucrose and 2 mL 5% (w/v)
sucrose, both prepared in buffer lacking Triton X-100 The
samples were centrifuged at 39 000 r.p.m (200 000 g) in
an SW41 rotor (Beckman Instruments) for 16–20 h,
fractionated into 11 1 mL fractions sequentially from the
top of the gradient, and concentrated by precipitation with
trichloroacetic acid or by ultracentrifugation following
dilution
Assay for phosphorylation of ERK1/2
Forty-eight hours after transfection in a 6-well plate, the
CHO cells were cultured in serum-free medium for 24 h,
preincubated with 1 or 2 lgÆmL)1filipin III, or vehicle for
10 min at 37C, and then incubated with 100 nMET-1 or
vehicle for 5 min at 37C After stimulation, the cells were
washed twice with ice-cold NaCl/Pi, and were lysed with
100 lL RIPA buffer (50 mM Tris/HCl pH 7.5, 150 mM
NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium
deoxycholate, 0.5 mMNa3VO4, 50 mMNaF, 5 mMEDTA
and protease inhibitors) at 4C The lysates were
trans-ferred into centrifuge tubes, sonicated for 15 s to shear the
DNA and reduce the sample viscosity, and then boiled for
5 min at 95C The cooled samples were subjected to SDS/
PAGE and immunoblotted with anti-phosphoERK1/2 or
anti-ERK1 Igs
Other methods
Protein concentrations were measured by the modified
Lowry method (DC-protein assay, Bio-Rad) with BSA as
standard The amounts of ETBR contained in the samples
were measured by the125I-labelled ET-1 (NEN Life Science
Products) binding assay as described previously [29] The
relative intensities of indicated proteins were measured by
comparative densities of reactive bands on immunoblots
with IMAGEMASTER VDS-CL and TOTALLAB version 1.10 software (Amersham Biosciences Inc.)
Results
ETBR directly binds to caveolin-1 in an ET-1-dependent manner
To examine the interaction of ETBR with caveolin-1, we took advantage of an insect cell expression system, in which
ETAR and ETBR are expressed well, with KDvalues for ET-1 of 55 ± 8 and 83 ± 11 pM, the Bmax values of
21 ± 1.7 and 60 ± 7.8 pmol per mg membrane protein, respectively [28] In addition, it has also been shown that the caveolin-1 expressed in insect cells forms oligomers, and is incorporated into caveolae-sized vesicles [30] The human
ETAR-1D4 (ETAR fused with the 1D4 epitope at the Cterminus) and ETBR were each coexpressed with caveolin-1 in insect Sf9 cells, incubated with or without ET-1, and immunoprecipitated with the 1D4 and 2A5
anti-ETBR mAbs, respectively, as shown in Fig 1A We extensively used 60 mM OG/1% Triton X-100 mixed-micelle conditions for the membrane solubilization before immunoprecipitation, which allowed the recovery of ligand-free ETBR, as described later, and the solubilization of many Triton-insoluble proteins [31] The eluates from the antibody-immobilized resin were analysed for caveolin-1,
ETAR-1D4, and ETBR by immunoblotting The caveolin-1 coimmunoprecipitated with the ETAR-1D4, regardless of the absence or presence of ET-1 (lanes 2 and 3), but not without the expression of the receptor (lane 1) This observation is consistent with those of the previous report [21] In contrast, ETBR coimmunoprecipitated caveolin-1 only when the receptor was ligand-free (lane 5) The addition of ET-1 significantly reduced the amount of coimmunoprecipitated caveolin-1 (lane 6) This coprecipi-tation of caveolin-1 was not detected in the absence of
ETBR expression (lane 4) These results suggest that ETAR binds to caveolin-1 regardless of ET-1 binding, whereas
ETBR binds to it in an ET-1-sensitive manner
Although several GPCRs have been suggested to interact with caveolin, it is not clear if the interactions occur directly
