Regulated interaction of endothelin B receptor with caveolin-1

Regulated interaction of endothelin B receptor with caveolin-1

Tomohiro Yamaguchi1, Yasunobu Murata1, Yoshinori Fujiyoshi1,2 and Tomoko Doi1

1Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Japan; 2Japan Biological Information Research

Centre, Tokyo, Japan

The peptide hormone endothelin transmits various signalsthrough G protein-coupled receptors, the endothelin type A(ETAR) and B (ETBR) receptors. Caveolae are specializedlipid rafts containing polymerized caveolins. We examinedthe interaction of ETBR with caveolin-1, expressed in Sf9,COS-1, and HEK293 cells, and its effects on the subcellulardistribution and the signal transduction of ETBR. ETBRformed a complex with caveolin-1 in cells in which thesetwo proteins were coexpressed and in the mixture afterpurification and reconstitution (as examined by immuno-precipitation) suggesting the direct binding of ETBR withcaveolin-1. The complex formed efficiently only when theETBR 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, theETAR 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 toETBR, as for other signalling molecules. Furthermore, theamount ofETBR localized in caveolae increased significantlywith the expression of caveolin-1 and decreased with theaddition of ET-1. The disruption of caveolae by filipinreduced the ET-1-derived phosphorylation of ERK1/2.These results suggest the possibility that the binding tocaveolin-1 retains the ligand-free ETBR in caveolae andregulates the ET signal.

Keywords: caveolae; caveolin; endothelin; endothelin type Breceptor; lipid raft.

Endothelins (ETs) are 21-amino acid peptides that mediatediverse physiological effects on vasoconstriction, cellulardevelopment, 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 ETBRmediates the release of nitric oxide to stimulate vasodilata-tion in vascular endothelial cells. The ET ligand–receptorcomplex exhibits a slow dissociation rate and almostirreversible binding, which could explain the prolongedvasoconstriction by ET [3]. This property highlights theimportance of understanding ET signal regulation. BothETAR and ETBR undergo rapid desensitization [4–6] andinternalize differently in transfected cells, as shown formanyGPCRs [7]. Concerning the intracellular trafficking path-

ways, ETAR is internalized rapidly, either via caveolae orclathrin-coated pits, upon ligand binding [3,8,9] and followsa recycling pathway, whereas ETBR follows a degradativepathway after internalization via coated-pits, implicated forclearance of plasma ET-1 [10]. Moreover, the ETBR in theplasma membrane is constitutively transported to thispathway independently of ligand stimulation, indicatingthat ETBR is hardly retained in the plasma membrane atsteady state [9]. Differences in the final fates and subcellularlocalization of the ETRs are determined by the C-terminalsequences of the two receptors [9,11]. However, the effects ofthe cell surface localization of the ETRs on the ET signalshave not been studied carefully.Lipid rafts are microdomains of cell membranes that are

rich in cholesterol and sphingolipid and serve as sites forgathering signalling molecules [12–14]. Caveolae are vesi-cular invaginations of the plasma membrane, formed fromlipid rafts with polymerized caveolins [15]. The threecaveolin genes have been cloned (caveolin-1, caveolin-2,and caveolin-3). The caveolin-1 and caveolin-2 show thesame distribution pattern, whereas caveolin-3 is specific tomuscle cells. Caveolins act as scaffolding proteins to clusterand regulate signalling molecules targeted to the caveolae,such as Src-family tyrosine kinases, Ha-Ras, G proteina subunits, endothelial nitric oxide synthase, protein kinaseC, and epidermal growth factor (EGF) receptor, amongothers [16]. Caveolins are cholesterol-binding integralmembrane proteins that are thought to form an unusualhairpin-like structure, with the central hydrophobic domain(residues 102–134 of caveolin-1) in the membrane and theN- (residues 1–101) and C-terminal (residues 135–178)domains inside the cell. In this structure, the scaffoldingdomain 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: [email protected]

Abbreviations: ET, endothelin; ETR, endothelin receptor; ETAR and

ETBR, 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, His6-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)

Eur. J. Biochem. 270, 1816–1827 (2003) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03544.x

mediate the interactions with the signalling moleculesdescribed above [17]. On the other hand, the caveolin-binding sequencemotif (FXFXXXXF orFXXXXFXXF,where F is an aromatic amino acid, Trp, Phe, or Tyr) hasbeen suggested to exist in the signalling molecules, by whichthey bind to the caveolin scaffolding domain [18].Several GPCRs, including the B2 bradykinin, b-adrener-

gic, cholecystokinin, ETA, angiotensin II type 1, andadenosine A1 receptors, are localized within caveolae[19–25]. Particularly, the B2 bradykinin, angiotensin II type1, and m2 muscarinic acetylcholine receptors are translo-cated to caveolae upon agonist binding, whereas adenosineA1 and b2-adrenergic receptors are translocated out ofcaveolae upon activation, and the binding of ETAR withcaveolins is not affected by agonist binding. Furthermore,them2muscarinic acetylcholine andETA receptors could beinternalized via caveolae [8,26]. The mitogenic signalthrough ETBR in primary astrocytes, where the ETBR isrelatively well expressed, originates from caveolae micro-domains [27]. In addition, a number of proteins mediatingintracellular Ca2+ signalling are concentrated in caveolae[15,16]. Thus, the localization to caveolae and the inter-action with caveolins appear to play fundamental roles inthe 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, westudied the interactions of ETBR with caveolins in vivo andin vitro, in terms of agonist-regulated localization on the cellsurface and signal transduction downstream. The resultsrevealed that only the ETBR in the ground-state structurebound to caveolin-1, through the caveolin scaffolding andC-terminal domains, and that a fraction of ETBR wastargeted to caveolae by the expression of caveolin-1 and wasgradually released from caveolae by ET-1 stimulation. Inaddition, the disruption of caveolae impaired the down-stream signals from ETBR. These results suggest feasibleregulations for ETBR signal transduction by the interactionwith caveolin-1. The residence of ETBR in caveolae mightbe a way of ensuring the cell surface localization of ETBRagainst rapid internalization.

