Biotechnology Letters 26: 293–299, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

293

Characterization and application of monoclonal antibodies against humanendothelin B receptor expressed in insect cells

Tomohiro Yamaguchi, Ikuyo Arimoto-Tahara, Yoshinori Fujiyoshi & Tomoko Doi∗Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto606-8502, Japan∗Author for correspondence (Fax: +81-75-753-4218; E-mail: doi@em.biophys.kyoto-u.ac.jp)

Received 28 October 2003; Revisions requested 27 November 2003; Revisions received 9 December 2003; Accepted 9 December 2003

Key words: cell surface staining, endothelin, endothelin B receptor, immunoaffinity chromatography, monoclonalantibody

Abstract

Endothelin B receptor (ETBR) is a G protein-coupled receptor that mediates a variety of signals by bindingto vasoconstrictive peptides, endothelins. Monoclonal antibodies were prepared against human ETBR using thefull-length protein expressed in Sf9 cells. Five typical monoclonal antibodies were characterized further for theirrecognition. The epitopes for the 2A5, 9A3 and 21A1 antibodies were mapped within the N-terminal extracellularsequences, V71-I85 and E27-Q41, respectively, which differ between the human and mouse ETBRs. All of theseantibodies labeled cell surface ETBR expressed in COS cells, suggesting that their recognition sites exist in theextracellular domain. In addition, the immobilized antibodies could purify ETBR expressed in Sf9 cells to themajority under mild conditions. Thus, immunization with the recombinant full-length membrane protein providesa strategy to produce monoclonal antibodies recognizing the native protein.

Introduction

Endothelins (ETs) are secreted by a variety of cellsin different tissues, including vascular endothelium,kidney, liver, heart, adrenals, and the central and peri-pheral nervous systems (Rubanyi & Polokoff 1994,Highsmith 1998). Similarly, the endothelin recept-ors (ETR) are also distributed throughout the body,and regulate not only vasoconstriction but also devel-opment, mitogenesis, cell proliferation, homeostasis,and so on (Ruffolo 1995). They are also implicatedin nociceptive signaling in the peripheral neurons ofanimals and humans (Khodorova et al. 2003). In mam-mals, two receptor subtypes, ETAR and ETBR havebeen identified. They displayed different rank ordersof affinity for the endothelin isopeptides, ET-1, ET-2 and ET-3. They are often found in different celltypes and support different functions, and in somecases they modulate each other’s function. While con-siderable biochemical and biological data on the ETsand the ETRs have been accumulated, the roles of the

ETBR in peripheral tissues and the regulation of itssignal transduction are not fully understood. Mono-clonal antibodies (mAbs) recognizing ETBR would beimportant tools for biological studies to understand theregulation by ETs and for the clinical pathology.

Synthetic peptides corresponding to the hydro-philic region of membrane proteins, conjugated withsuitable carriers, have yielded efficient and successfulresults in raising antibodies against integral membraneproteins. However, sometimes antibodies do not ef-ficiently recognize the native structure of the proteinin the membrane. In contrast, the use of bovine rod,outer-segment disc membranes or purified rhodopsinsuccessfully generated specific monoclonal antibod-ies (mAbs) against rhodopsin, a typical abundantly-occurring G protein-coupled receptor in photoreceptorrod cells (Adamus et al. 1991, MacKenzie et al.1984). Further characterization of the mAbs had ledto the establishment of a mild purification procedurefor rhodopsin (Oprian et al. 1987), thus support-ing extensive biological and biochemical studies of

294

Fig. 1. A secondary structure model of human ETBR. The putative seven α-helices are boxed. The residues differing from those in mouseETBR are represented by bold italic letters. The arrow indicates the signal peptidase cleavage site, and the asterisk indicates the N-linkedglycosylation site. The underlined N-1 sequence (71–85) is recognized by the 2A5 antibody, and the underlined N-3 and N-6 sequences arerecognized by the 9A3 and/or 21A1 antibodies.

rhodopsin and photoreceptor cells (Khorana 1992, Taiet al. 1999).