or indirectly To clarify the interaction of ETBR with caveolin-1, we purified ETBR and caveolin-1 in detergent-micelles ETBR, expressed in Sf9 cells, was purified on a ligand-affinity column eluted with 2M NaSCN to yield ligand-free ETBR [29] (Fig 1B, lane 1) Caveolin-1, with a His6-tag fusion at the Cterminus (Cav.1-H6), was also expressed in Sf9 cells and purified by nickel-affinity chromatography, by which Cav.1-H6 became the predom-inant protein, as described in Experimental procedures (Fig 1B, lane 2) The purified Cav.1-H6 could interact with
a purified G protein ai subunit in detergent micelles, as detected by immunoprecipitation, and as described previ-ously (data not shown) [32] The purified ETBR was reconstituted into phospholipid vesicles, into which the purified Cav.1-H6 was added, and the interaction between them was assessed by immunoprecipitation (Fig 1C) While
no Cav.1-H6 was immunoprecipitated from the phospho-lipid vesicles without ETBR (lane 1), it coimmunoprecipi-tated from the ETBR-containing vesicles (lane 2) Moreover, the addition of ET-1 significantly reduced the amount of the
Trang 5precipitated Cav.1-H6 (lane 3) These results using the
heterologously expressed and purified proteins completely
reproduced the observations in the Sf9 membranes
(Fig 1A), suggesting the direct interaction of ETBR with
caveolin-1 Interestingly, ETBR and caveolin-1 did not form
the complex in a detergent/micelle solution, but they bound
to each other only when the receptor was reconstituted into
vesicles This could be due to that either the interaction of
ETBR and caveolin-1 occurs by two-dimensional molecular movements along the membranes, or that the integration of
ETBR and caveolin-1 into the lipid bilayer stabilizes the structures required for the interaction, or that the detergent molecules binding to the ETBR interfere with caveolin-1 binding
Fig 1 ET B R directly interacts with caveolin-1 and dissociates from it upon ET-1 binding (A) Sf9 membranes containing ET A R-1D4 (lanes 1, 2 and 3) or ET B R (lanes 4, 5 and 6) expressed with caveolin-1 were incubated with (lanes 3 and 6) or without 2 l M ET-1 (lanes 1, 2, 4 and 5) for 1 h at room temperature, solubilized, and immunoprecipitated with the 1D4 or 2A5 mAbs The eluates from the resin were analysed by SDS/PAGE, followed by immunoblotting with the anti-(caveolin-1) mAb (upper panel), the 1D4 mAb (lower panel, lanes 1–3), or the 2A5 mAb (lower panel, lanes 4–6) ET A R-1D4 coimmunoprecipitated with caveolin-1, regardless of the absence or presence of ET-1 (lanes 2 and 3), whereas ET B R coimmunoprecipitated with caveolin-1 only when the receptor was ligand-free (lanes 5 and 6) Immunoprecipitation of caveolin-1 was not detected
in the absence of the receptor (lanes 1 and 4) (B) ET B R and Cav.-1-H6 were individually expressed and purified from infected Sf9 cells, as described
in the Experimental procedures The purified ET B R (lane 1) and Cav.1-H6 (lane 2) were visualized by silver staining, following SDS/PAGE (C) Phospholipid vesicles reconstituted with (lanes 2 and 3) or without (lane 1) the purified ET B R (1–2 pmol) were incubated with vehicle (lanes 1 and 2)
or 1 l M ET-1 for 1 h at room temperature, and were then incubated with the purified Cav.1-H6 overnight at 4 C The binding was analysed by immunoprecipitation with the 2A5 mAb after solubilization The eluates from the resin were analysed by immunoblotting with the anti-(caveolin-1) mAb (upper panel) or the 2A5 mAb (lower panel) While the phospholipid vesicles without ET B R did not show any coimmunoprecipitated Cav.1-H6 (lane 1), the Cav.1-H6 coimmunoprecipitated from the ET B R-containing vesicles (lane 2) The addition of ET-1 significantly reduced the amount of coprecipitated Cav.1-H6 (lane 3).