Experimental procedures

Materials

The cyclic-peptide antagonist for ETBR, RES701-1, wasgenerously provided byM.Yoshida (KyowaHakkoKogyoCo., Ltd, Tokyo, Japan). The cDNAs encoding humanETAR 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 byR. Molday (University of British Columbia, Vancouver,Canada). The antagonist for ETBR, BQ788, was fromPhoenix Pharmaceuticals, Inc. The anti-ETBR mAb, 2A5,was generated against Sf9-expressed human ETBR (T.Yamaguchi, I.Arimoto,Y. Fujiyoshi&T.Doi, unpublisheddata). Goat anti-mouse and anti-(rabbit IgG)–alkalinephosphatase and –horseradish peroxidase conjugates werefrom Promega Biotech.

DNA construction

The caveolin-1 cDNA was produced from human lungmRNA by RT-PCR using appropriate oligonucleotideprimers, and was subsequently subcloned into the BamHI–NotI sites of the mammalian expression vector pcDNA3.1(Invitrogen). The ETBR cDNA was subcloned into theBamHI–NotI sites of pcDNA3.1. Mutagenesis of the ETBRcDNA was carried out with appropriate oligonucleo-tide primers by a PCR-based site-directed mutagenesisapproach. In each ETBR mutant, the aromatic amino acidresidue (Tyr127, Tyr200, Trp206, Trp217, Phe326, Tyr387,Phe393, Phe397, Trp404, or Phe408) was replaced withalanine. These cDNAs were subcloned into the BamHI–NotI sites of pcDNA3.1.In the Cav.1-H6 cDNA, a His6-tag was attached to the

C terminus of caveolin-1 for affinity purification usingNi2+–NTA agarose (Qiagen). The 1D4 epitope sequence(KTETSQVAPA, an epitope for the anti-rhodopsin mAB)was fused to the C terminus of ETAR. For expression ininsect cells, the cDNAs encoding caveolin-1, Cav.1-H6,ETBR, and ETAR-1D4 were subcloned into a transfervector, pVL1393 (BD Biosciences), and the isolation ofrecombinant 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 [aminoacids 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), andGST-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 weresubcloned into the BamHI–NotI sites. After expressionin 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 inDulbecco’s modified Eagle’s medium (Sigma) containing10% foetal bovine serum (Invitrogen) and penicillin/strep-tomycin at 37 �C in a 5% CO2 atmosphere. For transientexpression, LipofectAmine Plus reagent (Invitrogen) wasused to transfect COS, HEK293, and CHO cells in a100-mm diameter plate or in a 6-well plate with 1–8 lgpcDNA3.1 encoding the wild type or mutant ETBRs,caveolin-1, or vector only. These cells were subjected to eachstudy 36 h after transfection.For the isolation of stably transfected HEK293 cell lines

expressing ETBR, the cells were transfectedwith the plasmidpcDNA3.1 containing the ETBR cDNA, using the calciumphosphate precipitation technique (Invitrogen). Two daysafter transfection, the cells were plated in selective mediumcontaining 500 lgÆmL)1 G418 (Invitrogen). The G418-resistant colonies were selected, and the single colonies werepurified further. The ETBR expression level by the HEK293cell lines isolated for further studies was 1–2 pmol per mgmembrane protein.

� FEBS 2003 Endothelin-1 dissociates the ETBR–caveolin-1 complex (Eur. J. Biochem. 270) 1817

The culture of Sf9 insect cells and the expression of ETBRin Sf9 cells were performed as described previously [28]. Theexpression of caveolin-1 and His6-tagged caveolin-1 (Cav.1-H6) was also performed similarly. Co-expression of ETBRand caveolin-1 in Sf9 cells was carried out by a doubleinfection with ETBR and caveolin-1 recombinant viruses.

Purification of ETBR and caveolin-1 from Sf9 cells

All operations were carried out at 4 �C. Purification ofETBR by ligand-affinity chromatography using biotinylatedET-1, and reconstitution of the purified ligand-free ETBRinto phospholipid vesicles were performed as describedpreviously [28,29].Cav.1-H6 was purified from infected Sf9 cells using

Ni2+–NTA-agarose beads. The membranes were preparedas described (� 100 mg protein from a 500-mL suspensionculture) and were solubilized in 20 mL NaCl/Tris (20 mMTris/HCl pH 7.5, 150 mM NaCl) with protease inhibitors(1 mM phenylmethylsulfonyl fluoride, 10 lgÆmL)1 aproti-nin, 10 lgÆmL)1 leupeptin, 10 lgÆmL)1 pepstatin), 60 mMn-octyl-b-D-glucopyranoside (OG; Dojindo), and 1% Tri-ton X-100 (Wako). After 30 min solubilization, the lysateswere ultracentrifuged for 1 h at 100 000 g. The super-natants were adjusted to 10 mM imidazole and wereincubated with 200 lL Ni2+-nitrilotriacetic acid-agarosebeads overnight. After the incubation, the beads werewashed extensively with washing buffer (NaCl/Tris con-taining 20 mM imidazole, 30 mMOG, and 0.03%Triton X-100) four times by rotating for 5 min. After washing, thebound proteins were eluted with 400 lL elution buffer(NaCl/Tris containing 100 mM imidazole, 30 mM OG, and0.03% Triton X-100) by rotating for 1 h.

