Báo cáo khoa học: Regulated interaction of endothelin B receptor with caveolin-1 potx

Regulated interaction of endothelin B receptor with caveolin-1 Tomohiro Yamaguchi 1 , Yasunobu Murata 1 , Yoshinori Fujiyoshi 1,2 and Tomoko Doi 1 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 (ET A R) and B (ET B R) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ET B R with caveolin-1, expressed in Sf9, COS-1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ET B R. ET B R 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 ET B Rwith caveolin-1. The complex formed efficiently only when the ET B R 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 ET B R by caveolin-1. In contrast, the ET A R 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 ET B R, as for other signalling molecules. Furthermore, the amount of ET B R 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 ET B R 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 (ET A R) and B (ET B R) [1,2]. For example, an ET A R mediates vasocon- striction in vascular smooth muscle cells, whereas an ET B R 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 ET A RandET B R undergo rapid desensitization [4–6] and internalize differently in transfected cells, as shown for many GPCRs [7]. Concerning the intracellular trafficking path- ways, ET A R is internalized rapidly, either via caveolae or clathrin-coated pits, upon ligand binding [3,8,9] and follows a recycling pathway, whereas ET B R follows a degradative pathway after internalization via coated-pits, implicated for clearance of plasma ET-1 [10]. Moreover, the ET B Rinthe plasma membrane is constitutively transported to this pathway independently of ligand stimulation, indicating that ET B R 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 Rand 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,fulllength. (Received 27 November 2002, revised 11 February 2003, accepted 27 February 2003) Eur. J. Biochem. 270, 1816–1827 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03544.x mediate the interactions with the signalling molecules described above [17]. On the other hand, the caveolin- binding sequence motif (FXFXXXXF or FXXXXFXXF, where F 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, ET A , angiotensin II type 1, and adenosine A 1 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 A 1 and b 2 -adrenergic receptors are translocated out of caveolae upon activation, and the binding of ET A Rwith caveolins is not affected by agonist binding. Furthermore, the m2 muscarinic acetylcholine and ET A receptors could be internalized via caveolae [8,26]. The mitogenic signal through ET B R in primary astrocytes, where the ET B Ris relatively well expressed, originates from caveolae micro- domains [27]. In addition, a number of proteins mediating intracellular Ca 2+ 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 ET B R 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 ET B R in the ground-state structure bound to caveolin-1, through the caveolin scaffolding and C-terminal domains, and that a fraction of ET B Rwas 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 ET B R. These results suggest feasible regulations for ET B R signal transduction by the interaction with caveolin-1. The residence of ET B R in caveolae might be a way of ensuring the cell surface localization of ET B R against rapid internalization. Experimental procedures Materials The cyclic-peptide antagonist for ET B R, RES701-1, was generously provided by M. Yoshida (Kyowa Hakko Kogyo Co., Ltd, Tokyo, Japan). The cDNAs encoding human ET A RandET B R 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 ET B R, BQ788, was from Phoenix Pharmaceuticals, Inc. The anti-ET B R mAb, 2A5, was generated against Sf9-expressed human ET B R(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 ET B R cDNA was subcloned into the BamHI–NotI sites of pcDNA3.1. Mutagenesis of the ET B R cDNA was carried out with appropriate oligonucleo- tide primers by a PCR-based site-directed mutagenesis approach. In each ET B R 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. IntheCav.1-H6cDNA,aHis 6 -tag was attached to the C terminus of caveolin-1 for affinity purification using Ni 2+ –NTA agarose (Qiagen). The 1D4 epitope sequence (KTETSQVAPA, an epitope for the anti-rhodopsin mAB) was fused to the C terminus of ET A R. For expression in insect cells, the cDNAs encoding caveolin-1, Cav.1-H6, ET B R, and ET A R-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 37 °C in a 5% CO 2 atmosphere. 