Trang 6The scaffolding domain and the C-terminal domain
of caveolin-1 both interact with ETBR
The caveolin scaffolding domain (residues 82–101 of
caveolin-1) is responsible for the binding with the
aforementioned signalling molecules, protein kinase A
catalytic subunit, connexin 43, and others [16,33,34]
Conversely, these proteins contain the caveolin-binding
motifs (UXUXXXXU and UXXXXUXXU, where U is an
aromatic amino acid residue), which have been suggested
to be responsible for the binding to caveolin [18] To
investigate the interaction between ETBR and caveolin-1,
we constructed a set of GST fusion proteins with various
caveolin-1 domains, as shown in Fig 2A These fusion
proteins were expressed in E coli and purified by
GST-affinity chromatography, as shown in Fig 2B Sf9 cell
membranes containing ETBR were incubated with these
purified fusion proteins, and immunoprecipitataed with
the 2A5 mAb While the ETBRs in each eluate were
recovered to similar extents (data not shown), certain
GST-fusions, including GST–Cav.1-FL, GST–Cav.1
(1–101), GST–Cav.1(61–101), and GST–Cav.1(136–178)
coimmunoprecipitated, whereas GST–Cav.1(1–81) and
GST itself did not (Fig 2C) GST–Cav.1(101–136) also
did not coimmunoprecipitate (data not shown) These
results suggest that caveolin-1 also utilizes the scaffolding
and C-terminal domains to interact with ETBR, as with
other signalling molecules The binding of the C-terminal
domain fusion, GST–Cav.1(136–178), appeared to be
weaker, as compared with that of the scaffolding domain,
in repeated experiments
Structure of ETBR recognized by caveolin
Since caveolin interacts with ETBR via the scaffolding and
C-terminal domains, the caveolin-binding motifs could be
the sites of these interactions in ETBR However, these
motifs are not present in the cytoplasmic and
transmem-brane domains of ETBR, at least in the primary structure,
which would contain the caveolin-binding region, because
caveolin resides inside the cell In fact, we mutated the
aromatic residues in the cytoplasmic and transmembrane
regions close to the cytoplasmic side, one by one
(Fig 3A) The ETBRs expressed in C OS cells, shown in
Fig 3B, were observed as two bands in the
immunoblot-ting because of N-terminal proteolysis, which was also
found with HEK293 and CHO cells, as described later
[35–37] These mutated ETBRs expressed in COS cells
bound caveolin-1 in the ligand-free form (Fig 3B), and
dissociated from caveolin-1 following ET-1 binding,
similar to the wild type (Fig 3C) The measurement of
band intensities showed no obvious differences between
the wild type and mutant ETBRs in the ligand-sensitive
caveolin-1 binding The C-terminal truncated ETBR
(residues 408–442 deleted) also showed these properties
in the COS cell system (data not shown) Therefore, single
mutations of these residues in the ETBR do not
substan-tially affect the caveolin-1 binding
The fact that the addition of ET-1 reduced the amount of
caveolin-1 bound to ETBR (Fig 1) indicates that caveolin-1
could distinguish the structure of the ligand-free ETBR from
that of the ligand-bound form To examine the
contribu-tions of the ETBR tertiary structure to the recognition by caveolin-1, we further analysed the interactions of caveo-lin-1 with ETBR in the presence of two types of antagonists, RES-701-1 [38] or BQ788 [39] (Fig 4) Previously, we showed that RES-701-1 displayed an inverse-agonist acti-vity that stabilizes ETBR structure in the ground-state, but BQ788 did not [28] The HEK293 cells stably expressing
ETBR were transfected with the caveolin-1 gene The membranes prepared from these cells were incubated with ET-1, RES-701-1 or BQ788 and were immunoprecipitated with the 2A5 mAb The eluates were analysed for caveolin-1
Fig 2 The scaffold and C-terminal domains of caveolin-1 recognize
ET B R (A) Schematic diagram summarizing the construction of a set
of GST–Cav.1 fusion proteins: GST–Cav.1-FL, GST–Cav.1(1–101), GST–Cav.1(1–81), GST–Cav.1(61–101), GST–Cav.1(136–178) and GST–Cav.1(101–136) (B) GST–Cav.1 fusion proteins, purified
by GST-affinity chromatography, were resolved by SDS/PAGE and visualized by Coomassie blue staining (C) Sf9 membranes containing
ET B R were incubated with each GST–Cav.-1 fusion protein (50 lg) overnight at 4 Cand then were subjected to immunoprecipitation with the 2A5 mAb after solubilization The eluates from the resin were analysed by immunoblotting with an anti-GST mAb GST–Cav.1-FL retained binding activity to ET B R While GST alone and GST– Cav.1(1–81) were not coimmunoprecipitated with ET B R, GST– Cav.1(1–101), GST–Cav.1(61–101) and GST–Cav.1(136–178) were coimmunoprecipitated (bands marked by asterisks) GST–Cav.1(101– 136) did not coimmunoprecipitate (data not shown) Among these constructs, the amount of coimmunoprecipitated GST–Cav.1(136– 178) appeared to be less than the others.