Immunoprecipitation

All preparations were carried out at 4 �C unless statedotherwise.

ETRs expressed with caveolin-1 in Sf9 membranes. Sf9membranes (10 mg membrane proteins) were prepared asdescribed 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)1 aprotinin, 10 lgÆmL)1 leupeptin,10 lgÆmL)1 pepstatin), and incubated with or without 2 lMET-1 for 1 h at room temperature. After the incubation, themembranes were solubilized with 60 mM OG and 1%Triton X-100 for 30 min, and were centrifuged at 100 000 gfor 30 min. The supernatants were incubated with 30 lL ofeither 1D4 or 2A5 monoclonal antibody-immobilized resin(1 mg antibody per mL resin) overnight. After thisincubation, the resins were washed extensively at least fourtimes with NaCl/Tris/EDTA containing the aforemen-tioned detergents, and then the bound proteins were elutedwith 100 lM of either the 1D4 or 2A5 epitope peptides for1 h at room temperature.

Purified ETBR and Cav.1-H6. Reconstituted phospho-lipid vesicles with or without ETBR (1–2 pmol) wereincubated 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 andTriton X-100 derived from the Cav.1-H6 solution were farbelow the critical micelle concentrations (OG, 2–3 mM;Triton X-100, 0.002–0.003%) so that the membranes werenot solubilized. After incubation, the membranes weresolubilized with 60 mM OG and 1% Triton X-100,incubated with 2A5-immobilized resin, washed, and elutedwith 2A5-epitope peptides.

GST–Cav.1 fusion proteins. Sf9 membranes containingETBR (2.5 mg protein per sample) were incubated with therespective GST–Cav.1 fusion proteins (50 lg) overnightand then centrifuged. The concentrations of detergents inthese mixtures derived from the GST–Cav.1 fusion proteinswere far below the critical micelle concentration. Themembranes were solubilized in 1 mL NaCl/Tris containing1% n-decyl-b-D-maltopyranoside (Dojindo), and werecentrifuged 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 hoursafter transfection with the wild type ETBR cDNA, eachmutant ETBR cDNA or the vector only, COS cells (one100-mm diameter plate per sample) were washed twice withNaCl/Pi, lysed with 1 mL NaCl/Tris/EDTA containing60 mM OG and 1% Triton X-100, and centrifuged at100 000 g (Fig. 3B). In addition, the washed cells werecollected in1 mLhypotonicbuffer (20 mMTris/HClpH 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, andcentrifuged at 100 000 g (Fig. 3C). The supernatants wereincubated with 2A5-immobilized resin, washed, and elutedwith 2A5-epitope peptides.

Effects of antagonists on the interaction. The membranesprepared fromHEK293 cells, which expressed ETBR stablyand caveolin-1 transiently (1–2 mgÆprotein per sample),were resuspended in NaCl/Tris/EDTA containing proteaseinhibitors, and incubated with 1 lM ET-1, 10 lM RES-701-1, or 10 lMBQ788 for 1 h at room temperature. Afterthis incubation, the samples were solubilized with 60 mMOGand 1%TritonX-100, incubatedwith 2A5-immobilizedresin, washed, and eluted with 2A5-epitope peptides.

Electrophoresis and immunoblotting

Samples prepared in Laemmli sample buffer were subjectedto SDS/PAGE and transferred to nitrocellulose membranes(Schleicher and Schuell). The nitrocellulose membraneswere 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, TransductionLaboratories), 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-phosphoERK1/2mAb (E10) (1 : 2000; CellSignaling Technology) overnight at 4 �C. The membranes

1818 T. Yamaguchi et al. (Eur. J. Biochem. 270) � FEBS 2003

were washed with buffer (5 mM Tris/HCl pH 7.5, 150 mMNaCl, 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+ plusWestern blotting detection system (Promega Biotech andAmersham Biosciences Inc., respectively).

Preparation of caveolin-enriched, low buoyantmembrane fractions

Thirty-six hours after the transfection with the Cav.1 gene,the HEK293 cells stably expressing ETBR were cultured inserum-free medium for 6 h, and were then incubated withor without 100 nM ET-1 for 30 min at 37 �C. Thefollowing operations, except for the elution, were carriedout at 4 �C. After the incubation, the confluent HEK293cells of two 100-mm diameter plates (� 8 mg total protein)per sample were washed twice with NaCl/Pi, scraped into2 mL buffer (25 mM Mes pH 6.5, 150 mM NaCl andprotease inhibitors) containing 1% Triton X-100, andhomogenized 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 lackingTriton X-100, placed in the bottom of ultracentrifugetubes, and then overlaid with a discontinuous sucrosegradient of 5 mL 30% (w/v) sucrose and 2 mL 5% (w/v)sucrose, both prepared in buffer lacking Triton X-100. Thesamples were centrifuged at 39 000 r.p.m. (200 000 g) inan SW41 rotor (Beckman Instruments) for 16–20 h,fractionated into 11 1 mL fractions sequentially from thetop of the gradient, and concentrated by precipitation withtrichloroacetic acid or by ultracentrifugation followingdilution.