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 ET B Rs, 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 ET B R, the cells were transfected with the plasmid pcDNA3.1 containing the ET B R 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 ET B R expression level by the HEK293 cell lines isolated for further studies was 1–2 pmol per mg membrane protein. Ó 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 ET B R in Sf9 cells were performed as described previously [28]. The expression of caveolin-1 and His 6 -tagged caveolin-1 (Cav.1- H6) was also performed similarly. Co-expression of ET B R and caveolin-1 in Sf9 cells was carried out by a double infection with ET B R and caveolin-1 recombinant viruses. Purification of ET B R and caveolin-1 from Sf9 cells All operations were carried out at 4 °C. Purification of ET B R by ligand-affinity chromatography using biotinylated ET-1, and reconstitution of the purified ligand-free ET B R into phospholipid vesicles were performed as described previously [28,29]. Cav.1-H6 was purified from infected Sf9 cells using Ni 2+ –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 m M Tris/HCl pH 7.5, 150 m M NaCl) with protease inhibitors (1 m M phenylmethylsulfonyl fluoride, 10 lgÆmL )1 aproti- nin, 10 lgÆmL )1 leupeptin, 10 lgÆmL )1 pepstatin), 60 m M 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 m M imidazole and were incubated with 200 lLNi 2+ -nitrilotriacetic acid-agarose beads overnight. After the incubation, the beads were washed extensively with washing buffer (NaCl/Tris con- taining 20 m M imidazole, 30 m M OG, 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 m M imidazole, 30 m M OG, and 0.03% Triton X-100) by rotating for 1 h. Immunoprecipitation All preparations were carried out at 4 °C unless 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 m M Tris/HCl pH 7.5, 150 m M NaCl, 1 m M EDTA) containing protease inhibitors (1 m M phenylmethanesulfo- nyl fluoride, 10 lgÆmL )1 aprotinin, 10 lgÆmL )1 leupeptin, 10 lgÆmL )1 pepstatin), and incubated with or without 2 l M ET-1 for 1 h at room temperature. After the incubation, the membranes were solubilized with 60 m M 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 lLof 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 l M of either the 1D4 or 2A5 epitope peptides for 1 h at room temperature. Purified ET B R and Cav.1-H6. Reconstituted phospho- lipid vesicles with or without ET B R(1–2pmol)were incubated with 1 l M 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 m M ; Triton X-100, 0.002–0.003%) so that the membranes were not solubilized. After incubation, the membranes were solubilized with 60 m M 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 ET B R (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 ET B Rs expressed in COS cells. Thirty six hours after transfection with the wild type ET B R cDNA, each mutant ET B R cDNA or the vector only, COS cells (one 100-mm diameter plate per sample) were washed twice with NaCl/P i , lysed with 1 mL NaCl/Tris/EDTA containing 60 m M 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 m M Tris/HCl pH 7.5, 1m M EDTA, and protease inhibitors) to burst the cells, incubated with 1 l M ET-1 for 1 h at room temperature, solubilized with 60 m M 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 ET B Rstably and caveolin-1 transiently (1–2 mgÆprotein per sample), were resuspended in NaCl/Tris/EDTA containing protease inhibitors, and incubated with 1 l M ET-1, 10 l M RES- 701-1, or 10 l M BQ788 for 1 h at room temperature. After this incubation, the samples were solubilized with 60 m M 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 4 °C. The membranes 1818 T. Yamaguchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 were washed with buffer (5 m M Tris/HCl pH 7.5, 150 m M 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 ET B Rwereculturedin serum-free medium for 6 h, and were then incubated with or without 100 n M ET-1 for 30 min at 37 °C.The following operations, except for the elution, were carried out at 4 °C. After the incubation, the confluent HEK293 cells of two 100-mm diameter plates ( 8 mg total protein) per sample were washed twice with NaCl/P i , scraped into 2 mL buffer (25 m M Mes pH 6.5, 150 m M 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 )1 filipin III, or vehicle for 10 min at 37 °C, and then incubated with 100 n M ET-1 or vehicle for 5 min at 37 °C. After stimulation, the cells were washed twice with ice-cold NaCl/P i , and were lysed with 100 lL RIPA buffer (50 m M Tris/HCl pH 7.5, 150 m M NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 0.5 m M Na 3 VO 4 ,50m M NaF, 5 m M EDTA and protease inhibitors) at 4 °C. 