Trang 7and ETBR by immunoblotting (Fig 4A) The ETBR
expressed in HEK293 cells was also observed as two bands
in immunoblotting Fig 4B shows the extents of caveolin-1
bound to ETBR, as shown in Fig 4A, relative to the binding
to the ligand-free ETBR (lane 1) as 1.0 As observed with the
Sf9 membranes (Fig 1), the extent of caveolin-1 binding to
ETBR was reduced to 0.35 ± 0.03 by the addition of ET-1
(lane 2) However, the inverse-agonist, RES-701-1-bound
ETBR retained caveolin-1-binding activity (0.98 ± 0.13,
lane 3) similar to that of the ligand-free form, whereas the
BQ788-bound ETBR reduced the activity (0.42 ± 0.13,
lane 4) The results suggest that the ETBR, in the ligand-free
or ground-state structure, exhibits higher affinity to
cave-olin-1 than that in the BQ788-bound or an activated
structure, and that the recognition by caveolin-1 is
influ-enced highly by structure
Caveolin-1 targets ETBR to the caveolae membrane and ET-1 releases ETBR from caveolae
To examine the effects of caveolin-1 on the localization of
ETBR, a cell line stably expressing ETBR was isolated using HEK 293 cells, which do not express endogenous caveolin The expression level of ETBR in this cell line is approxi-mately 1–2 pmolÆmg)1 membrane protein The ETBR distribution in these cell membranes was analysed before
or after transfection with the caveolin-1 gene Fig 5A shows the results of sucrose-density gradient centrifugation of the Triton-insoluble fractions of the caveolin-1-transfected cells The low buoyant density and bottom fractions have been shown to contain the caveolae membranes and the Triton-soluble membrane proteins, respectively [31] Indeed, cave-olin-1 and another caveolae marker, flotillin, were present within the low-density fractions around fraction 3, whereas
a noncaveolae marker, the transferrin receptor, was fract-ionated to the bottom, high-density fractions When cave-olin-1 was transfected, the ETBR was fractionated to the low-density and the bottom fractions, suggesting that a fraction of ETBR was targeted to the caveolae membranes Since the treatment with Triton X-100 denatured the ETBR that was solubilized from the nonlipid raft membranes, the approximate distribution of ETBR, as assessed by ligand binding, was examined using fractions prepared by cell disruption with sodium carbonate and sucrose density-gradient centrifugation Approximately 7% of the cell surface ETBR was fractionated to the low-density fraction, based on the ligand binding activities of ETBR in the low-density fraction and in the plasma membrane fraction (data not shown)
The ETBR found in the detergent-resistant, low-density fraction (DRM, combined fractions 2 and 3 in Fig 5A) is shown in Fig 5B, while Fig 5Crepresents the averaged band intensities of ETBR observed in Fig 5B from five repeated experiments, relative to the band intensity of total ET R [band I (full-length isoform) plus band II
Fig 3 Single mutations of aromatic amino acids in or close to the cytoplasmic domain of ET B R did not affect the interaction with caveolin-1 (A) Secondary structure model of human ET B R, showing the 10 aromatic residues (marked with circles) that were each mutated
to Ala (Tyr127, Tyr200, Trp206, Trp217, Phe326, Tyr387, Phe393, Phe397, Trp404, and Phe408) The putative seven helices are boxed (B) COS cells transiently expressing the wild type or each mutant ET B R were subjected to immunoprecipitation with the 2A5 mAb The eluates from the resin were analysed by immunoblotting with the anti-caveolin polyclonal Ig (upper panel) or the 2A5 mAb (lower panel) The band intensities of caveolin-1 per ET B R were compared to that of the wild type ET B R as 1.0 The three independent experiments were averaged All of the mutant ET B Rs interacted with caveolin-1, in a similar manner to that of the wild type (C) COS cells transiently expressing the wild type or each mutant ET B R were lysed with a hypotonic buffer, incubated with 1 l M ET-1 for 1 h at room temperature, and then subjected to immunoprecipitation with the 2A5 mAb The eluates from the resin were analysed by immunoblotting with the anti-caveolin polyclonal Ig (upper panel) or the 2A5 mAb (lower panel) The band intensities of caveolin-1 were compared as in (B) The two independent experiments were averaged All ligand-bound mutant ET B Rs disso-ciated from caveolin-1 significantly, similar to the wild type.