Assay for phosphorylation of ERK1/2

Forty-eight hours after transfection in a 6-well plate, theCHO cells were cultured in serum-free medium for 24 h,preincubated with 1 or 2 lgÆmL)1 filipin III, or vehicle for10 min at 37 �C, and then incubated with 100 nM ET-1 orvehicle for 5 min at 37 �C. After stimulation, the cells werewashed twice with ice-cold NaCl/Pi, and were lysed with100 lL RIPA buffer (50 mM Tris/HCl pH 7.5, 150 mMNaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodiumdeoxycholate, 0.5 mM Na3VO4, 50 mM NaF, 5 mM EDTAand protease inhibitors) at 4 �C. The lysates were trans-ferred into centrifuge tubes, sonicated for 15 s to shear theDNA and reduce the sample viscosity, and then boiled for5 min at 95 �C. The cooled samples were subjected to SDS/PAGE and immunoblotted with anti-phosphoERK1/2 oranti-ERK1 Igs.

Other methods

Protein concentrations were measured by the modifiedLowry method (DC-protein assay, Bio-Rad) with BSA asstandard. The amounts of ETBR contained in the sampleswere measured by the 125I-labelled ET-1 (NEN Life ScienceProducts) binding assay as described previously [29]. Therelative intensities of indicated proteins were measured bycomparative densities of reactive bands on immunoblots

with IMAGEMASTER VDS-CL and TOTALLAB version 1.10software (Amersham Biosciences Inc.).

Results

ETBR directly binds to caveolin-1 in an ET-1-dependentmanner

To examine the interaction of ETBR with caveolin-1, wetook advantage of an insect cell expression system, in whichETAR and ETBR are expressed well, with KD values forET-1 of 55 ± 8 and 83 ± 11 pM, the Bmax values of21 ± 1.7 and 60 ± 7.8 pmol per mg membrane protein,respectively [28]. In addition, it has also been shown that thecaveolin-1 expressed in insect cells forms oligomers, and isincorporated into caveolae-sized vesicles [30]. The humanETAR-1D4 (ETAR fused with the 1D4 epitope at theC terminus) and ETBR were each coexpressed withcaveolin-1 in insect Sf9 cells, incubated with or withoutET-1, and immunoprecipitated with the 1D4 and 2A5 anti-ETBR mAbs, respectively, as shown in Fig. 1A. Weextensively used 60 mM OG/1% Triton X-100 mixed-micelle conditions for the membrane solubilization beforeimmunoprecipitation, which allowed the recovery of ligand-free ETBR, as described later, and the solubilization ofmany Triton-insoluble proteins [31]. The eluates from theantibody-immobilized resin were analysed for caveolin-1,ETAR-1D4, and ETBR by immunoblotting. The caveolin-1coimmunoprecipitated with the ETAR-1D4, regardless ofthe absence or presence of ET-1 (lanes 2 and 3), but notwithout the expression of the receptor (lane 1). Thisobservation is consistent with those of the previous report[21]. In contrast, ETBR coimmunoprecipitated caveolin-1only when the receptor was ligand-free (lane 5). Theaddition of ET-1 significantly reduced the amount ofcoimmunoprecipitated caveolin-1 (lane 6). This coprecipi-tation of caveolin-1 was not detected in the absence ofETBR expression (lane 4). These results suggest that ETARbinds to caveolin-1 regardless of ET-1 binding, whereasETBR 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 directlyor indirectly. To clarify the interaction of ETBR withcaveolin-1, we purified ETBR and caveolin-1 in detergent-micelles. ETBR, expressed in Sf9 cells, was purified on aligand-affinity column eluted with 2 M NaSCN to yieldligand-free ETBR [29] (Fig. 1B, lane 1). Caveolin-1, with aHis6-tag fusion at the C terminus (Cav.1-H6), was alsoexpressed in Sf9 cells and purified by nickel-affinitychromatography, 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 witha purified G protein ai subunit in detergent micelles, asdetected by immunoprecipitation, and as described previ-ously (data not shown) [32]. The purified ETBR wasreconstituted into phospholipid vesicles, into which thepurified Cav.1-H6 was added, and the interaction betweenthemwas assessed by immunoprecipitation (Fig. 1C).Whileno 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


precipitated Cav.1-H6 (lane 3). These results using theheterologously expressed and purified proteins completelyreproduced the observations in the Sf9 membranes(Fig. 1A), suggesting the direct interaction of ETBR withcaveolin-1. Interestingly, ETBR and caveolin-1 did not formthe complex in a detergent/micelle solution, but they boundto each other only when the receptor was reconstituted into

vesicles. This could be due to that either the interaction ofETBR and caveolin-1 occurs by two-dimensional molecularmovements along the membranes, or that the integration ofETBR and caveolin-1 into the lipid bilayer stabilizesthe structures required for the interaction, or that thedetergent molecules binding to the ETBR interfere withcaveolin-1 binding.