Previously, we established a milligram-scale puri-fication procedure for human ETBR expressed in in-sect Sf9 cells, by taking advantage of the slow dis-sociation of the receptor-ligand binding (Doi et al.1997). Using the purified, full-length human ETBRin a detergent-micelle solution, we raised antibod-ies against the receptor in mice. Although the mouseand human ETBRs share high sequence homology(∼80%), the N-terminal tail and the extracellular loopscontain considerable differences, as shown in Fig-ure 1. The cloned mAbs are capable of labeling thecell surface ETBR expressed in COS cells, and are ap-plicable to immunoaffinity chromatography. Thus, therecombinant full-length membrane protein facilitatesthe production of mAbs recognizing the native protein.

Materials and methods

Materials

The wild-type and mutant ETBRs (6hN-ETBR and�6h-ETBR) were expressed in insect Sf9 cells andpurified in the ligand-bound form by ligand-affinityand nickel-affinity chromatography in a 0.1% (w/v)digitonin-micelle solution, as described previously(Doi et al. 1997). The genes encoding the chimericreceptors, ETA/ETB and ETB/ETA, were constructedby mutating TTT (Phe 229) to ATG (Met) in the ETARgene to make an Nco I site. Afterwards, the genescorresponding to each of the half molecules were con-nected to each other at the Nco I sites of ETAR andETBR, corresponding to the 245–247 MDY sequence.The Sf9 membranes containing the wild-type or chi-meric receptors were prepared as described (Doi et al.1997).

295

Production of ETBR mAbs

All animal immunizations, enzyme-linked immun-osorbent assay (ELISA) screenings of mouse sera,and splenocyte-myeloma cell fusions were performedin conjunction with Immuno-Biological Laboratories(IBL) Co. Ltd. (Gunma, Japan). BALB/c mice wereimmunized subcutaneously with a 1:1 (v/v) emulsionof 20 µg purified 6hN-ETBR in adjuvant. After boost-ing with 10 µg purified 6hN-ETBR five times every7 d, the spleen from the hyperimmunized mouse wasfused to X63-Ag8.653 myeloma cells. The result-ing hybridomas were screened for specific mAbs byELISA coated with 50 ng 6hN-ETBR per well. Outof 17 hybridomas, five were selected and cloned bytwo-rounds of single-cell limiting dilutions.

The isolated hybridomas were cultured in TIL me-dia (IBL) supplemented with 10% fetal bovine serum.To collect the ascitic fluids, 1–2 × 107 hybridomacells per mouse were injected into the peritoneum ofpristane-pretreated mice, and after 3 weeks, the asciticfluids (3–5 ml/mouse) were collected. The mAbs werepurified from the ascitic fluids by protein G affinitychromatography. Approx. 0.3–6 mg mAbs per ml ofascitic fluid were obtained.

ELISAs for affinity comparison of the mAbs and forpeptide competition

The purified 6hN-ETBR in 0.1% digitonin, was dilutedwith PBS to 0.1 mg ml−1, and 25 µl (2.5 µg well−1)were used to coat 96 well-plates, which were in-cubated at 4 ◦C overnight and at 60 ◦C for 2 h todry. After the wells were blocked with 3% (w/v)BSA in PBS/0.02% NaN3, at 37 ◦C for 2 h or at4 ◦C overnight, the serially diluted mAbs in PBS/1%BSA were added to the wells and incubated at roomtemperature for 2 h. After washing with PBS, thesecondary antibody labeling and signal amplificationwere performed using a Vectastain Elite ABC kit(Vector Laboratories, Inc.) according to the manu-facturer’s procedures. For the peptide competition,mAbs (0.6 µg well−1) and the serially diluted pep-tides (0–1 mM) were incubated in microtiter wells withthe 6hN-ETBR coated as above. After 2 h at roomtemperature, the plates were treated as above.

Immunoaffinity purification

Each of the purified 2A5, 9A3, 16B3, and 21A1antibodies (2 mg) was incubated overnight with0.5 ml CNBr-activated Sepharose 4B (Amersham

Biosciences), washed, and blocked according tothe manufacturer’s procedures. The Sf9 membranes(200 mg protein) containing the wild-type ETBR weresolubilized in 20 ml 20 mM Tris/HCl (pH 7.5), 0.2 M