Trang 8(N-terminally cleaved isoform)] without the expression of
caveolin-1 (lane 1) The intensities of the total and the
band II ETBR in each lane are shown separately Similar
amounts of proteins were recovered in the low-density
fractions in each experiment When the caveolin-1 gene was
not transfected, the ETBR was scarcely recovered in the
low-density fraction (lane 1) On the other hand, when
caveolin-1 was expressed, the total amount of ETBR found
in the low-density fraction (lane 2) increased about
three-fold Furthermore, when the caveolin-1-expressing cells
were treated with ET-1 for 30 min at 37C, the total amount of ETBR was slightly reduced In addition, the amount of N-terminally cleaved ETBR (band II) in the low-density fraction was reduced to about 64% (lane 3), as compared to that of the ET-1-untreated cells (lane 2) Two isoforms of ETBR corresponding to bands I and II, observed in mammalian tissue culture cells and in native tissues [35,36], have been shown to be caused by proteases activated or released from cells during membrane prepar-ation The increased band II ETBR intensities in Fig 5B compared to those in Fig 5A could be due to the proteolysis during further ultracentrifugation to concentrate the fraction 2 and 3 membranes It was also shown that the stably expressed ETBR in HEK293 cells is not cleaved at the cell surface without agonist stimulation, and that metallo-proteases cleave the N terminus of agonist-bound ETBR at the cell surface [37] We assume that the band II isoform in ET-1-untreated cells was derived from proteolysis during the membrane preparation, whereas the band II in ET-1-treated cells was derived from metalloprotease cleavage, in addition to proteolysis during the membrane preparation Furthermore, the band II might predominantly contain the ET-1-bound form as compared to the band I isoform, because ET-1 binding to full-length ETBR might not be quantitative at the cell surface in a 30-min assay at 37C Therefore, the decrease in the band II intensity of ET-1-treated cells suggests that a fraction of the ET-1-bound
ETBR is gradually exiting out from caveolae The reason why the band I isoform did not decrease is not clear at present Thus, some of the ETBR is targeted to the caveolae membranes by the expression of caveolin-1, and is gradually excluded from the caveolae by agonist stimulation in HEK293 cells
Disruption of caveolae impairs ET-1-induced ERK activation
Cholesterol binding agents such as filipin have been shown
to disrupt lipid rafts, probably by altering biophysical characteristics [40,41] The ETBR activates mitogen-activa-ted protein kinases, such as ERK, c-Jun N-terminal kinase and p38 kinase, to mediate mitogenic and cell-proliferation signals [42–44] To study the significance of the compart-mentalization to caveolae membranes, the ETBR was expressed transiently in CHO cells, which expressed cave-olin-1 abundantly, and the ET-1-induced phosphorylation
of ERK was examined Fig 6 shows that the addition of ET-1 greatly increased the amount of phosphorylated ERK1/2 in CHO cells, while the amounts of the recovered ERK1/2 remained unchanged, as shown by the immuno-blotting However, pretreatment with increasing amounts of filipin III before the addition of ET-1 significantly reduced the amount of accumulated phosphorylated ERK1/2 In untransfected CHO cells, no ET-1-induced phosphorylation