Fig. 1. ETBR directly interacts with caveolin-1 and dissociates from it upon ET-1 binding. (A) Sf9 membranes containing ETAR-1D4 (lanes 1, 2 and

3) or ETBR (lanes 4, 5 and 6) expressed with caveolin-1 were incubated with (lanes 3 and 6) or without 2 lM ET-1 (lanes 1, 2, 4 and 5) for 1 h atroom 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). ETAR-1D4 coimmunoprecipitated with caveolin-1, regardless of the absence or presence of ET-1 (lanes 2 and 3), whereas ETBR

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) ETBR andCav.-1-H6were individually expressed and purified from infected Sf9 cells, as described

in the Experimental procedures. The purified ETBR (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 ETBR (1–2 pmol) were incubated with vehicle (lanes 1 and 2)

or 1 lM 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 byimmunoprecipitation with the 2A5mAb after solubilization. The eluates from the resin were analysed by immunoblotting with the anti-(caveolin-1)

mAb (upper panel) or the 2A5mAb (lower panel).While the phospholipid vesicleswithoutETBRdidnot showany coimmunoprecipitatedCav.1-H6

(lane 1), the Cav.1-H6 coimmunoprecipitated from the ETBR-containing vesicles (lane 2). The addition of ET-1 significantly reduced the amount of

coprecipitated Cav.1-H6 (lane 3).


The scaffolding domain and the C-terminal domainof caveolin-1 both interact with ETBR

The caveolin scaffolding domain (residues 82–101 ofcaveolin-1) is responsible for the binding with theaforementioned signalling molecules, protein kinase Acatalytic subunit, connexin 43, and others [16,33,34].Conversely, these proteins contain the caveolin-bindingmotifs (UXUXXXXU and UXXXXUXXU, where U is anaromatic amino acid residue), which have been suggestedto be responsible for the binding to caveolin [18]. Toinvestigate the interaction between ETBR and caveolin-1,we constructed a set of GST fusion proteins with variouscaveolin-1 domains, as shown in Fig. 2A. These fusionproteins were expressed in E. coli and purified by GST-affinity chromatography, as shown in Fig. 2B. Sf9 cellmembranes containing ETBR were incubated with thesepurified fusion proteins, and immunoprecipitataed withthe 2A5 mAb. While the ETBRs in each eluate wererecovered to similar extents (data not shown), certainGST-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) andGST itself did not (Fig. 2C). GST–Cav.1(101–136) alsodid not coimmunoprecipitate (data not shown). Theseresults suggest that caveolin-1 also utilizes the scaffoldingand C-terminal domains to interact with ETBR, as withother signalling molecules. The binding of the C-terminaldomain fusion, GST–Cav.1(136–178), appeared to beweaker, 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 andC-terminal domains, the caveolin-binding motifs could bethe sites of these interactions in ETBR. However, thesemotifs 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, becausecaveolin resides inside the cell. In fact, we mutated thearomatic residues in the cytoplasmic and transmembraneregions close to the cytoplasmic side, one by one(Fig. 3A). The ETBRs expressed in COS cells, shown inFig. 3B, were observed as two bands in the immunoblot-ting because of N-terminal proteolysis, which was alsofound with HEK293 and CHO cells, as described later[35–37]. These mutated ETBRs expressed in COS cellsbound caveolin-1 in the ligand-free form (Fig. 3B), anddissociated from caveolin-1 following ET-1 binding,similar to the wild type (Fig. 3C). The measurement ofband intensities showed no obvious differences betweenthe wild type and mutant ETBRs in the ligand-sensitivecaveolin-1 binding. The C-terminal truncated ETBR(residues 408–442 deleted) also showed these propertiesin the COS cell system (data not shown). Therefore, singlemutations 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-1could distinguish the structure of the ligand-free ETBR fromthat of the ligand-bound form. To examine the contribu-

tions of the ETBR tertiary structure to the recognition bycaveolin-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, weshowed that RES-701-1 displayed an inverse-agonist acti-vity that stabilizes ETBR structure in the ground-state, butBQ788 did not [28]. The HEK293 cells stably expressingETBR were transfected with the caveolin-1 gene. Themembranes prepared from these cells were incubated withET-1, RES-701-1 or BQ788 and were immunoprecipitatedwith the 2A5mAb. The eluates were analysed for caveolin-1

Fig. 2. The scaffold and C-terminal domains of caveolin-1 recognize

ETBR. (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

ETBR were incubated with each GST–Cav.-1 fusion protein (50 lg)overnight at 4 �C and 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 ETBR. While GST alone and GST–

Cav.1(1–81) were not coimmunoprecipitated with ETBR, 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.


and ETBR by immunoblotting (Fig. 4A). The ETBRexpressed in HEK293 cells was also observed as two bandsin immunoblotting. Fig. 4B shows the extents of caveolin-1bound to ETBR, as shown in Fig. 4A, relative to the bindingto the ligand-free ETBR (lane 1) as 1.0. As observed with theSf9 membranes (Fig. 1), the extent of caveolin-1 binding toETBR was reduced to 0.35 ± 0.03 by the addition of ET-1(lane 2). However, the inverse-agonist, RES-701-1-boundETBR retained caveolin-1-binding activity (0.98 ± 0.13,lane 3) similar to that of the ligand-free form, whereas theBQ788-bound ETBR reduced the activity (0.42 ± 0.13,lane 4). The results suggest that the ETBR, in the ligand-freeor ground-state structure, exhibits higher affinity to cave-olin-1 than that in the BQ788-bound or an activatedstructure, and that the recognition by caveolin-1 is influ-enced highly by structure.