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 95 °C. 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 ET B R contained in the samples were measured by the 125 I-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 ET B R directly binds to caveolin-1 in an ET-1-dependent manner To examine the interaction of ET B R with caveolin-1, we took advantage of an insect cell expression system, in which ET A RandET B R are expressed well, with K D values for ET-1 of 55 ± 8 and 83 ± 11 p M ,theB max 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 ET A R-1D4 (ET A R fused with the 1D4 epitope at the C terminus) and ET B R were each coexpressed with caveolin-1 in insect Sf9 cells, incubated with or without ET-1, and immunoprecipitated with the 1D4 and 2A5 anti- ET B R mAbs, respectively, as shown in Fig. 1A. We extensively used 60 m M OG/1% Triton X-100 mixed- micelle conditions for the membrane solubilization before immunoprecipitation, which allowed the recovery of ligand- free ET B R, as described later, and the solubilization of many Triton-insoluble proteins [31]. The eluates from the antibody-immobilized resin were analysed for caveolin-1, ET A R-1D4, and ET B R by immunoblotting. The caveolin-1 coimmunoprecipitated with the ET A R-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, ET B R 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 ET B R expression (lane 4). These results suggest that ET A R binds to caveolin-1 regardless of ET-1 binding, whereas ET B R 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 ET B Rwith caveolin-1, we purified ET B R and caveolin-1 in detergent- micelles. ET B R, expressed in Sf9 cells, was purified on a ligand-affinity column eluted with 2 M NaSCN to yield ligand-free ET B R [29] (Fig. 1B, lane 1). Caveolin-1, with a His 6 -tag fusion at the C terminus (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 a i subunit in detergent micelles, as detected by immunoprecipitation, and as described previ- ously (data not shown) [32]. The purified ET B Rwas 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 ET B R (lane 1), it coimmunoprecipi- tated from the ET B R-containing vesicles (lane 2). Moreover, the addition of ET-1 significantly reduced the amount of the Ó FEBS 2003 Endothelin-1 dissociates the ETBR–caveolin-1 complex (Eur. J. Biochem. 270) 1819 precipitated 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 ET B Rwith caveolin-1. Interestingly, ET B R 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 ET B R and caveolin-1 occurs by two-dimensional molecular movements along the membranes, or that the integration of ET B R and caveolin-1 into the lipid bilayer stabilizes the structures required for the interaction, or that the detergent molecules binding to the ET B R 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). 1820 T. Yamaguchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The scaffolding domain and the C-terminal domain of caveolin-1 both interact with ET B R 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,whereU is an aromatic amino acid residue), which have been suggested to be responsible for the binding to caveolin [18]. To investigate the interaction between ET B R 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 ET B R were incubated with these purified fusion proteins, and immunoprecipitataed with the 2A5 mAb. While the ET B Rs 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 ET B R, 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 ET B R recognized by caveolin Since caveolin interacts with ET B R via the scaffolding and C-terminal domains, the caveolin-binding motifs could be the sites of these interactions in ET B R. However, these motifs are not present in the cytoplasmic and transmem- brane domains of ET B R, 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 ET B RsexpressedinCOScells,shownin 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 ET B Rs 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 ET B Rs in the ligand-sensitive caveolin-1 binding. The C-terminal truncated ET B R (residues 408–442 deleted) also showed these properties in the COS cell system (data not shown). Therefore, single mutations of these residues in the ET B R 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 ET B R (Fig. 1) indicates that caveolin-1 could distinguish the structure of the ligand-free ET B Rfrom that of the ligand-bound form. To examine the contribu- tions of the ET B R tertiary structure to the recognition by caveolin-1, we further analysed the interactions of caveo- lin-1 with ET B R 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 ET B R structure in the ground-state, but BQ788 did not [28]. The HEK293 cells stably expressing ET B R 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 °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 ET B R.WhileGSTaloneandGST– 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. Ó FEBS 2003 Endothelin-1 dissociates the ETBR–caveolin-1 complex (Eur. J. Biochem. 