NaCl, 5 mM EDTA, and 1% digitonin with proteaseinhibitors at 4 ◦C for 1 h (Doi et al. 1997). The solubil-ized supernatant after ultracentrifugation was dividedinto four aliquots. Each of the antibody-immobilizedresins was incubated with an aliquot of the solubil-ized solution (approx. 5 ml) at 4 ◦C overnight. Afterextensive washing with 20 mM Tris/HCl (pH 7.5),0.15 M NaCl, 1 mM EDTA, and 0.1% digitonin, theETBR was eluted from the 2A5-Sepharose resin with0.5 ml 100 µM peptide N-1 (71V-85I) by three roundsof elution in washing buffer at room temperature for20 min each. The 9A3- and the 21A1-conjugated res-ins were eluted similarly, by three rounds of elutionwith 0.5 ml 100 µM peptide N-3 (27E-41Q). The16B3-Sepharose resin was eluted by two incubationswith 0.5 ml 50 mM sodium acetate (pH 4.2) at 4 ◦C for10 min, and the eluates were neutralized with 50 µl2 M Tris/HCl (pH 8) immediately after elution. Theligand binding activity of ETBR in detergent solutionwas measured using [125I-ET-1] (PerkinElmer LifeSciences) and Hydroxyapatite Bio-gel HT resin (Bio-Rad Laboratories), as described (Doi et al. 1997). Onealiquot of the solubilized supernatant contained 2,848pmol of [125I-ET-1] binding activity. The eluates fromthe 2A5-, 9A3-, 16B3-, and 21A1-immobilized res-ins contained 1,057.6, 697.6, 186, and 1001.8 pmolof [125I-ET-1] binding activity, and 0.5, 0.5, 0.2, and0.7 mg total protein, respectively.

Immunofluorescence staining of COS cells

COS-1 cells (3 × 105 cells per well) were seeded in a6-well plate containing coverslips coated with bovinefetal serum, and were cultured in Dulbecco’s modi-fied Eagle medium supplemented with 10% (w/v) fetalbovine serum for 14–16 h. The cells were transfec-ted with a plasmid containing the wild-type ETBRcDNA cloned in the pcDNA3.1 vector (Invitrogen LifeTechnology), using Lipofectamine reagent accordingto the manufacturer’s procedure, and were culturedfor another 40–48 h. For cell-surface staining, thecells were washed three times with 20 mM Hepes(pH 7.5)-buffered Hanks’ balanced salt solution at4 ◦C, blocked with 1% BSA in the above washingbuffer on ice for 30 min, and incubated with mAbs(50 µg ml−1) in the blocking buffer at 4 ◦C for 2 h.After three washes with the blocking buffer, the cells

296

were further incubated with rhodamine-conjugatedgoat anti-mouse IgG antibody in the washing buffer at4 ◦C for 1 h. After washing with the blocking buffer,the cell-attached coverslips were placed in methanolat −20 ◦C for 5 min and then in acetone at −20 ◦Cfor 2 min, mounted on glass slides, and observed witha fluorescence microscope. For staining permeabil-ized cells, after transfection the cells were fixed andpermeabilized with methanol/acetone at −20 ◦C, asabove. After rehydration in Hepes-buffered Hanks’balanced salt solution, the cells were incubated withmAbs (10 µg ml−1) at room temperature for 2 h,washed as above, and incubated further with therhodamine-conjugated second antibody at room tem-perature for 30 min. After washing, the coverslipswere mounted and observed.

Results and discussion

Human ETBR as an antigen in mice

The proteins involved in signal transduction are of-ten well-conserved among mammalian species. Thiscould be one of the reasons why useful monoclonalantibodies for these proteins are difficult to generatein mice. In addition, synthetic peptide-based immun-ization sometimes resulted in antibodies that lackedhigh affinity for the native proteins, and particularlyfor membrane proteins. The human and mouse ETBRsshare high sequence homology. However, their uniqueresidues are clustered in the extracellular domains(Figure 1). Therefore, we attempted to generate mono-clonal antibodies that recognize the receptor in thenative structure, using the full-length, ligand-boundhuman ETBR as an antigen in mice. As the anti-gen, we used 6hN-ETBR, which contained mutationsin the N-terminal tail to introduce hexahistidines andto avoid the glycosylation and proteolysis observedin mammalian cultured cells (Doi et al., 1997). Fig-ure 2A shows the 6hN-ETBR protein, which wasexpressed in Sf9 cells and purified to near homogen-eity by the ligand- and nickel-affinity chromatography,as described (Doi et al. 1997).