Caveolin-1 targets ETBR to the caveolae membraneand ET-1 releases ETBR from caveolae

To examine the effects of caveolin-1 on the localization ofETBR, a cell line stably expressing ETBR was isolated usingHEK 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 ETBRdistribution in these cell membranes was analysed beforeor after transfectionwith the caveolin-1 gene. Fig. 5A showsthe results of sucrose-density gradient centrifugation of theTriton-insoluble fractions of the caveolin-1-transfected cells.The low buoyant density and bottom fractions have beenshown to contain the caveolae membranes and the Triton-soluble membrane proteins, respectively [31]. Indeed, cave-olin-1 and another caveolae marker, flotillin, were presentwithin the low-density fractions around fraction 3, whereasa 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 thelow-density and the bottom fractions, suggesting that afraction of ETBR was targeted to the caveolae membranes.Since the treatment with Triton X-100 denatured the ETBRthat was solubilized from the nonlipid raft membranes, theapproximate distribution of ETBR, as assessed by ligandbinding, was examined using fractions prepared by celldisruption with sodium carbonate and sucrose density-gradient centrifugation. Approximately 7% of the cellsurface 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 (datanot shown).The ETBR found in the detergent-resistant, low-density

fraction (DRM, combined fractions 2 and 3 in Fig. 5A) isshown in Fig. 5B, while Fig. 5C represents the averagedband intensities of ETBR observed in Fig. 5B from fiverepeated experiments, relative to the band intensity oftotal ETBR [band I (full-length isoform) plus band II

Fig. 3. Single mutations of aromatic amino acids in or close to the

cytoplasmic domain of ETBR did not affect the interaction with

caveolin-1. (A) Secondary structure model of human ETBR, 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 thewild type or eachmutant ETBR

were subjected to immunoprecipitation with the 2A5mAb. 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 ETBR were compared to that of the wild

type ETBR as 1.0. The three independent experiments were averaged.

All of the mutant ETBRs interacted with caveolin-1, in a similar

manner to that of thewild type. (C)COS cells transiently expressing the

wild type or each mutant ETBR were lysed with a hypotonic buffer,

incubated with 1 lM ET-1 for 1 h at room temperature, and then

subjected to immunoprecipitationwith the 2A5mAb. 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 ETBRs disso-

ciated from caveolin-1 significantly, similar to the wild type.


(N-terminally cleaved isoform)] without the expression ofcaveolin-1 (lane 1). The intensities of the total and theband II ETBR in each lane are shown separately. Similaramounts of proteins were recovered in the low-densityfractions in each experiment. When the caveolin-1 gene wasnot transfected, the ETBR was scarcely recovered in thelow-density fraction (lane 1). On the other hand, whencaveolin-1 was expressed, the total amount of ETBR foundin 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 37 �C, the totalamount of ETBR was slightly reduced. In addition, theamount of N-terminally cleaved ETBR (band II) in thelow-density fraction was reduced to about 64% (lane 3), ascompared to that of the ET-1-untreated cells (lane 2). Twoisoforms of ETBR corresponding to bands I and II,observed in mammalian tissue culture cells and in nativetissues [35,36], have been shown to be caused by proteasesactivated or released from cells during membrane prepar-ation. The increased band II ETBR intensities in Fig. 5Bcompared to those in Fig. 5A could be due to theproteolysis during further ultracentrifugation to concentratethe fraction 2 and 3 membranes. It was also shown that thestably expressed ETBR inHEK293 cells is not cleaved at thecell surface without agonist stimulation, and that metallo-proteases cleave the N terminus of agonist-bound ETBR atthe cell surface [37]. We assume that the band II isoform inET-1-untreated cells was derived from proteolysis duringthe membrane preparation, whereas the band II in ET-1-treated cells was derived from metalloprotease cleavage, inaddition to proteolysis during the membrane preparation.Furthermore, the band II might predominantly contain theET-1-bound form as compared to the band I isoform,because ET-1 binding to full-length ETBR might not bequantitative at the cell surface in a 30-min assay at 37 �C.Therefore, the decrease in the band II intensity of ET-1-treated cells suggests that a fraction of the ET-1-boundETBR is gradually exiting out from caveolae. The reasonwhy the band I isoform did not decrease is not clear atpresent. Thus, some of the ETBR is targeted to the caveolaemembranes by the expression of caveolin-1, and is graduallyexcluded from the caveolae by agonist stimulation inHEK293 cells.