270) 1821 and ET B R by immunoblotting (Fig. 4A). The ET B R expressed in HEK293 cells was also observed as two bands in immunoblotting. Fig. 4B shows the extents of caveolin-1 bound to ET B R, as shown in Fig. 4A, relative to the binding to the ligand-free ET B R (lane 1) as 1.0. As observed with the Sf9 membranes (Fig. 1), the extent of caveolin-1 binding to ET B R was reduced to 0.35 ± 0.03 by the addition of ET-1 (lane 2). However, the inverse-agonist, RES-701-1-bound ET B R retained caveolin-1-binding activity (0.98 ± 0.13, lane 3) similar to that of the ligand-free form, whereas the BQ788-bound ET B R reduced the activity (0.42 ± 0.13, lane 4). The results suggest that the ET B R, 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 ET B R to the caveolae membrane and ET-1 releases ET B R from caveolae To examine the effects of caveolin-1 on the localization of ET B R,acelllinestablyexpressingET B R was isolated using HEK 293 cells, which do not express endogenous caveolin. The expression level of ET B R in this cell line is approxi- mately 1–2 pmolÆmg )1 membrane protein. The ET B R 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 ET B R was fractionated to the low-density and the bottom fractions, suggesting that a fraction of ET B R was targeted to the caveolae membranes. Since the treatment with Triton X-100 denatured the ET B R that was solubilized from the nonlipid raft membranes, the approximate distribution of ET B R, 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 ET B R was fractionated to the low-density fraction, based on the ligand binding activities of ET B Rinthelow- density fraction and in the plasma membrane fraction (data not shown). The ET B R found in the detergent-resistant, low-density fraction (DRM, combined fractions 2 and 3 in Fig. 5A) is shown in Fig. 5B, while Fig. 5C represents the averaged band intensities of ET B RobservedinFig.5Bfromfive repeated experiments, relative to the band intensity of total ET B 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. 1822 T. Yamaguchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (N-terminally cleaved isoform)] without the expression of caveolin-1 (lane 1). The intensities of the total and the band II ET B R 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 ET B R was scarcely recovered in the low-density fraction (lane 1). On the other hand, when caveolin-1 was expressed, the total amount of ET B R 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 37 °C, the total amount of ET B R was slightly reduced. In addition, the amount of N-terminally cleaved ET B R (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 ET B R 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 ET B R 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 ET B R in HEK293 cells is not cleaved at the cell surface without agonist stimulation, and that metallo- proteases cleave the N terminus of agonist-bound ET B Rat 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 ET B R might not be quantitative 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-bound ET B R 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 ET B R 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 ET B R 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 ET B Rwas 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 ET B R, although the effects of filipin III on caveolae and the ET B R are not exactly clear. In addition, these results are consistent with the report showing impaired ERK and focal adhesion kinase signal transduction via the ET B R in filipin III-treated primary astrocytes [27]. Fig. 4. Effects of antagonists on the interaction of ET B Rwith 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. Ó FEBS 2003 Endothelin-1 dissociates the ETBR–caveolin-1 complex (Eur. J. Biochem. 