Five typical monoclonal antibodies

The screening of antibodies specific for ETBR coatedon microtiter plates yielded 17 positive hybridomas.The wild-type ETBR, purified in a detergent-micellesolution by affinity-chromatography, gradually de-graded from the termini when stored at 4 ◦C, resulting

Fig. 2. (A) The purified 6hN-ETBR used for immunization. Thepurified 6hN-ETBR was analyzed by SDS-12.5 % PAGE andvisualized by silver staining. The arrow indicates the band for6hN-ETBR. (B) Three typical blotting patterns (lanes 1–3) wereobserved when immunoblotting the proteolyzed wild-type ETBRwith the hybridoma culture supernatants. The arrow indicates thewild-type ETBR, and the arrowheads indicate proteolyzed species.In immunoblotting, alkaline phosphatase-conjugated anti-mouseIgG (Promega Biotech) was used as the second antibody.

Fig. 3. The affinities of mAbs to the ETBR. Serially diluted mAbswere measured for their binding to ETBR-coated microtiter plates.Three independent experiments were averaged. The 2A5, 9A3,16B3, and 21A1 antibodies showed similar affinities.

in many species. The cultured supernatants from 17hybridomas were used for immunoblotting againstETBR that had been stored for three months and gen-erated the three typical patterns shown in Figure 2B.One or two hybridomas showing strong signals foreach pattern were chosen and cloned in two-rounds.As a result, the 2A5 and 8Z11 antibodies showed thetype-1 pattern, the 16B3 antibody showed the type-2pattern, and the 9A3 and 21A1 antibodies showed thetype-3 pattern.

297

Fig. 4. Recognition of the mutant ETBR by the mAbs. (A) Thepurified wild-type ETBR (lane 1) and �6h-ETBR (lane 2) wereimmunoblotted with the 2A5, 9A3, 16B3, 21A1, and 8Z11 mAbs.The arrow indicates the band for the wild-type ETBR, and the ar-rowhead indicates the band for �6h-ETBR. The 9A3 and 21A1antibodies did not recognize the �6h-ETBR. (B) The wild-typeETBR (lane 1), the chimeric ETB/ETA receptor (lane 2), the chi-meric ETA/ETB receptor (lane 3), and the wild-type ETAR (lane4) were immunoblotted with the 2A5, 16B3, and 8Z11 antibodies.Fifteen to eighteen µg Sf9 membranes containing each receptorwere analyzed. The expression of each receptor was confirmed byimmunoblotting with the 1D4 antibody, whose recognition sequencewas attached to the C-terminus of each receptor (data not shown).All three mAbs recognized ETBR and ETB/ETA, but not ETA/ETBand ETAR, indicating that their recognition sites exist within theN -terminal half of the ETBR molecule.

The immunoglobulin subtype of the 2A5 antibodywas IgG1, while the other antibodies were IgG2a. ThemAbs were purified from the ascites cells, and theiraffinities for ETBR were compared by ELISA. TheELISA with serially diluted 2A5, 9A3, 16B3, and21A1 antibodies revealed their similar affinities forETBR (Figure 3). Each effective concentration thatdisplays by 50% the binding of mAbs to immobilizedETBR is ca. 70 ng ml−1, namely 0.5 nM under thecondition employed. The affinity of the 8Z11 antibodyfor ETBR was approximately one tenth of that of the2A5 antibody (data not shown).

The recognition sites of monoclonal antibodies

To investigate the recognition sites of the five mono-clonal antibodies, the N-terminal deletion mutant ofETBR (�6hETBR) (Figure 4A) and the chimeric

ETAR and ETBR receptors (Figure 4B) were immun-oblotted with these antibodies. While the 2A5, 8Z11,and 16B3 antibodies recognized the �6hETBR, inwhich the sequence between residues 29 to 64 wasreplaced with hexahistidines, the 9A3 and 21A1 anti-bodies did not recognize it (each lane 2 in Figure 4A),indicating that their recognition sites exist within thedeleted region. Using the chimeric receptors, in whichthe N- or the C-terminal half molecules of each re-ceptor were connected with the other half molecule atthe N-terminus of the second extracellular loop, the2A5, 16B3, and 8Z11 antibodies did not recognizethe ETA/ETB chimeric receptor (lane 3, Figure 4B)but they could recognize the ETB/ETA chimeric re-ceptor (lane 2, Figure 4B) indicating that their recog-nition sites lie within the N-terminal half of the ETBRmolecule.