Disruption of caveolae impairs ET-1-induced ERKactivation

Cholesterol binding agents such as filipin have been shownto disrupt lipid rafts, probably by altering biophysicalcharacteristics [40,41]. The ETBR activates mitogen-activa-ted protein kinases, such as ERK, c-Jun N-terminal kinaseand p38 kinase, to mediate mitogenic and cell-proliferationsignals [42–44]. To study the significance of the compart-mentalization to caveolae membranes, the ETBR wasexpressed transiently in CHO cells, which expressed cave-olin-1 abundantly, and the ET-1-induced phosphorylationof ERK was examined. Fig. 6 shows that the addition ofET-1 greatly increased the amount of phosphorylatedERK1/2 in CHO cells, while the amounts of the recoveredERK1/2 remained unchanged, as shown by the immuno-blotting. However, pretreatment with increasing amounts offilipin III before the addition of ET-1 significantly reducedthe amount of accumulated phosphorylated ERK1/2. Inuntransfected CHO cells, no ET-1-induced phosphorylationof ERK1/2 was observed (data not shown). These resultssuggest that the caveolae microdomain plays fundamentalroles in efficient signal propagation in the ERK pathway bythe ETBR, although the effects of filipin III on caveolae andthe ETBR are not exactly clear. In addition, these results areconsistent with the report showing impaired ERK and focaladhesion kinase signal transduction via the ETBR in filipinIII-treated primary astrocytes [27].

Fig. 4. Effects of antagonists on the interaction of ETBR with

caveolin-1. The membranes prepared from HEK293 cells expressing

ETBR and caveolin-1 (1–2 mg of proteins per sample) were incubated

with either vehicle, 1 lM ET-1, 10 lMRES-701-1, or 10 lM BQ788 for1 h at room temperature, and subsequently were subjected to immu-

noprecipitation with the 2A5mAb. (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 ETBR observed in (A) are represented by normalizing

the binding to the ligand-free ETBR (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 ETBR

retained an activity similar to that of the ligand-free form.


Discussion

We studied the interaction of ETBR and caveolin-1 in vitroand in vivo, using the expressed proteins in insect andmammalian cells. The ligand-free ETBR in the reconstitutedphospholipid vesicles formed a complex with caveolin-1,which dissociated upon agonist binding. The significance ofthis interaction could be perceived in a model cultured cellsystem. The expression of caveolin-1 targeted some of theETBR to the membrane microdomain, caveolae, and ET-1stimulation translocated the ETBR out of caveolae, whichwhen disrupted, diminished the ETBR-derived signal pro-pagation. This is the first report showing the caveolin-1- andET-1-regulated localization of the ETBR and the directinteraction of a GPCR with caveolin.

The heterologously expressed ETBR and caveolin-1formed a complex after purification and reconstitution intovesicles, suggesting their direct interaction. Interestingly, thisinteraction requires the existence of the ETBR within thelipid bilayer, and does not occur in the detergent micelle.Considering the fact that ETBR binding by caveolin-1involves the scaffolding domain, which is thought to beproximal to the membrane domain, a region close to orwithin the transmembrane domain of ETBR might beimportant for the interaction. However, we could notspecify the region of ETBR involved in the caveolin-1binding. At the very least, the conformational changes ofETBR affect this interaction, and the ground-state structureof ETBR is required for the interaction with caveolin-1.The discrimination by caveolin-1 of the RES-701-1-

bound and BQ788-bound forms of ETBR agrees well withthe previous observation, in which a cyclic peptideantagonist, RES-701-1, could antagonize an ETBR artifi-cially activated by a chaotropic reagent, NaSCN, butanother antagonist, BQ788, could not [28]. We suggestthat the inverse agonist activity of RES-701-1 led theETBR to an inactive (ground state) conformation. On theother hand, the BQ788-bound conformation is somewhatdifferent from both the ground state structure and theG protein-coupling structure. The interaction with caveo-lin may be a useful tool for molecular pharmacologicalstudies 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 ETBR to caveolae. HEK293 cells stably

expressing ETBR were transfected with either Cav.1-pcDNA3.1 or the

empty vector. The transfected cells were incubated with 100 nM ET-1

or vehicle and were lysed in 1%Triton X-100 at 4 �C. The lysates weresubjected 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 lLfor ETBR 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 ETBR partially

cofractionated with caveolin-1. The two arrowheads represent the

positions of the full-length and N-terminally cleaved ETBR. (B) The

ETBR 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, andwith (lane 3) or without (lanes 1 and 2) ET-1

treatment. Bands I and II indicate the full-length and N-terminally

cleaved ETBR, respectively. (C) The band intensities of ETBR recov-

ered in DRM in (B) are compared. The total (bands I and II) and

N-terminally cleaved ETBR (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

ETBR to caveolae, and the ET-1 treatment releases a fraction of ETBR,

particularly band II ETBR, from caveolae.


suggest that a fraction of ETBR is localized in caveolae,driven by the interaction with caveolin-1, although thelocalization efficiencywas only 7%of the cell surface ETBR.In primary astrocytes, substantial amounts of ETBR arelocalized in the caveolae fraction [27]. The localization ofligand-free GPCR to caveolae has been reported for theadenosine A1 and b2-adrenergic receptors, which interactwith caveolin-3 in cardiomyocytes, while the b2-adrenergicreceptor do not require caveolin-3 to target to caveolae,when expressed in HEK cells containing a functionalhomologue 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, themolecularmechanisms used in the targeting of b2-adrenergicreceptor and ETBR to caveolae might be different. Furtherstudies on the targeting mechanisms to caveolae arerequired.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 37 �C [23]. Mostof the b2-adrenergic receptors in cardiomyocytes are alsoexcluded from caveolae upon agonist stimulation by 30 minat 37 �C [20]. Although such a dramatic decrease in theabundance of ETBR in the caveolae of HEK293 cells wasnot observed, an � 12% reduction of the total ETBR wasobserved (Fig. 5). This slow reduction could be becausesignal transduction of the ETBR during the exit fromcaveolae might be required, or it may be due to incompleteET-1 binding at the cell surface, or to overexpression ofETBR in HEK293 cells. In addition, because most of theETBR at the cell surface is localized in nonlipid raftmembranes, the constitutive trafficking of ETBR fromnonlipid raft membranes or the newly synthesized ETBRmoving from inside the cells to the caveolae might mask afraction of the ETBR exiting from the caveolae. However,the decrease in the N-terminally cleaved ETBR (band II)was significant (Fig. 5C). The agonist-dependentN-terminal