270) 1823 Discussion We studied the interaction of ET B R and caveolin-1 in vitro and in vivo, using the expressed proteins in insect and mammalian cells. The ligand-free ET B R 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 ET B R to the membrane microdomain, caveolae, and ET-1 stimulation translocated the ET B R out of caveolae, which when disrupted, diminished the ET B R-derived signal pro- pagation. This is the first report showing the caveolin-1- and ET-1-regulated localization of the ET B Randthedirect interaction of a GPCR with caveolin. The heterologously expressed ET B R and caveolin-1 formed a complex after purification and reconstitution into vesicles, suggesting their direct interaction. Interestingly, this interaction requires the existence of the ET B R within the lipid bilayer, and does not occur in the detergent micelle. Considering the fact that ET B R 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 ET B R might be important for the interaction. However, we could not specify the region of ET B R involved in the caveolin-1 binding. At the very least, the conformational changes of ET B R affect this interaction, and the ground-state structure of ET B R is required for the interaction with caveolin-1. The discrimination by caveolin-1 of the RES-701-1- bound and BQ788-bound forms of ET B R agrees well with the previous observation, in which a cyclic peptide antagonist, RES-701-1, could antagonize an ET B Rartifi- 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 ET B R 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 ET B RtargetedtheET B R to the Triton-insoluble, low buoyant density fraction, caveolae. These results Fig. 5. Caveolin-1 targets ET B Rtocaveolae.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.Thelysateswere 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 Rand2lL 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 Rrecov- 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. 1824 T. Yamaguchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 suggest that a fraction of ET B R is localized in caveolae, driven by the interaction with caveolin-1, although the localization efficiency was only 7% of the cell surface ET B R. In primary astrocytes, substantial amounts of ET B Rare localized in the caveolae fraction [27]. The localization of ligand-free GPCR to caveolae has been reported for the adenosine A 1 and b 2 -adrenergic receptors, which interact with caveolin-3 in cardiomyocytes, while the b 2 -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 b 2 -adren- ergic receptor, but not in the case of ET B R. Therefore, the molecular mechanisms used in the targeting of b 2 -adrenergic receptor and ET B R to caveolae might be different. Further studies on the targeting mechanisms to caveolae are required. Upon agonist addition, the adenosine A 1 receptors in ventricular myocytes dissociate from caveolin-3 and trans- locate out of the caveolae within 15 min at 37 °C [23]. Most of the b 2 -adrenergic receptors in cardiomyocytes are also excluded from caveolae upon agonist stimulation by 30 min at 37 °C [20]. Although such a dramatic decrease in the abundance of ET B R in the caveolae of HEK293 cells was not observed, an  12% reduction of the total ET B Rwas observed (Fig. 5). This slow reduction could be because signal transduction of the ET B R 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 ET B R in HEK293 cells. In addition, because most of the ET B R at the cell surface is localized in nonlipid raft membranes, the constitutive trafficking of ET B Rfrom nonlipid raft membranes or the newly synthesized ET B R moving from inside the cells to the caveolae might mask a fraction of the ET B R exiting from the caveolae. However, the decrease in the N-terminally cleaved ET B R (band II) was significant (Fig. 5C). The agonist-dependent N-terminal cleavage of ET B R by metalloproteases on the cell surface has been shown, which yields band II, whose functional significance is not known [37]. While the full-length ET B R might be supplied from other domains in the cells, the N-terminally cleaved, agonist-bound ET B R may dissociate from caveolin-1 and be gradually excluded from the caveolae. The agonist-regulated localization of ET B Ron 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 ET B Rincaveolae seemed to be slow in HEK293 cells, the transient vasodil- atation due to the ET B R-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 ET B R [9–11]. The colocalization and coimmunoprecipitation of ET A R with caveolin-1 [21] and the internalization of