of ERK1/2 was observed (data not shown) These results suggest that the caveolae microdomain plays fundamental roles in efficient signal propagation in the ERK pathway by the ETBR, although the effects of filipin III on caveolae and the ETBR are not exactly clear In addition, these results are consistent with the report showing impaired ERK and focal adhesion kinase signal transduction via the ETBR in filipin III-treated primary astrocytes [27]
Fig 4 Effects of antagonists on the interaction of ET B R with
caveolin-1 The membranes prepared from HEK293 cells expressing
ET B R and caveolin-1 (1–2 mg of proteins per sample) were incubated
with either vehicle, 1 l M ET-1, 10 l M RES-701-1, or 10 l M BQ788 for
1 h at room temperature, and subsequently were subjected to
immu-noprecipitation with the 2A5 mAb (A) The eluates from the resin were
analysed by immunoblotting with the anti-caveolin-1 mAb (upper
panel) or the 2A5 mAb (lower panel) (B) The extents of caveolin-1
bound to ET B R observed in (A) are represented by normalizing
the binding to the ligand-free ET B R (lane 1) as 1 The data are
means ± SE of three independent experiments The extents of
caveolin binding was decreased significantly by the addition of either
ET-1 (lane 2) or BQ788 (lane 4), whereas the RES-701-1-bound ET B R
retained an activity similar to that of the ligand-free form.
Trang 9We studied the interaction of ETBR and caveolin-1 in vitro
and in vivo, using the expressed proteins in insect and
mammalian cells The ligand-free ETBR in the reconstituted
phospholipid vesicles formed a complex with caveolin-1,
which dissociated upon agonist binding The significance of
this interaction could be perceived in a model cultured cell
system The expression of caveolin-1 targeted some of the
ETBR to the membrane microdomain, caveolae, and ET-1
stimulation translocated the ETBR out of caveolae, which
when disrupted, diminished the ETBR-derived signal
pro-pagation This is the first report showing the caveolin-1- and
ET-1-regulated localization of the ETBR and the direct
interaction of a GPCR with caveolin
The heterologously expressed ETBR and caveolin-1 formed a complex after purification and reconstitution into vesicles, suggesting their direct interaction Interestingly, this interaction requires the existence of the ETBR within the lipid bilayer, and does not occur in the detergent micelle Considering the fact that ETBR binding by caveolin-1 involves the scaffolding domain, which is thought to be proximal to the membrane domain, a region close to or within the transmembrane domain of ETBR might be important for the interaction However, we could not specify the region of ETBR involved in the caveolin-1 binding At the very least, the conformational changes of
ETBR affect this interaction, and the ground-state structure
of ETBR is required for the interaction with caveolin-1 The discrimination by caveolin-1 of the RES-701-1-bound and BQ788-RES-701-1-bound forms of ETBR agrees well with the previous observation, in which a cyclic peptide antagonist, RES-701-1, could antagonize an ETBR artifi-cially activated by a chaotropic reagent, NaSCN, but another antagonist, BQ788, could not [28] We suggest that the inverse agonist activity of RES-701-1 led the
ETBR to an inactive (ground state) conformation On the other hand, the BQ788-bound conformation is somewhat different from both the ground state structure and the
G protein-coupling structure The interaction with caveo-lin may be a useful tool for molecular pharmacological studies of GPCR
The expression of caveolin-1 in HEK293 cells stably expressing ETBR targeted the ETBR to the Triton-insoluble, low buoyant density fraction, caveolae These results
Fig 5 Caveolin-1 targets ET B R to caveolae HEK293 cells stably expressing ET B R were transfected with either Cav.1-pcDNA3.1 or the empty vector The transfected cells were incubated with 100 n M ET-1