To further localize the recognition sites of themAbs, synthetic peptides corresponding to part ofthe N-terminal tail or the first extracellular loop wereused to compete against the receptor antibody inter-action in the ELISA assay. Figure 5 shows that the2A5 antibody was competed with the peptide N-1(residues 71–85), whereas the 9A3 and 21A1 antibod-ies were competed with the peptide N-3 (residues 27–41). However, the 9A3 and 21A1 antibodies showeddifferent reactivities to the peptide N-6 (residues 27–35). Namely the 21A1 binding was not inhibited bythe peptide N-6, suggesting that the recognition siteof the 21A1 antibody exists within residues 36–41 ofthe N-terminal tail. In addition, these three antibodiesdid not bind to other synthetic peptides correspondingto the other region of N-terminal tail or the extracel-lular or cytoplasmic loops in the ELISA assay. Theseresults indicate that the recognition sites of the 2A5,9A3, and 21A1 antibodies were within the competingpeptide sequence, which differed between the humanand mouse ETBRs. In contrast, the interactions of the8Z11 and 16B3 antibodies with the ETBR were notcompeted by these peptides, and thus could not beattributed to a specific sequence.

Immunoaffinity column chromatography

To investigate whether the isolated mAbs were use-ful for immunoaffinity column chromatography, thefive mAbs were produced in ascites fluids and purifiedby protein-G affinity chromatography. The purified2A5, 9A3, 16B3, and 21A1 antibodies were immobil-ized on the resin and were mixed with solubilized Sf9membranes containing the wild-type ETBR. After the

298

Fig. 5. Competitive inhibition of the 2A5, 9A3, and 21A1 antibody binding to the ETBR by synthetic peptides. The synthetic peptides, N-1 andN-3, efficiently inhibited the binding of the 2A5, 9A3, and 21A1 antibodies to the ETBR. The N-6 peptide weakly inhibited the 9A3 antibodybinding to the ETBR.

Fig. 6. Immunopurification of the ETBR. The solubilized su-pernatant of Sf9 cell membranes was purified with anti-body-immobilized resins. The eluates from four resins were ana-lyzed by SDS-12.5% PAGE and visualized by silver staining. Thearrow indicates the ETBR purified to the majority. The recovery(marked by an asterisk) was calculated based on the ligand bindingactivities before and after purification.

resins were incubated and washed, the bound ETBRswere eluted with 100 µM of the competing peptidesdescribed above for the 2A5, 9A3, and 21A1 antibod-ies. The 16B3-bound ETBR was eluted with sodiumacetate buffer (pH 4.2) at 4 ◦C. Figure 6 shows thepurities of each eluate and their recovery, based onthe ligand binding activity of the ETBR. Each resinwas able to purify the expressed ETBR to the major-

ity, and the 2A5-resin gave 37% recovery. Thus, inaddition to the efficient binding of the ETBR to theresin, the elution from the resin with competing pep-tides assists in the mild and specific purification, asshown for bovine rhodopsin (Oprian et al. 1987). Be-cause G protein-coupled receptors are easy to denatureafter solubilization, mild conditions are required dur-ing their purification. Utilization of this purificationmethod, combined with hexahistidine-tagged receptorand nickel-affinity chromatography allows to obtainbetter purity and to evaluate the receptor function.Mutant ETBRs were purified from Sf9 membranes,reconstituted into phospholipid vesicles, and evalu-ated for their G protein coupling properties (Doi et al.1999, Imamura et al. 2000). In addition, the 2A5-immobilized resin was successfully used in an immun-oprecipitation to investigate the interaction of ETBRwith caveolin-1 (Yamaguchi et al. 2003).