ligand-bound ET A R via caveolae have been shown [8]. These properties are explained well by the fact that ET A Rinteractswith caveolin-1, regardless of agonist binding. For ET B R, 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 ET B R is present on the cell surface to transmit ET-1 signals. The reduced ERK signalling via the ET B R in filipin-treated cells might be due to rapid ET B R 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 ET B R action may be well balanced, depending upon the cell and tissue types. ET B R couples to multiple G proteins, mainly G q and G i in native tissues but also to G s and G o in a few tissues, cultured cells, and in vitro [2,28,45,46], and this could be regulated by the intracellular conditions. While the G q a subunit has been shown to bind caveolin and to be concentrated in caveolae, the heterotrimeric G i and G s are localized in lipid rafts [47]. In the case of b 2 -adrenergic receptor in neonatal cardiomyocytes, the localization in caveolae seems to regulate signal transduction by the limiting access to G s [48]. Although caveolae appear to facilitate the ERK signalling activated by ET B RinCHO cells, other signalling pathways through ET B R could exist in nonlipid rafts, because substantial amounts of ET B Rare 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 ET B R. Furthermore, the recent report that EGF receptors in noncaveolar lipid rafts show lower EGF binding (B max ), than those in nonlipid rafts, suggests that the receptor properties are regulated by the lipid environment [49]. Based on the pharmacological heterogeneity of ET B R, the exist- ence of the ET B1 RandET B2 R (tentatively termed) subtypes has been suggested; nevertheless, there is no molecular biological evidence supporting this. The ET B1 R located on the vascular endothelium mediates vasodilatation through the release of nitric oxide, which is sensitive to the mixed ET A R/ET B R antagonist, PD 142893, bosentan, RES-S701-1, BQ788, etc. The other subtype (ET B2 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 )1 filipin 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. Ó FEBS 2003 Endothelin-1 dissociates the ETBR–caveolin-1 complex (Eur. J. Biochem. 270) 1825 [...]... (Eur J Biochem 270) Fig 7 A model for regulated < /b> localization of < /b> ETBR by caveolin-1 and agonist stimulation This figure illustrates the regulated < /b> localization of < /b> ETBR by caveolin-1 and ET-1, according to our findings in this report A fraction of < /b> ETBR bound to caveolin-1 is targeted to caveolae, where Ca2+ signalling and other signalling molecules are concentrated Upon agonist stimulation, the ETBR dissociates... subtypes with different affinities (super-high and high affinity sites) to endothelins have been reported in the rat brain and atrium [52] This pharmacological heterogeneity of < /b> ETBR might be 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 binding to caveolin-1, ... 6439–6446 9 Abe, Y., Nakayama, K., Yamanaka, A., Sakurai, T & Goto, K (2000) Subtype-specific trafficking of < /b> endothelin < /b> receptors J Biol Chem 275, 8664–8671 10 Bremnes, T., Paasche, J.D., Mehlum, A., Sandberg, C., Bremnes, B & Attramadal, H (2000) Regulation and intracellular trafficking pathways of < /b> the endothelin < /b> receptors J Biol Chem 275, 17596–17604 11 Paasche, J.D., Attramadal, T., Sandberg, C., Johansen,... of < /b> caveolins with the catalytic subunit of < /b> protein kinase A J Biol Chem 274, 26353–26360 34 Schubert, A.L., Schubert, W., Spray, D.C & Lisanti, M.P (2002) Connexin family members target to lipid raft domains and interact with caveolin-1 Biochemistry 41, 5754–5464 35 Takasuka, T., Adachi, M., Miyamoto, C., Furuichi, Y & Watanabe, T (1992) Characterization of < /b> endothelin < /b> receptors ETA and ETB expressed... 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Schweighoffer, F., Strosberg, A.D & Couraud, P.O (1994) Endothelin < /b> induces tyrosine phosphorylation and GRB2 association of < /b> Shc in astrocytes J Biol Chem 269, 24805–24809 Shapiro, P.S., Evans, J.N., Davis, R.J & Posada, J.A (1996) The seven-transmembrane-spanning receptors for endothelin < /b> and thrombin cause proliferation of < /b> airway smooth muscle cells and activation of < /b> the extracellular regulated < /b> kinase and . 4216, E-mail: doi@em.biophys.kyoto-u.ac.jp Abbreviations: ET, endothelin; ETR, endothelin receptor; ET A Rand ET B R, endothelin receptor type A and type B; GPCR,. 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