or vehicle and were lysed in 1% Triton X-100 at 4 C The lysates were subjected to subcellular fractionation using a 5/30% discontinuous sucrose gradient, as described in Experimental procedures (A) A 100-lL aliquot of each fraction prepared from the caveolin-1-trans-fected cells was precipitated with trichloroacetic acid and resuspended
in 50 lL of Laemmli sample buffer Aliquots of each fraction (20 lL for ET B R and 2 lL for caveolin-1, transferrin receptor and flotillin) were analysed by immunoblotting with the respective antibodies Fractions 2 and 3 correspond to the 5/30% sucrose interface The transfected caveolin-1 and endogenous flotillin, which are both caveolae proteins, were enriched in fractions 2 and 3, whereas the transferrin receptor, which is distributed in nonlipid raft membranes, was fractionated to the bottom fractions The ET B R partially cofractionated with caveolin-1 The two arrowheads represent the positions of the full-length and N-terminally cleaved ET B R (B) The
ET B R in fractions 2 and 3 (DRM) in (A) was concentrated by ultra-centrifugation following dilution and was compared by immunoblot-ting with the 2A5 mAb, with (lanes 2 and 3) or without (lane 1) caveolin-1 expression, and with (lane 3) or without (lanes 1 and 2) ET-1 treatment Bands I and II indicate the full-length and N-terminally cleaved ET B R, respectively (C) The band intensities of ET B R recov-ered in DRM in (B) are compared The total (bands I and II) and N-terminally cleaved ET B R (band II) are shown separately The data are means ± SE of five independent experiments The amounts of total proteins recovered in DRM are more or less constant, as shown under the columns The caveolin-1 expression drives the targeting of
ET B R to caveolae, and the ET-1 treatment releases a fraction of ET B R, particularly band II ET B R, from caveolae.
Trang 10suggest that a fraction of ETBR is localized in caveolae,
driven by the interaction with caveolin-1, although the
localization efficiency was only 7% of the cell surface ETBR
In primary astrocytes, substantial amounts of ETBR are
localized in the caveolae fraction [27] The localization of
ligand-free GPCR to caveolae has been reported for the
adenosine A1 and b2-adrenergic receptors, which interact
with caveolin-3 in cardiomyocytes, while the b2-adrenergic
receptor do not require caveolin-3 to target to caveolae,
when expressed in HEK cells containing a functional
homologue of caveolin, flotillin/ESA [20,23] The Flotillin/
ESA might be able to compensate in the case of b2
-adren-ergic receptor, but not in the case of ETBR Therefore, the
molecular mechanisms used in the targeting of b2-adrenergic
receptor and ETBR to caveolae might be different Further
studies on the targeting mechanisms to caveolae are
required
Upon agonist addition, the adenosine A1 receptors in
ventricular myocytes dissociate from caveolin-3 and
trans-locate out of the caveolae within 15 min at 37C[23] Most
of the b2-adrenergic receptors in cardiomyocytes are also
excluded from caveolae upon agonist stimulation by 30 min
at 37C[20] Although such a dramatic decrease in the
abundance of ETBR in the caveolae of HEK293 cells was
not observed, an 12% reduction of the total ETBR was
observed (Fig 5) This slow reduction could be because
signal transduction of the ETBR during the exit from
caveolae might be required, or it may be due to incomplete
ET-1 binding at the cell surface, or to overexpression of
ETBR in HEK293 cells In addition, because most of the
ETBR at the cell surface is localized in nonlipid raft
membranes, the constitutive trafficking of ETBR from
nonlipid raft membranes or the newly synthesized ETBR
moving from inside the cells to the caveolae might mask a
fraction of the ETBR exiting from the caveolae However,
the decrease in the N-terminally cleaved ETBR (band II)
was significant (Fig 5C) The agonist-dependent N-terminal