Cell surface labeling with mAbs

To analyze the accessibilities of the mAbs to ETBRin the cell surface membrane, the ETBR expressed inCOS cells was labeled with mAbs before permeabiliz-ation, accompanied by secondary antibody labeling.All of the 2A5, 8Z11, 9A3, 16B3, and 21A1 an-tibodies bound to the cell surface ETBR, as shownin Figure 7, panels A, B, C, E, and F, respectively.Figure 7D shows COS cells labeled with the 2A5 anti-body, after fixation and permeabilization. The otherantibodies also produced similar patterns to that ofpanel D in Figure 7 for the ETBR in permeabilizedcells. In addition, paraformaldehyde-treated ETBRsamples for negative staining or freeze fracture, weresuccessfully labeled by the 2A5 antibody (K. Nishi-

299

Fig. 7. Immunofluorescence staining of the ETBR. The COS cells expressing the wild-type ETBR were immunofluorescently labeled bythe 2A5 (panels A and D), 8Z11 (panel B), 9A3 (panel C), 16B3 (panel E), and 21A1 (panel F) antibodies. The COS cells were labeledbefore (panels A–C, E and F) or after (panel D) permeabilization. The cell edges were stained distinctly in the unpermeabilized cells, andthe permeabilized cells showed strong signals in the nuclear and perinuclear regions. The 8Z11, 9A3, 16B3, and 21A1 antibodies generatedstaining patterns for permeabilized cells similar to that in panel D, produced by the 2A5 antibody (data not shown). The bar represents 50 µm.

kawa and Y. Fujiyoshi, personal communication).Thus, these cloned mAbs are capable of recogniz-ing ETBR in the membrane and are applicable forimmuno-cytochemical studies.

Acknowledgement

This work was supported by the Japan New Energyand Industrial Technology Development Organization(NEDO).

References

Adamus G, Zam ZS, Arendt A, Palczewski K, McDowell JH,Hargrave PA (1991) Anti-rhodopsin monoclonal antibodies ofdefined specificity: characterization and application. Vision Res.31: 17–31.

Doi T, Hiroaki Y, Arimoto I, Fujiyoshi Y, Okamoto T, Satoh M,Furuichi Y (1997) Characterization of human endothelin B re-ceptor and mutant receptors expressed in insect cells. Eur. J.Biochem. 248: 139–148.

Doi T, Sugimoto H, Arimoto I, Hiroaki Y, Fujiyoshi Y (1999) In-teractions of endothelin receptor subtypes A and B with Gi, Go,and Gq in reconstituted phospholipid vesicles. Biochemistry 38:3090–3099.

Highsmith RF, ed. (1998) Endothelin: Molecular Biology,Physiology, and Pathology. New Jersey: Humana Press.

Imamura F, Arimoto I, Fujiyoshi Y, Doi T (2000) W276 mutation inthe endothelin receptor subtypes B impairs Gq coupling but notGi or Go coupling. Biochemistry 39: 686–692.

Khodorova A, Navarro B, Jouaville LS, Murphy LE, Rice FL,Mazurkiewicz JE, Long-Woodward D, Stoffel M, Strichartz GR,Yukhananov R, Davar G (2003) Endothelin-B receptor activationtriggers an endogenous analgesic cascade at sites of peripheralinjury. Nat. Med. 9: 1055–1061.

Khorana HG (1992) Rhodopsin, photoreceptor of the rod cell. J.Biol. Chem. 267: 1–4.

MacKenzie D, Arendt A, Hargrave P, McDowell JH, Molday RS(1984) Localization of binding sites for carboxy terminal specificanti-rhodopsin monoclonal antibodies using synthetic peptides.Biochemistry 23: 6544–6549.

Oprian D, Molday RS, Khorana GH (1987) Expression of a syn-thetic bovine rhodopsin gene in monkey kidney cells. Proc. Natl.Acad. Sci. USA 84: 8874–8878.

Rubanyi GM, Polokoff A (1994) Endothelins: molecular bio-logy, biochemistry, pharmacology, physiology, pathophysiology.Pharmacol. Rev. 48: 325–415.

Ruffolo Jr RR, ed. (1995) Endothelin Receptors: From the Gene tothe Human. Boca Raton, FL: CRC Press.

Tai AW, Chuang JZ, Bode C, Wolfrum U, Sung CH (1999) Rhodop-sin’s carboxy-terminal cytoplasmic tail acts as a membranereceptor for cytoplasmic dynein by binding to the dynein lightchain Tctex-1. Cell 97: 877–887.

Yamaguchi T, Murata Y, Fujiyoshi Y, Doi T (2003) Regulated inter-action of endothelin B receptor with caveolin-1. Eur. J. Biochem.270: 1816–1827.