cleavage of ETBR by metalloproteases on the cell surfacehas been shown, which yields band II, whose functionalsignificance is not known [37]. While the full-length ETBRmight be supplied from other domains in the cells, theN-terminally cleaved, agonist-bound ETBR may dissociatefrom caveolin-1 and be gradually excluded from thecaveolae. The agonist-regulated localization of ETBR onthe cell surface should be studied further using native tissuesor primary cultures of astrocytes, and endothelial cells,among others. Although a decrease of the ETBR in caveolaeseemed to be slow in HEK293 cells, the transient vasodil-atation due to the ETBR-induced nitric oxide release fromendothelial cells [1,2] might be regulated by interactions withcaveolins, 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-boundETAR via caveolae have been shown [8]. These propertiesare explained well by the fact that ETAR interacts withcaveolin-1, regardless of agonist binding. For ETBR, rapidinternalization to a degradative pathway upon agonistbinding in CHO cells [10,11] and constitutive internalizationto lysosomes in HeLa and Clone 9 cells [9] have beenreported. In contrast, the localization to caveolae micro-domains by interaction with caveolin-1 ensures that asubfraction of the ETBR is present on the cell surface totransmit ET-1 signals. The reduced ERK signalling via theETBR in filipin-treated cells might be due to rapid ETBRinternalization/degradation or simply due to the lack ofcaveolae, where signalling molecules are concentrated, asfilipin reduces the cell-surface caveolin [27]. Both extrememechanisms of ETBR action may be well balanced,depending upon the cell and tissue types.ETBR couples to multiple G proteins, mainly Gq and 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 beregulated by the intracellular conditions. While the Gqasubunit has been shown to bind caveolin and to beconcentrated in caveolae, the heterotrimeric Gi and Gs arelocalized in lipid rafts [47]. In the case of b2-adrenergicreceptor in neonatal cardiomyocytes, the localization incaveolae seems to regulate signal transduction by thelimiting access to Gs [48]. Although caveolae appear tofacilitate the ERK signalling activated by ETBR in CHOcells, other signalling pathways through ETBR could exist innonlipid rafts, because substantial amounts of ETBR arelocalized here. Therefore, localization to specific membranemicrodomains, such as caveolae, lipid rafts and nonlipidrafts, may also contribute toward specifying the signaltransduction 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 receptorproperties are regulated by the lipid environment [49]. Basedon the pharmacological heterogeneity of ETBR, the exist-ence of the ETB1R and ETB2R (tentatively termed) subtypeshas been suggested; nevertheless, there is no molecularbiological evidence supporting this. The ETB1R located onthe vascular endothelium mediates vasodilatation throughthe release of nitric oxide, which is sensitive to themixed ETAR/ETBR antagonist, PD 142893, bosentan,RES-S701-1, BQ788, etc. The other subtype (ETB2R),

Fig. 6. Filipin treatment impairs ETBR-mediated ERK1/2 signalling.

CHO cells transiently expressing ETBR were cultured in FBS-free

medium for 24 h. After a pretreatment with 0, 1 or 2 lgÆmL)1 filipinIII for 10 min at 37 �C, these cells were stimulated with 100 nM ET-1for 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-ERKmAb (lower panel). Pretreatment

with filipin attenuated the phosphorylation of ERK1/2 mediated by

the activation of ETBR. The bands for ERK1 (44 kDa) and ERK2

(42 kDa) were not separated in these gels.


located on the vascular smooth muscle cells, directlymediates vasoconstriction and is sensitive to BQ788, butinsensitive to PD 142893 [2,50,51]. Similarly, two subtypeswith different affinities (super-high and high affinity sites) toendothelins have been reported in the rat brain and atrium[52]. This pharmacological heterogeneity of ETBR mightbe due to the membrane lipid raft environments.In conclusion, the present study shows that ETBR

interacts with caveolin-1 in an ET-1-sensitive manner,suggesting that ETBR is targeted to caveolae by bindingto caveolin-1, and is excluded from caveolae by agonistbinding (Fig. 7). Although the physiological significance ofET-1-sensitive dissociation of the ETBR/caveolin-1 complexis not exactly clear at present, we suggest that thiscompartmentalization within caveolae ensures signal trans-duction by ETBR, in spite of the rapid internalization/degradation process. Further studies of ETBR in caveolaeand in nonlipid rafts using native tissues would providefurther insights into the properties of the subtypes, thepromiscuous coupling with G proteins, and the desensiti-zation mechanisms of ETBR.

Acknowledgements

We thank S. Satoh for useful suggestions and discussions through this

work. This work was supported by Grant-in-Aid for Specially

Promoted Research, Japan, and by the Japan New Energy and

Industrial Technology Development Organization.

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Regulated interaction of endothelin B receptor with caveolin-1