cleavage of ETBR by metalloproteases on the cell surface has been shown, which yields band II, whose functional significance is not known [37] While the full-length ETBR might be supplied from other domains in the cells, the N-terminally cleaved, agonist-bound ETBR may dissociate from caveolin-1 and be gradually excluded from the caveolae The agonist-regulated localization of ETBR on the cell surface should be studied further using native tissues
or primary cultures of astrocytes, and endothelial cells, among others Although a decrease of the ETBR in caveolae seemed to be slow in HEK293 cells, the transient vasodil-atation due to the ETBR-induced nitric oxide release from endothelial cells [1,2] might be regulated by interactions with caveolins, in addition to the rapid desensitization/internal-ization of ETBR [9–11]
The colocalization and coimmunoprecipitation of ETAR with caveolin-1 [21] and the internalization of ligand-bound
ETAR via caveolae have been shown [8] These properties are explained well by the fact that ETAR interacts with caveolin-1, regardless of agonist binding For ETBR, rapid internalization to a degradative pathway upon agonist binding in CHO cells [10,11] and constitutive internalization
to lysosomes in HeLa and Clone 9 cells [9] have been reported In contrast, the localization to caveolae micro-domains by interaction with caveolin-1 ensures that a subfraction of the ETBR is present on the cell surface to transmit ET-1 signals The reduced ERK signalling via the
ETBR in filipin-treated cells might be due to rapid ETBR internalization/degradation or simply due to the lack of caveolae, where signalling molecules are concentrated, as filipin reduces the cell-surface caveolin [27] Both extreme mechanisms of ETBR action may be well balanced, depending upon the cell and tissue types
ETBR couples to multiple G proteins, mainly Gqand Gi
in native tissues but also to Gs and Go in a few tissues, cultured cells, and in vitro [2,28,45,46], and this could be regulated by the intracellular conditions While the Gqa subunit has been shown to bind caveolin and to be concentrated in caveolae, the heterotrimeric Giand Gsare localized in lipid rafts [47] In the case of b2-adrenergic receptor in neonatal cardiomyocytes, the localization in caveolae seems to regulate signal transduction by the limiting access to Gs [48] Although caveolae appear to facilitate the ERK signalling activated by ETBR in C HO cells, other signalling pathways through ETBR could exist in nonlipid rafts, because substantial amounts of ETBR are localized here Therefore, localization to specific membrane microdomains, such as caveolae, lipid rafts and nonlipid rafts, may also contribute toward specifying the signal transduction by ETBR
Furthermore, the recent report that EGF receptors in noncaveolar lipid rafts show lower EGF binding (Bmax), than those in nonlipid rafts, suggests that the receptor properties are regulated by the lipid environment [49] Based
on the pharmacological heterogeneity of ETBR, the exist-ence of the ETB1R and ETB2R (tentatively termed) subtypes has been suggested; nevertheless, there is no molecular biological evidence supporting this The ETB1R located on the vascular endothelium mediates vasodilatation through the release of nitric oxide, which is sensitive to the mixed ETAR/ETBR antagonist, PD 142893, bosentan, RES-S701-1, BQ788, etc The other subtype (ET R),
Fig 6 Filipin treatment impairs ET B R-mediated ERK1/2 signalling.
CHO cells transiently expressing ET B R were cultured in FBS-free
medium for 24 h After a pretreatment with 0, 1 or 2 lgÆmL)1filipin
III for 10 min at 37 C, these cells were stimulated with 100 n M ET-1
for 5 min at 37 C The cells were lysed in RIPA buffer and were
analysed by immunoblotting with the anti-(phosphorylated ERK)
mAb (upper panel) or the anti-ERK mAb (lower panel) Pretreatment
with filipin attenuated the phosphorylation of ERK1/2 mediated by
the activation of ET B R The bands for ERK1 (44 kDa) and ERK2
(42 kDa) were not separated in these gels.